Nonproliferative and Proliferative
Lesions of the Rat and Mouse
Hematolymphoid System

Toxicologic Pathology
2019, Vol. 47(6) 665-783ª The Author(s) 2019
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DOI: 10.1177/0192623319867053
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Cynthia L. Willard-Mack, (Chair)1, Susan A. Elmore2,c ,William C. Hall3, Johannes Harleman4,e,g, C. Frieke Kuper5,f,Patricia Losco6,a, Jerold E. Rehg7,d,g, Christine Rühl-Fehlert8,g,Jerrold M. Ward9,d,g, Daniel Weinstock10,b, Alys Bradley11,
Satoru Hosokawa12, Gail Pearse13, Beth W. Mahler14,
Ronald A. Herbert2, and Charlotte M. Keenan15

Abstract
The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a
joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America(STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The
purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoidorgans, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues(serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of thelesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout
the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature forthese organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The
rationale for this approach and guidance for its application to toxicologic pathology are described in detail below.
Keywords
INHAND, nomenclature, diagnostic criteria, lymphoid, bone marrow, thymus, spleen, lymph node, MALTa

General hematolymphoid subgroup lead
Bone marrow subgroup leadcThymus subgroup leaddSpleen subgroup leadseLymph node subgroup leadf
Associated lymphoid organs subgroup leadg
Neoplasm subgroup leadsb

1

Covance, Somerset, NJ, USA
National Toxicology Program, National Institute of Environmental Health
Sciences, Research Triangle Park, NC, USA
3
Hall Consulting, Inc, Mt Airy, MD, USA
4
Independent Consultant, Darmstadt, Germany
5
Independent Consultant, Utrecht, the Netherlands
6
Independent Consultant, West Chester, PA, USA

7Saint Jude Children’s Research Hospital, Memphis, TN, USA
Bayer AG, Wuppertal, Germany
9
Global VetPathology, Montgomery Village, MD, USA
10
Janssen, Spring House, PA, USA
11Charles River Laboratories, Tranent, Scotland, United Kingdom
12Eisai Co, Ltd, Drug Safety Research Laboratories, Ibaraki, Japan
13
GlaxoSmithKline, Ware, United Kingdom
14
Experimental Pathology Laboratories, Research Triangle Park, NC, USA
15
CM Keenan ToxPath Consulting, Doylestown, PA, USA
8

2Corresponding Author:
Cynthia L. Willard-Mack, Covance, PO Box 2360 Mettlers Road, East Millstone,
NJ New Jersey 08875-2360, USA.
Email: cynthia.willard-mack@covance.com

666
Introduction
Best Practices and Diagnostic Challenges
General Hematolymphoid
Amyloid
Aplasia/hypoplasia
Apoptosis, increased, lymphocyte
Cellularity, increased, mast cell
Extramedullary hematopoiesis (EMH)
Infiltrate
Inflammation
Metaplasia, osseous
Mineralization
Necrosis
Phospholipidosis
Pigment, macrophage
Tingible body macrophage, increased
Vacuolation, macrophage
Bone Marrow
Organization
Function
Development
Histology
Sampling and Diagnostic Considerations
Nonproliferative Changes
Angiectasis
Cellularity, decreased, adipocyte
Cellularity, decreased, bone marrow
Dyshematopoiesis
FibrosisHypersegmentation, granulocyte
Inflammation—see General Hematolymphoid
Necrosis—see General Hematolymphoid
Serous atrophy of fat
Proliferative Changes (Non-Neoplastic)
Cellularity, increased, adipocyte
Cellularity, increased, bone marrow
Cellularity, increased, macrophage
Cellularity, increased, mast cellThymusOrganization
Function
Development
Histology
Sampling and Diagnostic Considerations
Nonproliferative Changes
Apoptosis, increased, lymphocyte
Cellularity, decreased, lymphocyte
Corticomedullary ratio, decreased
Corticomedullary ratio, increased
Cyst, epithelial
Ectopic tissue, parathyroid
Ectopic tissue, (specify tissue)
Ectopic tissue, thymus
Hypoplasia
Inflammation—see General Hematolymphoid
Involution, age-related

Toxicologic Pathology 47(6)Loss of corticomedullary distinction
Necrosis, lymphocyte
Tingible body macrophage, increased
Proliferative Changes (Non-Neoplastic)
Cellularity, increased, epithelial cell
Cellularity, increased, lymphocyte
Epithelium-free areas, increased
Thymic corpuscles, increased
SpleenOrganization
Function
Development
Histology
Sampling and Diagnostic Considerations
Nonproliferative Changes
Aplasia/hypoplasia—see General hematolymphoid
White Pulp
Apoptosis, increased, lymphocyte
Cellularity, decreased, white pulp
Necrosis, lymphocyte—see General Hematolymphoid
Tingible body macrophage, increased—see General
Hematolymphoid

Red Pulp
Angiectasis
Cellularity, decreased, red pulp
Congestion
Contraction
Ectopic tissue, spleen
Erythrophagocytosis
Fibrosis
Pigment, macrophage—see General Hematolymphoid
Vacuolation, macrophage—see General Hematolymphoid
Proliferative Changes (Non-Neoplastic)
White Pulp
Aggregates, macrophage, increased
Cellularity, increased, plasma cell, white pulp
Cellularity, increased, white pulp
Red Pulp
Cellularity, increased, adipocyte
Cellularity, increased, macrophage
Cellularity, increased, mast cell
Cellularity, increased, mesothelial
Cellularity, increased, plasma cell, red pulp
Cellularity, increased, stromal cell
Extramedullary hematopoiesis, increased
Hyperplasia, nodular
Lymph Node
Organization
Function
Development
Histology
Sampling and Diagnostic Considerations
Nonproliferative Changes
Cortex, Paracortex, and Medullary Cords
Aplasia/hypoplasia—see General Hematolymphoid
Apoptosis, increased, lymphocyte

Willard-Mack et al.
Cellularity, decreased, lymphocyte
Necrosis—see General Hematolymphoid
Pigment, macrophage—see General Hematolymphoid
Tingible body macrophage, increased—see General
Hematolymphoid

Inflammation—see General Hematolymphoid
Sinuses and Lymphatics
Dilatation, sinus
Erythrocytes, intrasinusoidal
Lymphangiectasis
Pigment, macrophage—see General Hematolymphoid
Vacuolation, macrophage—see General Hematolymphoid
Proliferative Changes (Non-Neoplastic)
Cortex, Paracortex, and Medullary Cords
Aggregates, macrophage, increased
Cellularity, increased, interdigitating dendritic cell
Cellularity, increased, lymphocyte
Cellularity, increased, plasma cell
Cellularity, increased, stromal cell
Hyperplasia, angiomatous
Hypertrophy/hyperplasia, high endothelial venules (HEVs)
Sinuses and Lymphatics
Cellularity, increased, macrophage, intrasinusoidal
Cellularity, increased, mast cell
Fibrosis
Mucosa-Associated Lymphoid Tissue (MALT)
Organization
Definition and Function of MALT
Development
Histology
Sampling and Diagnostic Issues
Nonproliferative Changes
Aplasia/hypoplasia—see General Hematolymphoid
Apoptosis, increased, lymphocyte—see General Hematolymphoid
Cellularity, decreased, lymphocyte
Degeneration, follicle-associated epithelium
Hyaline material
Inflammation—see General Hematolymphoid
Lymphangiectasis—see General Hematolymphoid
Mineralization—see General Hematolymphoid
Necrosis—see General Hematolymphoid
Pigment, macrophage—see General Hematolymphoid
Tingible body macrophage, increased—see General
Hematolymphoid

667
Proliferative Changes (Non-Neoplastic)
Aggregates, macrophage
Cellularity, increased, lymphocyte
Cellularity, increased, macrophage
Hyperplasia, follicle-associated epithelium
Hyperplasia, goblet cell, follicle-associated epithelium
Hypertrophy/hyperplasia, high endothelial venules (HEV)
Metaplasia, squamous, follicle-associated epithelium
Other Lymphoid Tissues
Tertiary Lymphoid Structures (TLSs)
Organization
Function
Development
Histology
Sampling and diagnostic considerations
Proliferative Changes (Non-Neoplastic)
Tertiary lymphoid structures (TLSs)
Serosa-Associated Lymphoid Clusters (SALCS)
Organization
Function
Development
Histology
Sampling and diagnostic considerations
Proliferative Changes (Non-Neoplastic)
SALCs, increased
Hematolymphoid Neoplasms
Hematopoietic Neoplasms
Leukemia, erythroid
Leukemia, megakaryocytic
Leukemia, myeloid
Leukemia, NOS
Lymphoid Neoplasms
Lymphoma
Histiocytic Neoplasm
Histiocytic sarcoma
Mast Cell Neoplasms
Leukemia, mast cell
Tumor, mast cell, benign
Tumor, mast cell, malignant
Thymus Neoplasms
Thymoma, benign
Thymoma, malignant

668

Introduction
The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) isa joint initiative of the Societies of Toxicologic Pathology from
Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North
America (STP) to develop an internationally accepted nomenclature for proliferative and non-proliferative changes in rats
and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in thehematolymphoid organs, including the bone marrow, thymus,
spleen, lymph nodes, mucosa-associated lymphoid tissues(MALT) and other lymphoid tissues (serosa-associated lymphoid clusters [SALCs] and tertiary lymphoid structures[TLSs]). The nomenclature for these organs is divided into
three terminologies; descriptive, conventional and enhanced.
Three terms are listed for each diagnosis. The rationale for thisapproach and guidance for its application to toxicologic pathology are described in detail below.
The hematolymphoid organs produce and maintain the cellsof acquired and innate immunity (lymphocytes, plasma cells,
monocytes, macrophages, dendritic cells, and granulocytes)
and they also produce the cells that carry blood gases (erythrocytes) and maintain vascular integrity (megakaryocytes). The
modifier “hematolymphoid” acknowledges both the hematopoietic role of the bone marrow (and spleen in rodents) and thepresence of lymphoid cells (lymphocytes, lymphoblasts,
plasma cells) in the bone marrow, thymus, spleen, lymph
nodes, MALT, and other lymphoid tissues (SALCs and TLSs).
The hematolymphoid organs are the organs of the immune
system and they collectively produce the lymphocyte repertoire, conduct immune surveillance, and mount immunologic
reactions. These functions are distributed among the organs
which are classified as primary/central and secondary/peripheral. The classic primary or central organs are the bone marrow
and thymus where lymphocyte proliferation and maturation
take place independent of stimulation by exogenous antigens.
The spleen, lymph nodes, MALT, and SALC are secondary
lymphoid organs where exogenous antigen-dependent lymphocyte development and proliferation take place. Tertiary lymphoid structures are tertiary lymphoid tissues that are induced
in nonlymphoid organs.
The hematolymphoid organs share basic stromal and parenchymal features that enable them to function together as anintegrated system. Each organ has a sponge-like fibroblastic
reticular stroma (reticular meshwork) that divides the organ
into morphologically and functionally distinct compartments.1-3 The interstices in the meshwork are populated byblood cells which serve as the organ’s parenchymal cells. The
reticular meshwork is composed of fibroblastic reticular cells(FRCs) and their reticular fibers (except in thymus which has
reticular cells of predominantly epithelial origin which do not
produce reticular fibers). Reticular cells provide surfaces forblood cell adherence and produce trophic factors (chemokines)that direct lymphocyte movement (trafficking by means of
haptotaxis) to B-cell and T-cell regions for further development

Toxicologic Pathology 47(6)and function.1 Cell trafficking occurs in primary, secondary,and tertiary lymphoid organs. Reticular fibers act as conduitsthat conduct soluble mediators to specific locations in the secondary lymphoid organs (eg, high endothelial venules [HEVs]
in the lymph node) in order to enhance recruitment and migration of specific lymphocytes into the lymphoid organ for further development, function, and interaction with other cell
types.4-6 The reticular meshwork is distensible and can expand
to accommodate increased numbers of blood cells, as can beseen in an antigenically stimulated lymph node or a congested
spleen. When depleted of blood cells, the meshwork can collapse and contract, as can be seen in lymphoid depletion in thethymus and contraction of the spleen. The reticular meshwork
is difficult to appreciate in routinely stained tissues because its
fine cytoplasmic processes are typically obscured by blood
cells in its interstices, particularly leukocytes, but it can bereadily observed in lymph node sinuses that are not crowdedwith cellular traffic (Figure 1). The distinctive morphologicalappearances of the hematolymphoid organs are a function of
the arrangement of their compartments and the number, types
and distribution of the blood cells within the compartments.
A key feature of the hematolymphoid organs is that blood
cells can move from one organ to another using the blood andlymph for transportation. Over the course of their long andcomplex life histories, lymphocytes move in and out of all thehematolymphoid organs via specialized adaptations in vascularand lymphatic endothelium (Figure 2). Mature naive lymphocytes are particularly mobile and constantly cycle through secondary lymphoid organs in their continual search for cognate
antigens. The life histories of all the blood cell types include
some time spent as constituents of organ parenchyma and sometime spent as constituents of the blood. Even erythrocytes andplatelets spend time in organ parenchyma, first during development in the bone marrow and then during periodic filtration
through the spleen when they pass through the interstices of the
red pulp. Erythrocytes, monocytes, and platelets are also stored
in the red pulp for ready release. Because of their mobility,
blood cells can increase or decrease in a given location in
response to demand. Lymph nodes can swell with newly
recruited lymphocytes within hours of antigenic stimulationand the spleen can contract and expel erythrocytes into theblood within minutes in response to a drop in blood pressureor an increase in epinephrine. (Splenic contraction, a wellknown feature of muscular spleens, can also occur to a lesser
degree in the mouse and rat spleen by virtue of contractile
proteins in the FRCs.)7,8 These types of rapid shifts in blood
cell populations are due to migration and redistribution ofexisting blood cells and are not the result of a change in theabsolute numbers of blood cells in the short term.
Blood cell migration creates special nomenclature challenges
for the hematolymphoid system. Descriptive terminology(increased/decreased cellularity), now widely used in otherorgan systems, is particularly applicable to mobile blood cellsof bone marrow origin (which include macrophages, mast cells,
and dendritic cells in addition to lymphocytes, erythrocytes, andgranulocytes) because it is often not possible to distinguishWillard-Mack et al.

669

Table 1. Recommended Use of Terminologies.
Terminology Study Type

ExamplesDescriptive

Increased cellularity,
cortex, thymus

All general toxicity studies(≤ 3 months) except
immunotoxicity
Conventional Chronic toxicity or
carcinogenicity studies
Enhanced
Immunotoxicity and shortterm (≤ 3 months) studies

Hyperplasia, lymphoid,
thymusLymphocytes,
increased, cortex,
thymuslocally produced blood cells from those that arrived recently or
to determine with certainty if missing blood cells died or emigrated elsewhere. Conventional terminology (hyperplasia/atrophy), historically the diagnostic standard, remains useful for
diagnosing changes in carcinogenicity studies and is also used
to diagnose changes in structures (eg, HEVs). Enhanced terminology (cell type, increased/decreased, compartment) can beused in short-term studies when a precise mechanistic approachto describing and evaluating the effect of exogenous substanceson the hematolymphoid system is desired. This document
defines and aligns these 3 terminologies to allow applicationof appropriate nomenclature based upon the needs of the individual study.

Best Practices and Diagnostic Challenges
In 2005, the STP Immunotoxicology Working Group publisheda “best practice” concept for examination of hematolymphoidorgans using enhanced histopathology 9 which has been
described extensively elsewhere.10-14 The enhanced histopathology approach evaluates the compartments of each lymphoid organ individually in order to identify specific cellularand architectural changes.15-19 Descriptive diagnostic terms areused in a specifically proscribed way to quantify changes inindividual cell types and localize these changes to the specificcompartments of the organ(s) in which they occur (details
about the compartments and their cellular constituents appear
in tables in each organ section). Changes are reported using
semiquantitative descriptive terminology rather than interpretive terminology. This methodology cannot directly measure
immune function but it is a sensitive method for detecting
subtle changes and has the potential to determine whether or
not a specific treatment causes suppression or stimulation of
the immune system.11 Moreover, evaluating the different cell
types and changes within compartments may suggest the possible cause(s) or mechanisms for the findings. For example, adiagnosis of increased lymphocytes in the lymph node paracortex or increased follicles and germinal centers in the lymph
node cortex provides more mechanistic information than adiagnosis of lymphoid hyperplasia.
This document regards descriptive terminology, conventional terminology, and enhanced terminology as separate but
complementary and evolving terminologies for thehematolymphoid organs. For each diagnostic entity, the desdescriptive term is presented first, followed by the conconventional term, and then by the enhenhanced term. This approachacknowledges the common practice of descriptive terminology along with the utility of standard interpretive diagnostic
terms, such as hyperplasia and atrophy, and recognizes thescientific basis for enhanced descriptive diagnostic terms,
such as increased or decreased cell type, which are moreclosely aligned with how the various cell types and compartments function. Table 1 indicates the recommended use of thedescriptive, conventional and enhanced terminologies.
Descriptive terminology is recommended for short-term (3months) general toxicity studies. Conventional terminology isrecommended for long-term (chronic) studies such as 2-yearbioassays (carcinogenicity studies). Enhanced terminology isrecommended for characterizing immunomodulatory effects
in short-term studies, especially immunotoxicology studies.
The level of detail generated by an enhanced histopathology
evaluation is generally considered unnecessary or undesirable
in chronic studies.
The choice of which terminology to use is made at the discretion of the pathologist. Factors to consider include the lengthof the study, the expectation of an immunomodulatory effect,
compliance with regulatory guidelines (eg, Organisation for
Economic Co-operation and Development [OECD] 407), additional studies to be performed, availability of ancillary data (eg,immunohistochemistry [IHC], flow cytometry, antidrug antibody assessment, concurrent disease processes, etc), and thequestions the study is addressing. Compartment locators andcell type are optional with descriptive and conventional terminology but should always be used with enhanced terminology.15-18,20 Regardless of which terminology is used, it isgood practice to evaluate hematolymphoid organs in a compartment-aware manner. When a full enhanced histopathology
evaluation is performed, the final interpretations and conclusions should be presented within the pathology narrative.
The selection of INHAND terms for the hematolymphoidorgans was guided by the knowledge that these organs sharesimilar structural and functional features and react in similar
ways to physiological challenges. Terms were chosen that
could generally be applied similarly across the organs. Synonyms and other closely related diagnostic terms in current use
or of historical significance are listed under Other Terms andare provided as a bridge from former diagnostic practices to thecurrent recommended INHAND terminologies. Descriptive
terminology and conventional terminology use common baseterms that can be augmented with cell type and compartment
modifiers when necessary. Enhanced terminology combines
information about the process, the cell type(s) involved, andthe compartment(s) in which the process occurs.
All morphologically distinct areas are referred to as compartments, even when one compartment is nested within anothercompartment. In the spleen, for example, germinal centers arecontained within follicles which are in turn contained within thewhite pulp. The spleen and lymph node are unique because theyeach have a nonlymphoid compartment that filters a body fluid;

670
blood is filtered in the red pulp of the spleen and lymph isfiltered in the sinuses of the lymph node. Changes in thesefiltration compartments are presented under the subheadings“Red Pulp” in the spleen and “Sinuses and Lymphatics” in thelymph node. Changes in lymphoid compartments are presented
under the subheadings “White Pulp” in the spleen and “Cortex,
Paracortex, and Medullary Cords” in the lymph node.
Macrophages present unique diagnostic challenges
because they phagocytize, degrade, and/or store cellularmaterial. These physiological activities produce a wide arrayof cytoplasmic characteristics. Macrophage cytoplasm may
contain apoptotic bodies (tingible body macrophages), erythrocytes (erythrophagocytosis), hemosiderin, lipofuscin, ceroid or other pigments (pigmented macrophages), or vacuoles(vacuolation), as well as granules, crystals, exogenous pigments, or other manifestations of ingested xenobiotics.
Macrophages can also become enlarged (hypertrophy) and
can adhere together in clusters (macrophage aggregates).
Macrophages are present in every hematolymphoid compartment but they may be difficult to identify when scatteredamong dense lymphocyte populations. Some populations areeasily recognized, such as those in lymph node sinuses (traditionally referred to as sinus histiocytes). In this document,
the term “macrophage” is applied to macrophages in all locations to emphasize the similarity of the cell type across theorgans. Because of the inherent variability of macrophages,
their diagnoses are provided with a menu of modifiers andlocators that can be selected to best describe a particularlesion. Macrophage diagnoses are listed in the General Hematolymphoid section and some are also listed under specificorgans.
Lymphocytes are the most visible and abundant blood cells
in the hematolymphoid organs. They present unique diagnostic challenges because the different lymphocyte subsets arefunctionally distinct but morphologically similar. They havediffering sensitivities to toxicity and they can give rise todifferent subtypes of lymphomas, but the different lymphocyte subtypes generally cannot be identified in routine hematoxylin and eosin (H&E) slide preparations. Lymphocytes are
best distinguished, when necessary, by using IHC to identify
cellular markers (surface, cytoplasmic, nuclear).21 Information about using IHC is included under Diagnostic Features
for many diagnoses. Although lymphocyte diagnoses arecommon to all the lymphoid organs, they are listed under eachorgan individually because of their central importance in thehematolymphoid system.
Immature lymphocytes (especially double-positive lymphocytes [CD4þ/CD8þ]) are sensitive to stress because endogenous cortisol triggers them to undergo apoptosis, especially inthe thymus. Stress-related changes should be differentiatedfrom immunomodulatory effects based on a combination ofclinical signs (such as decreased body weight gain and activity), complete blood count results (increase in circulating neutrophils, decrease in circulating lymphocytes), increase in
adrenal gland weight, decrease in thymus weight, decrease inthymic cortical cellularity with associated lymphocyte

Toxicologic Pathology 47(6)apoptosis, and changes in spleen and lymph node cellularity.22
Because the hematolymphoid organs and circulating blood
cells are intimately intertwined, a complete evaluation of thehematolymphoid organs should always include clinical pathology (hematology) evaluation of the blood.
A background level of immune surveillance and responseis always present in the hematolymphoid organs. Increases incell numbers are generally reactive and are part of the normal
physiological responses of these organs to acute and chronic
insults or physiologic stimulation. Hyperplastic changes inthese organs do not, therefore, infer preneoplastic or precancerous lesions. However, in unusual circumstances of severeor persistent hyperplasia, cell proliferation may increase therisk of neoplastic transformation, presumably due to accumulation of random mutations associated with DNA replication.23 Assessment of a hyperplastic change should include
a thorough evaluation of body condition to rule out underlying
conditions such as infection and inflammation and should
consider whether or not the location, stage of maturation,
and/or morphology of the cells in question are unusual or
unexpected. If there is a concern, clonality studies should beconsidered.21
The level of background activity for each strain and groupof animals is influenced by nutritional status, antigen load,age, genetics (rodent strain and genetically engineered mice
[GEM]), spontaneous lesions, steroid hormone status, andinfectious agents (opportunistic, incidental, or concurrent).24-26 As with all screening tests, comparison with concurrent control tissues is critically important in order toestablish the range of normal tissue changes for a particulargroup of animals. It is therefore essential to compare treatedanimals to concurrent controls to accurately distinguishbetween background activity and changes attributable toxenobiotics. One recommended method of evaluation is toreview all concurrent control tissues to determine the “range
of normal” for overall tissue architecture and cellularity
within that group of animals. The high-dose group is evaluated next, followed by the low- and mid-dose groups, constantly referring back to tissues from the control group toprevent diagnostic drift. Once all tissues from one lymphoidorgan have been reviewed, the evaluation of each of the otherorgans is done in the same manner.27 Another acceptable
method, following examination of controls and high dose, isto combine slides from all dose groups and controls in ablinded fashion and reexamine all slides to determine if the
hypothetical change cleanly segregates into treatment andcontrol groups.
In summary, histopathologic evaluation of the hematolymphoid system requires a mechanistic understanding of normal
histology and physiology and a holistic assessment of the entire
distributed multi-organ hematolymphoid system. Differentiation and identification of background, individual, local, or systemic effects requires accurate description and interpretation ofhistologic findings in conjunction with ancillary data, such as
clinical history, clinical pathology, organ weights, and grossobservations. If available, flow cytometry, immune function

Willard-Mack et al.
assays, and antidrug antibody assessment provide additional
valuable ancillary data that may impact interpretation of morphologic assessments. Integration of all available data should
result in an interpretive narrative in the written report. The goalof this document is to provide defined sets of terminology toenable clear communication of the histopathologic changes
present in hematolymphoid organs.

GENERAL HEMATOLYMPHOID
Some changes may occur in one or more hematolymphoidorgans as part of a localized or systemic condition. To avoid
repetition in the individual organ sections, they are described inthis section with the most commonly affected lymphoid tissues
noted. In some cases, these shared diagnoses also appear inindividual organs. Compartment location of the findings may
be important and can be used to modify the process term at thediscretion of the pathologist.
Vascular findings such as hemorrhage and inflammation ofblood vessels occur in lymphoid tissues but are not included inthis document because they are covered in detail in the
INHAND cardiovascular document. Congestion, a common
finding in the spleen, is included in the spleen section of thisdocument.

671


Mesenteric lymph node.
 Common site in mice.
 Subcapsular sinus area often affected first withextension to paracortex later.
 Medulla usually not involved.

Spleen.

 Red pulp can be replaced by amyloid and white pulp Congo red—stains pink or red with H&E—shows
green birefringence under polarized light.

 Thioflavin T—fluoresces under UV light.
 Crystal violet or methyl violet—metachromasia.
Amyloid (N) (Figure 3) General
hematolymphoid

con
enh

Amyloid

Amyloid(indicate compartment)

Species
Mouse; rat.

Electron microscopy.

 In humans, nonbranching fibrils with indefinite
length and a diameter of about 7.5 to 10 nm.

 In mice, 100 Å wide, rigid nonbranching strands
twisted into 2 filaments.

Differential Diagnoses
Deposition of Collagen or Fibrindesmay exhibit pressure atrophy.
Staining properties.

Negative for Congo red.

Comment
Amyloid is not a chemically distinct entity. In experimental
animals, amyloid protein is mostly of the AA type. Amyloid was
a common spontaneous finding in certain strains of mice (CD-1
and C57B6) in the past,28 but the incidence has decreased overtime and it now occurs as an incidental finding in occasional
animals. It is uncommon in BALB/c mice and is rarely observed
as a spontaneous finding in rats. The kidney, ileum, and adrenal
gland are most often affected.28 C57BL6 mice are susceptible toboth senile (AApoAll) and secondary amyloid.

Other Terms
Amyloidosis; amyloid accumulation.

desAplasia/Hypoplasia (N) (Figures 4 and 5)
General hematolymphoid

Pathogenesis/Cell of Origin

con

Deposition of twisted b-pleated sheet fibrils due to abnormal
assembly of various proteins.

enhDiagnostic Features

SpeciesDense masses of eosinophilic hyaline material.
Deposits efface normal architecture and cause pressure
atrophy.
Distribution.
 Systemic deposition most common but localized
deposits also occur.
 May occur in any tissue.
 Predilection for perivascular distribution.

Aplasia/Hypoplasia

Aplasia/Hypoplasia

Mouse; rat.

Synonym
AgenesisOther Terms
Congenital decreased lymphocytes.

672

Toxicologic Pathology 47(6)Pathogenesis/Cell of Origin
Loss of specific gene function resulting in lack of normal
development.

Diagnostic FeaturesComplete lack of development of a lymphoid organ.
Absence of tissue or organ.

Differential Diagnoses
Normal Development
Marginal zone is absent in mouse spleen until 4 weeks ofage.

Normal Aging

Age-related involution in thymus.

AtrophyLoss of lymphocytes due to age, toxicity, or disease.
At the gross and subgross level, the entire organ is small
compared to concurrent controls.
Decreased lymphocyte cellularity.
Lymphocyte necrosis or apoptosis may be present.
Underlying stromal cells may be more prominent.

Comment
Aplasia of the thymus, spleen, and Peyer’s patches (PP) has
been reported in the mouse. These conditions in the mouse are
generally congenital genetic disorders which can arise spontaneously or be found in strains of GEM. Aplasia may be difficult
to distinguish morphologically from severe lymphoid hypoplasia and decreased cellularity (atrophy), so age, species, history,
and changes observed in other tissues should be consideredduring diagnostic differentiation. If there is a congenital
decrease in the development of an organ, then the term hypoplasia may be used.
Aplasia of the thymus occurs in nude mice that are homozygous null for the Foxnu1 gene. These mice either completely
lack a thymus or have only severely small cystic thymic remnants. Affected mice are hairless from birth throughout life.
Absence of the thymus is due to a failure of the thymus anlage
to develop from the ectoderm of the third pharyngeal pouchand from premature degeneration of thymic epithelium inutero.29,30
In humans with DiGeorge syndrome, the third and fourthpharyngeal pouches fail to develop resulting in absence of thethymus and parathyroid glands. Lack of the thymus in homozygotes leads to many defects in the immune system, including
depletion of lymphocytes from thymus-dependent areas oflymph nodes, spleen, and PP, a much reduced lymphocyte
population composed almost entirely of B cells, very poor
response to thymic-dependent antigens, including failure to

reject relatively allogeneic and xenogeneic skin and tumor
grafts, and increased susceptibility to infection.
Aplasia of the spleen occurs in mice that are either
homozygous or heterozygous for the Dh gene. Asplenicmice have enlarged PP and absolute lymphocytosis, granulocytosis, and monocytosis and their serum protein concentrations and plasma high-density lipoprotein cholesterollevels are lower than normal. Homozygotes (Dh/Dh) are
imperforate and die within 3 days of birth due to associatedgastrointestinal anomalies. Heterozygotes (Dh/ þ) can live
for several months when housed in a specific pathogen-freeenvironment.
In the Sharpin null mouse, Peyer’s patch development
occurs during embryogenesis but regresses spontaneously
after birth resulting in a lack of distinct PP in the small intestine. The spleen, lymph nodes, and nasal-associated lymphoidtissues are present but have architectural changes. Serum
immunoglobulin G (IgG), IgA, and IgE concentrations are
significantly decreased, while serum IgM is normal. Inflammation involving multiple organs is a common feature in thisgenetic disorder.
In addition to these spontaneous mutant immune deficient
mice, several GEM strains are immune deficient such as NSG(NOD scid gamma; Nod. Cg-Prkdcscid IL2rgtm1Wjl/SzJ)31 andNRG (NOD rag1 gamma; NOD.Cg-Rag1tm1MomIL2rgtm1Wjl/
SzJ).32
Immunodeficient mice (nude, SCID [severe combined
immune deficient mice]) and rats (nude) have hypocellularfollicles and T-lymphocyte-dependent areas in spleen andlymph nodes. The MZ is retained in nude mice and rats. Neonatal thymectomy of normal rodents does not affect MZ lymphocyte colonization.
Severe combined immune deficient mice (Prkdc scid /Prkdscid) are deficient for protein kinase enzyme activity
involved in DNA repair, a deficiency that affects the functionof lymphoid stem cells. Because they cannot generate T and B
lymphocytes, SCID mice are lymphopenic and they cannot
activate some components of the complement system. They
have normal natural killer (NK) cells, macrophages, and granulocytes; however, the thymus has a rudimentary medulla andno cortex, the spleen has no follicles, and the lymph nodes andPP have undeveloped T zones and B zones. Some “leaky”
strains may produce small populations of functional B and/or T cells as they age.29,30,33

desApoptosis, Increased, Lymphocyte (N)(Figure 6) General hematolymphoid

con
enh

Apoptosis, Increased, Lymphocyte

Apoptosis, Increased, Lymphocyte(indicate compartment and diagnose decreased lymphocytes, decreased area, tingible body macrophages, etc, separately if applicable)

Willard-Mack et al.

Species
Mouse; rat.

Other Terms
Lymphocyte depletion; atrophy.

Pathogenesis/Cell of Origin
Lymphocyte apoptosis may result from direct lymphocyte toxicity or from endogenous factors such as diet or stress (glucocorticoid release).

Diagnostic Features











Single cells or small clusters of cells.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cytoplasm retained in apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis.
Nuclear fragmentation.
Intact cell membrane.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

673
normal processes.34 Apoptosis eliminates activated or autoaggressive immune cells during maturation; therefore, a low
level of lymphocyte apoptosis is considered within normal
physiological variation. Increased lymphocyte apoptosis may
result from direct lymphocyte toxicity or from endogenous
factors such as diet or stress (glucocorticoid release). Severe
ongoing apoptosis results in severe decreased lymphocyte
cellularity (lymphoid atrophy). Necrosis may occur together
with apoptosis. While it is preferable to identify and record
diagnoses of apoptosis and classic necrosis separately, this
distinction may not be possible when one type of cell death
histologically obscures the other. Also, necrotic cell debris
can have some similarities to apoptotic debris, such as pyknosis and karyorrhexis. Apoptosis may predominate with conversion to a necrotic phenotype, or necrosis may predominate
with scattered apoptosis. In these cases, it would be appropriate to use both terms together (apoptosis/necrosis) or only
diagnose the predominate type of cell death and discuss thepresence of the other type of cell death in the narrative.
desCellularity, Increased, Mast Cell(N) General Hematolymphoid

con
enh

Differential Diagnoses

Hyperplasia, Mast Cell

Mast Cells, Increased(indicate compartment)

Necrosis, Lymphocyte






Necrotic cells are often contiguous, but pattern can be
focal, multifocal, or diffuse.
Cell swelling.
Cell rupture.
Karyolysis, pyknosis, and karyorrhexis.
Inflammation usually present.

Species
Mouse; rat.

Pathogenesis/Cell of Origin
Develops from mast cells and their precursors present in thehematopoietic, mucosal, and/or connective tissues.

Involution, Age-Related (THYMUS)









Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells.
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined bycuboidal to squamous epithelium.

Diagnostic Features








Comment
Apoptosis is a coordinated and often energy-dependent modeof cell death that is considered a vital component of various

A loosely arranged collection of mature mast cells without nodule formation.
Mast cells are uniform, round, or polygonal, mediumsized and well differentiated.
Nuclei are uniformly round but may be obscured bycytoplasmic granules.
Cytoplasm is abundant, granular, and slightly to heavily
basophilic.
Cytoplasmic granules may or may not be visible withhematoxylin and eosin depending on the type offixation.
Cytoplasmic granules are metachromatic and generally
stain with Giemsa, toluidine blue, or other metachromatic stains.
No compression of adjacent tissues.
May involve one or more tissues or organs.

674




Toxicologic Pathology 47(6)May be reactive to a tumor or associated with otherinflammatory cells.
Mitotic figures are not present.
In lymph nodes, mast cells are located predominantly inthe sinuses.

desExtramedullary Hematopoiesis (EMH)(N) (Figures 7 and 8) General hematolymphoid

con
enh

Differential Diagnoses

Extramedullary Hematopoiesis

Extramedullary Hematopoiesis(indicate organ and compartment)

Mast Cell Tumor, Benign

A single, solitary, compact (dense) mast cell aggregate
or nodule.
Compression of adjacent tissue.

Mast Cell Tumor, Malignant











Compact solitary nodule, local sarcomatous growth or
sheet-like accumulation(s) of round, spindle shaped, or
immature mast cells.
Cytoplasm is often hypogranular, but may have typicalbasophilic granules.
May have atypical bilobed or polylobed nuclei.
Eosinophils may be associated with the mast cells.
Destructive growth pattern, may be locallyinfiltrative.
Multiple organs may be involved.
No bone marrow involvement.
No clear inflammatory stimulus.
Considered malignant.

Modifier
Erythroid; myeloid

Species
Mouse; rat.

Other Terms
Increased hematopoiesis; red pulp hyperplasia (spleen); erythroid hyperplasia (lymph nodes, gut-associated lymphoid tissue [GALT], thymus); erythropoiesis; granulopoiesis; myeloid
metaplasia.

Pathogenesis/Cell of Origin
Circulating hematopoietic progenitor cells from the bone marrow and/or spleen.

Mast Cell LeukemiaAtypical mast cells are present in the bone marrow and/or peripheral blood.
Mast cell accumulations with sheet-like or leukemic
pattern present in one or more hematolymphoidorgans.

Diagnostic FeaturesHistiocytic Sarcoma Medullary cords in lymph nodes.
 Perivascular sites in thymus.
 Sinusoids in liver (refer to the INHAND monograph

Nuclei are less regular.
Cytoplasm is eosinophilic.
Negative for metachromatic cytoplasmic granules.

on the hepatobiliary system, see general introduction, objective, and outline).
 Red pulp in spleen.
&
Extramedullary hematopoiesis is normal in therodent spleen&
Extramedullary hematopoiesis over background levels is diagnosed as EMH, Increased(see Spleen section)

Melanoma, Malignant, AmelanoticVarying proportions of mature and immature forms of
myeloid, erythroid, and megakaryocytic lineages, depending on etiology.
Extramedullary hematopoiesis sites.

Differentiate from mast cells with IHC for expression ofmelanin (HMB45, PEP8).

Comment
Increased mast cell cellularity may occur in lymphoid,mucosal, or connective tissues in response to cytokines
associated with parasitic, allergic, and other inflammatory
lesions. The mast cells are usually mature with many metachromatic granules and do not form nodules. This finding may beseen in some mouse lines as an aging change without obvious
cause.

Differential Diagnoses
Infiltrate, Neutrophil
Infiltration of a relatively pure population of neutrophils
into the tissue.
Presence of polymorphonuclear leukocytes but no otherhistological criteria of inflammation.

Willard-Mack et al.
Leukemia; Myeloid, Erythroid, or MegakaryocyticTumor cells are often all at one stage of differentiation(refer to neoplasia section).

LymphomaDistinguished by cell morphology and tissue
distribution.

Comment
Extramedullary hematopoiesis is a response to increased hematopoietic demand that occurs in sites outside the bone marrow,such as in lymph nodes, thymus, and some nonlymphoidorgans, and at increased levels in the spleen. In lymph nodes,
EMH shows a preference for the medullary cords. Extramedullary hematopoiesis in medullary cords should be distinguishedfrom mature and degenerating neutrophils draining into thesinuses from inflamed tissues. Extramedullary hematopoiesis
is commonly seen in the spleen in rodents where it may berecorded as “EMH, Increased” when it is increased above background levels.
desInfiltrate (indicate modifier) (N) (Figure 9)
General hematolymphoid

con
enh

675

Differential Diagnoses
Cellularity, Increased (Cell Type)

Increased normal cells with normal maturation in normal locations.
May be expansion of tissue architecture but without
degeneration or distortion.
Reflects normal activity of the tissue or organ.

Inflammation


Infiltrates are associated with degenerative and vascularchanges, such as necrosis, edema, hemorrhage, congestion, and/or fibrosis.

Hematopoietic Neoplasia

Homogenous population of lymphocytes or granulocytes infiltrating the tissue.
Architecture effaced.
Other sites usually involved.

Extramedullary hematopoiesis
Population of mature/immature hematopoietic cells.
Response to a systemic condition.

Infiltrate (indicate modifier)

Cell type(s), increased(indicate organ and compartment)

Species
Mouse; rat.

Modifier
Neutrophil; eosinophil; mast cell; monocyte; macrophage;
mixed cell.

Pathogenesis/Cell of Origin
Inflammatory cells from the circulating blood or local
tissues.

Diagnostic Features

Infiltration of a relatively pure population of neutrophils, eosinophils, mast cells, macrophages, or a mixtureof these cell types into the tissue.
Presence of mononuclear or polymorphonuclear
leukocytes but no other histological criteria ofinflammation.

Comment
Infiltrating inflammatory cells must be distinguished fromhyperplasia of inflammatory cell types arising and maturing
normally in hematolymphoid organs. Factors to consider whenevaluating the presence of inflammatory cells include thestages of maturation present, the inflammatory cells normallypresent in the affected organ, inflammation in the local drainage field (for lymph nodes) or systemically (spleen), whether
the cells are a pure or mixed population, and whether there aredegenerative changes present. In the bone marrow, orderly
maturation of increased numbers of benign cells in situ is
hyperplasia. Infiltrating cells are not associated with significant
tissue damage. The term “infiltrate” is preferred over the term
“inflammation” when the infiltrating cells are not accompanied
by degenerative or vascular changes. The base term “infiltrate”
is recommended, followed by the predominant cell type in theinfiltrate or by “mixed cell” if there is not a predominant celltype. Inflammatory changes in adjacent tissues should be considered when assessing infiltrates in lymph nodes. Neutrophils
or other inflammatory cells draining through the sinuses andgranulopoiesis in medullary cords do not constitute an infiltrate
in lymph nodes. Increased lymphocytes in a lymphoid organ
are not generally diagnosed as a lymphocyte infiltrate because
they are normal constituents of lymphoid organs. Systemic
inflammatory conditions should be considered when assessing
infiltrates in bone marrow, thymus, lymph nodes, and spleen.
The choice of terminology should be left to the judgment of thepathologist.

676

Toxicologic Pathology 47(6)des Accumulation of karyorrhectic debris.
 Localized foci of necrosis associated with cellular

Inflammation (indicate modifier) (N)(Figures 10 to 17) General hematolymphoid

con
enh

infiltrates.

Inflammation (indicate modifier)

Cell type(s)des(indicate organ and compartment and diagnose necrosis,
hemorrhage, edema etc, separately if applicable)con

Abscess (Figure 12)enh

Cell type(s)(indicate organ and compartment and diagnose necrosis,
hemorrhage, edema etc, separately if applicable)

Species
Mouse; rat.

Other Terms
See below for the different types of inflammation.

Modifier
Neutrophil, mononuclear cell, lymphocyte, monocyte, mixed
cell, lymphoplasmacytic, pyogranulomatous, granulomatous,
acute, subacute, chronic, and chronic active.

Pathogenesis/Cell of Origin
See below for the different types of inflammation.

Diagnostic Features

Abscess

Other Terms
Purulent inflammation; suppurative inflammation.

Pathogenesis/Cell of Origin
Localized neutrophilic inflammation that generally resultsfrom bacterial infection.

Diagnositic Features

See below for the different types of inflammation.
desInflammation, neutrophil (Figures 10 to 11)

con
enh



Localized focus of neutrophilic infiltrates.
Usually has a necrotic center with abundant karyorrhectic debris due to release of neutrophilic proteolytic
enzymes.
Outer rim composed of macrophages, lymphoplasmacytic cells, and/or connective tissue, depending on durationof lesion.
Considered acute or chronic, depending on the amountof surrounding connective tissue.

Inflammation, acute

Cell type(s)(indicate organ and compartment and diagnose necrosis,
hemorrhage, edema etc, separately if applicable)

Other Terms
Acute lymphadenitis, splenitis, myelitis, and so on; purulent,
fibrinopurulent, or suppurative inflammation.

Pathogenesis/Cell of Origin
Infectious agent or recent tissue damage.

Diagnostic Features






Neutrophilic cellular infiltrates.
Edema.
Congestion.
Serous or fibrinous eosinophilic exudate.
Necrosis.
 Increased single cell necrosis/apoptosis in germinal
centers.

desInflammation, mononuclear cell,
lymphocyte, monocyte, eosinophil,mixed cell, lymphoplasmacytic, or
pyogranulomatous (Figures 13 and 14)

con

enh

Inflammation, subacute, chronic, or
chronic active

Cell type(s)(indicate organ and compartment and diagnose necrosis,
hemorrhage, edema etc, separately if applicable)

Other Terms
Nonsuppurative lymphadenitis, splenitis, myelitis and so on.

Pathogenesis/Cell of Origin
Incomplete resolution of neutrophilic (acute) inflammation or
infection with a low-grade infectious agent that is not easily
cleared by the immune system.

Willard-Mack et al.

Diagnostic Features (includes some combination of thefollowing)






Mononuclear cell infiltrates of macrophages and lymphocytes with or without increased plasma cells.
Mixed cell inflammation may include neutrophils and/or
eosinophils in addition to mononuclear cells.
Normal architecture is distorted/replaced.
Fibroplasia with or without neovascularization.
Congestion, edema, and exudates minimal or absent.

677

Other terms
Granulomata, microgranuloma, pyogranuloma.

Pathogenesis/Cell of Origin
A chronic unresolved inflammatory stimulus that isolates orwalls off a poorly digestible biologic or infectious agent or
foreign material.

Diagnostic Features
desInflammation, Granulomatous (Figures 15
and 16)conenhInflammation, Granulomatous

Cell type(s)(indicate organ and compartment and diagnose necrosis,
hemorrhage, edema etc, separately if applicable)

Other Terms
Granulomatous lymphadenitis, splenitis, myelitis, and so on;
histiocytic inflammation; Potter’s lesion35



Pathogenesis/Cell of Origin
Infection or accumulation of a poorly digestible biologic agent
or foreign material.

Diagnostic Features










A chronic inflammatory response characterized by a significant component of activated macrophages, epithelioid
cells, and/or multinucleate giant cells (Langhans or foreign
body types) along with other inflammatory cell types.
Epithelioid macrophages have abundant, pigmented,
foamy, or vacuolated cytoplasm.
Etiologic agent, for example, fungi, mycobacteria, or
phagocytized foreign material, may be evident in macrophage or giant cell cytoplasm.
May occur as a response to implanted biomaterials.
May exhibit lymphoid hyperplasia adjacent to theinfiltrates.
May efface normal tissue architecture.
Can be characterized by additional modifiers as needed,
for example, pyogranulomatous, necrotizing, and so on.

Differential Diagnoses (for all types of inflammation)
InfiltrateGranuloma (Figure 17)

con

Granuloma

Necrosis is the primary diagnosis; inflammation may or
may not be present.
Localized foci of necrosis associated with cellular
infiltrates.
Increased single cell necrosis/apoptosis in germinal centers with accumulation of karyorrhectic debris.
Absence of organizing macrophages.

Cellularity, Increased, Plasma Cell

Cell type(s)(indicate organ and compartment and diagnose necrosis,
hemorrhage, edema etc, separately if applicable)enh

A relatively pure population of neutrophils, eosinophils,
mast cells, macrophages, or a mixture of these cell types
infiltrates the tissue.
Absence of accompanying degenerative or vascularchanges.

NecrosisdesWell demarcated, organized, focal lesion, often small
and innocuous.
Often encapsulated by fibroblasts, lymphocytes, andplasma cells.
Nodules or aggregates of enlarged macrophages(epithelioid cells) which may have a solid center or anecrotic center composed of cellular debris and/or
neutrophils.
Epithelioid macrophages have abundant, pigmented,
foamy, or vacuolated cytoplasm.
Multinucleated giant cells (Langhans or foreign bodytypes) often present.
May efface normal architecture of tissue.
Etiologic agent, for example, fungi, mycobacteria, or
phagocytized foreign material, may be evident in macrophage or giant cell cytoplasm.
May exhibit lymphoid hyperplasia adjacent to the
nodules.

Relatively pure population of plasma cells.
Mott cells with Russell bodies may be present.
Typically, but not always, in medullary cords of lymph
nodes.
Germinal centers may be hypertrophic/hyperplastic.

678


Toxicologic Pathology 47(6)May be associated with an acute or chronic disease process, including infectious etiology or neoplasm.

Cellularity, Increased, Macrophage






Increased abundance and/or size of macrophages.
Most commonly in splenic red pulp and lymph nodesinuses, but may occur in other compartments/organs.
Macrophages are generally individualized and have distinct cell borders.
Cytoplasm may or may not contain phagocytized material, pigment (commonly hemosiderin), or vacuoles.
May be associated with increased filtration andclearance.

devices36 which may involve draining lymph nodes or the spleen(in the case of intravascular injection). The histologic presentation of inflammation can vary greatly and the use of modifiers tocharacterize it is strongly recommended. The choice of terminology and the decision on whether or not to diagnose should be left
to the judgment of the pathologist.
desMetaplasia, Osseous (N) (Figure 18) General
hematolymphoid

con
enh

Metaplasia, Osseous

Aggregates, MacrophageAdherent macrophages clustered together to form variably sized aggregates.
Macrophages may contain pigment.
Absence of accompanying degenerative or vascularchanges.
Most commonly located in the PALS in spleen andmedullary cords and paracortex in lymph nodes.

Lymphoplasmacytic or Granulocytic Neoplasia

Homogenous population of lymphocytes or granulocytes infiltrating the tissue, effacing its architecture andusually involving other sites.
Atypical cells.
Mitosis may or may not be evident.

Metaplasia, Osseous(indicate organ and compartment)

Species
Mouse; rat.

Other Terms
Heterotopic ossification; ectopic bone; metaplastic bone.

Pathogenesis/Cell of Origin
Bone morphogenetic proteins are thought to stimulate thedevelopment of osseous metaplasia in association with focal
tissue degeneration and/or neoplastic foci. Osseous metaplasia
may also develop from foci of mineralization.

Diagnostic Features
Comment
The term “inflammation” is generally accompanied by a modifierthat characterizes the histologic features of the finding and isrelated to the duration of the pathologic process. The characteristics of the inflammation may also be described in the tissuecomment, the data table, and/or the report text. In the case of an
infectious etiology, neutrophils may be intermixed with macrophages in a chronic response and the term “pyogranulomatous”
inflammation may be appropriate. The term “chronic active” canbe used at the discretion of the pathologist when the histologic
features of the inflammation demonstrate variable duration indifferent areas of the affected tissue. The significance of inflammation in a single site in lymph nodes should always be considered in the context of histologic findings in tissues that the lymph
node drains. Transitory intrasinusoidal inflammatory cells originating from a draining site of inflammation (Figure 8) must be
differentiated from an intrinsic inflammatory process within thelymph node itself and can be indicated by using the modifier“reactive.” Abscesses occur occasionally in lymph nodes and
GALT and are rarely observed in the spleen. Granulomas inlymph nodes usually occur in the paracortex and medullary cords.
Granulomatous inflammation may be observed in response tosutures, catheters, nanoparticles, microspheres, and biomedical

Presence of osteoblasts.
Presence of bony trabeculae derived from a collagenous
matrix.
Bone marrow may develop in larger foci of osseous
metaplasia.

Differential Diagnoses
Mineralization


Mineralized foci are amorphous and lack osteoblasts andtypical bone structure.

Comment
Osseous metaplasia is an uncommon incidental finding.
desMineralization (N) (Figure 19) General
hematolymphoid

con
enh

Mineralization

Mineralization(indicate organ and compartment)

Willard-Mack et al.

679

Species

Pathogenesis/Cell of Origin

Mouse; rat.

Necrosis can be seen in areas of infarction or as a direct treatment-related effect.

Other terms
Calcification; mineral deposits.

Diagnostic Features

Pathogenesis/Cell of Origin
In lymphoid tissues, mineralization is generally dystrophic andoccurs as a sequel to tissue degeneration or necrosis.

Diagnostic Features






Basophilic extracellular amorphous granular material
and/or lamellated structures.
Rare sporadic finding within lymph nodes, spleen, thymus, or PP.
May be seen in infarcts, germinal center degeneration,paracortical lymphocyte necrosis, granulomas, ortumors.
Positive with von Kossa silver method and Alizarin
red.

Differential Diagnoses
Metaplasia, OsseousContains osteoblasts and has typical bone structure.

Comment
Mineralization is rare in lymph nodes but can be seen in thegerminal centers of PP, usually as an incidental finding.

desNecrosis (N) (Figure 20) General
hematolymphoid

con
enh

Necrosis

Necrosis(indicate compartment and diagnose decreased lymphocytes, decreased area, pigment, etc. separately if
applicable)

Species
Mouse; rat.

Other Terms
Necrotic cell death; oncotic necrosis; lymphocyte depletion.

Modifier
Lymphoid, lymphocyte.






Necrotic cells are often contiguous but pattern can be
focal, multifocal, or diffuse.
Cell swelling.
Cell rupture.
Karyolysis, pyknosis, and karyorrhexis.
Inflammation usually present.

Differential Diagnoses
Apoptosis, Lymphocyte, Increased











Single cells or small clusters of cells.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cytoplasm retained in apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis.
Nuclear fragmentation.
Intact cell membrane.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

Cellularity, decreased, lymphocyte; atrophy, lymphoid; Involution, age-related (thymus)









Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells.
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined bycuboidal to squamous epithelium.

Comment
Lymphocyte necrosis is considered to be the result of a toxic
process where the cell is a passive victim and follows anenergy-independent mode of cell death.34 Necrosis in the thymus is generally classic necrosis rather than single cell necrosis. Necrosis in the bone marrow can be seen as a direct
treatment-related effect or as a result of ischemia. Ischemic
necrosis of the distal femoral epiphysis has been associatedwith vascular necrosis in mice treated with corticosteroids.37
Necrotic cell injury is mediated by three main, potentially overlapping, mechanisms: interference with the energy supply of680

Toxicologic Pathology 47(6)the cell, direct damage to DNA, and direct damage to cell
membranes. If both necrosis and apoptosis are present, necrosis
may predominate with scattered apoptosis or apoptosis may
predominate with conversion to a necrotic phenotype. In suchcases, necrosis and apoptosis may be diagnosed separately or
may be diagnosed together as a single entity (apoptosis/necrosis). Alternatively, the predominant type of cell death can be
diagnosed and the presence of the other type of cell death canbe discussed in the narrative.

CeroidPhospholipidosis (N) General hematolymphoid
See Vacuolated Macrophages.

MelanindesPigment, Macrophage (N) (Figure 21) General
hematolymphoid

con
enh

Pigment, Macrophage














Pigmented Macrophages, Increased(indicate organ and compartment)

A wax-like, golden, or yellow-brown pigment similar incomposition to lipofuscin which is often used in conjunction with the term lipofuscin.
A storage pigment that accumulates along withlipofuscin.
Alcohol-insoluble.
Positive with Sudan black and acid-fast stains.
Autofluorescence under ultraviolet light.

Minute, rounded, light, or dark brown granules.
May be intracytoplasmic or extracellular.
Nonrefractile.
Black pigment may be found in the lymph nodes andspleen of pigmented rodents.
Positive with DOPA-oxidase, Fontana-Masson, andSchmorl’s stains.
Melanin bleaching can be used for confirmation.

Species

Differential Diagnoses

Mouse; rat.

Test Article-Associated Inert or Insoluble Pigments
Hematoidin

Other terms
Pigment deposits; pigment deposition; pigment accumulation;
hemosiderosis; lipofuscinosis; ceroidosis; melanosis.

Pathogenesis/Cell of Origin
Pigments such as hemosiderin (iron derived from degradationof erythrocytes) and lipofuscin and ceroid (degradation products of phospholipid from cell membranes) are phagocytizedand stored in macrophages. Melanin may also be an endogenous pigment of pigmented rodents.






Formalin pigment (acid hematin pigment, acid formaldehyde
hematin)

Diagnostic Features
Hemosiderin

Golden brown granular pigment within macrophages.
Positive with Perl’s iron stain or Prussian blue reaction.

Yellow, yellow-brown, or orange-red refractile (notbirefringent) granules.38
Derived from hemoglobin, chemically similar tobilirubin.
Does not contain iron.
Forms intracellularly but may be found extracellularly inareas of previous hemorrhage.



Dark brown extracellular birefringent granules or
crystals.
Formed by the action of formaldehyde on hemoglobin.
Forms when aqueous formaldehyde fixative solution isacidic (pH  6).
Commonly seen artifact in spleen, bone marrow, lung,
liver, blood vessels, and areas of hemorrhage.

Lipofuscin







Tan to golden brown, may be granular or amorphouswithin macrophages.
Breakdown products of cell membrane lipids.
Associated with cell turnover, degeneration, and/or
necrosis.
Positive with Sudan black, Schmorl’s, Oil red O, carbol
lipofuscin stain, periodic acid–Schiff, lysosomal acidphosphatase, esterase, and Ziehl-Neelsen acid fast
stains.
Orange autofluorescence under ultraviolet light.

Comment
Test article-associated pigment must be differentiated fromnaturally occurring pigments and artifactual pigments. Test
article-associated pigments vary widely in color, granularity,
and refractility. None of the endogenous pigments are anisotropic (birefringent). Lymph nodes associated with the routeof exposure should be given special attention. The associationof pigmented macrophages in lymph nodes with intrasinusoidal red blood cells is suggestive of hemosiderin. Hemosiderin

Willard-Mack et al.
may be increased in lymph nodes and thymus in association
with hemorrhage. Hemosiderin tends to accumulate in the
spleen and bone marrow of aged rodents as iron is recycled
during erythropoiesis at these sites. Females tend to havemore splenic hemosiderin than males. Hemosiderin in thethymus has been reported in rats fed an iron overload diet.
Lymph node melanosis has been reported in transgenic micewith the tyrosine promoter fused to SV40. Pigment from identification tattoos applied to the skin can sometimes beobserved in the local draining lymph nodes and is generally
not diagnosed.
desTingible body macrophage, increased (N)General hematolymphoid

con
enh

Tingible body macrophage, increased

Tingible body macrophage, increased(indicate compartment, lymphocyte apoptosis, increased/
decreased area, etc. separately if applicable)

681



Comment
Increased tingible body macrophages are typically seenanytime there is an increase in lymphocyte apoptosis. The
pathogenesis may be treatment related (ie, dexamethasone)or environmental (ie, stress related, diet, etc). The relative
proportions of apoptotic lymphocytes, apoptotic bodies(free and phagocytized), and tingible body macrophages
and the resulting decrease in lymphocyte cellularity willvary depending on the severity and timing of the insult.
This is a temporary condition that generally returns to background levels once excess apoptotic lymphocytes have beencleared, although a severe and sustained insult can result
in severe decreased lymphocyte cellularity (lymphoid
atrophy).
desSpecies

Vacuolation, Macrophage (N) (Figure 22)
General hematolymphoidcon

Mouse; rat.
enh

Other Terms
Increased macrophages; macrophage hyperplasia; increased
histiocytes; histiocyte hyperplasia.

Pathogenesis/Cell of Origin
Macrophages engaged in phagocytic clearance of apoptotic
cells.

Diagnostic Features







Large macrophages with abundant pale cytoplasm scattered among lymphocytes.
Pale cytoplasm contrasts with basophilic lymphocytes
creating a “starry sky” appearance.
Contain intracytoplasmic apoptotic bodies.
 Darkly stained condensed nuclear material (tingible
bodies) from apoptotic lymphocytes.
 Round to oval.
 Variable numbers and sizes.
 May be free or phagocytized depending on durationof process.
Tingible body macrophages increased compared tobackground levels in controls.
Positive for CD68 and lysozyme.

May be dispersed throughout compartment(s) or occur
focally in aggregates.
Do not contain phagocytized apoptotic bodies.

Vacuolation, macrophage

Vacuolation, macrophage(indicate organ and compartment)

Species
Mouse; rat.

Other Terms
Foamy macrophages; cytoplasmic vacuolation; foam cells;
vacuolated histiocytosis; vacuolated macrophage hyperplasia;
phospholipidosis.

Pathogenesis/Cell of Origin
Macrophages develop cytoplasmic vacuoles due to toxic or
physiologic effect.

Diagnostic Features

Macrophages have vacuolated cytoplasm.
Vacuoles may be microvesicular, macrovesicular, orboth.
Can be focal, multifocal, or diffuse.
Negative for lysosome-associated membrane protein(LAMP)-2 IHC.

Differential Diagnoses
Differential Diagnoses
Cellularity, Increased, Macrophage

Cellularity, Increased, Macrophage


Macrophage cytoplasm is not foamy.

682

Toxicologic Pathology 47(6)Phospholipidosis






Foamy macrophages with pale, finely vacuolated cytoplasm and eccentric nuclei.
Tissue architecture is preserved.
Mesenteric lymph nodes are a common site.
Nonlymphoid organs are involved, especially lungs,
liver, and kidneys.
Definitive diagnosis can only be made by positive identification of secondary lysosomes.
 Lysosomal inclusion bodies (myeloid bodies) are
visible by transmission electron microscopy.
 Lysosomal vacuoles are positive for LAMP-2 by
IHC.

Fatty Change

Vacuoles are usually large.
Positive for fat stains.
Negative for LAMP-2 IHC.

ErythrophagocytosisPhagocytized erythrocytes can have a ghost appearance imparting microvesicular appearance to themacrophages.
Diligent searching should identify pink erythrocytes or
nuclei of nucleated erythrocytes within the macrophages.

Genetic Storage Disease

Histochemical and IHC stains can be helpful in identifying the content within the vacuoles.

in the text. Vacuolation is used as a base term followed bymodifiers as appropriate.39 Refer to the INHAND monographon the hepatobiliary system for additional information (see General introduction, objective, and outline).

BONE MARROW
Organization
Bone marrow is located within medullary cavities of bone andis considered to be a single compartment. It is variably distributed within the medullary cavity of long and flat bones andmakes up approximately 3% of the body weight of adult rats.40
In rodents, it is most prominent and most easily evaluated insternum, ribs, humerus, and femur. The bone marrow is encapsulated by endosteum which lines the irregular scalloped innersurfaces and projecting cancellous bone spicules of the marrowcavities. The endosteum consists of osteoclasts, osteoblasts,
and flat “bone lining cells,” which exert regulatory influenceon adjacent hematopoietic cells.
Arteries and veins pierce cortical bone via nutrient canals
specific to each bone. In general, the nutrient artery connectswith the main central artery, and together with the central vein,
they traverse the central core of the marrow parallel to the axisof the bone. Branching from the central artery are radial arteries
that again branch into arterioles that either penetrate the innersurface of cortical bone and drain back into the marrow cavity
vasculature or directly anastomose with the extensive venous
sinus network. A network of venous sinuses drains into thecentral vein before exiting the marrow cavity. Nerves generally
follow vascular structures. Bone marrow does not have recognized lymphatic drainage.

Comment

Function

Cytoplasmic vacuolation occurs in mice with genetic lysosomal
storage disorders and in animals exposed to certain xenobiotics.
Phospholipidosis is a generalized lysosomal storage disorderinduced by a variety of chemicals that interfere with lipid turnover and result in massive phospholipid accumulation in secondary lysosomes (myeloid bodies), particularly in macrophages.
Cationic amphiphilic drugs that often contain a hydrophilic ring
and a hydrophobic side chain with a charged amine group maybind to phospholipids to form complexes that are resistant todegradation by phospholipases or they may inhibit phospholipases directly. Although phospholipidosis most commonly
affects tissues with an abundance of macrophages, almost every
tissue in the body can be affected. Lymphocytes in the peripheral
blood, spleen, and lymph nodes can also be affected by lysosomal inclusion bodies (myeloid bodies). Transmission electron
microscopy or immunohistochemical staining is necessary to
make a definitive diagnosis of phospholipidosis. The diagnosisof “vacuolation, macrophage” can be used when phospholipidosis is suspected but not confirmed, and it can also be used as a
descriptive diagnosis when phospholipidosis has been confirmed. In the latter case, positive results can be referenced in
the report and the presence of phospholipidosis can be discussed

Bone marrow is the major tissue for hematopoiesis and is
responsible for production of erythrocytes, granulocytes,
monocytes, platelets, and dendritic cells. It is a primary lymphoid tissue and produces lymphocytes and lymphocyte precursors. The B-lymphocyte precursors migrate and mature insecondary lymphoid organs. The T-lymphocyte precursors
migrate to the thymus (a primary lymphoid organ) where theymature and subsequently circulate to secondary lymphoidorgans such as the lymph nodes and spleen.

Development
Hematopoietic stem cells (HSCs) generate the cellular components of blood throughout the life span of the animal. Thisrequires self-renewal and regulated differentiation of multiple
cell lineages. Bone marrow serves as the primary microenvironment for this function in postnatal mammals. During embryogenesis in mice, hematopoietic progenitors arise within theextraembryonic yolk sac at *E8.25 and within the placenta andother sites at *E10. Hematopoietic stem cells are present in the
fetal liver at day *E11.0. Shortly before birth, HSCs are present
in bone marrow. Within the postnatal bone marrow, HSCs are

Willard-Mack et al.

683

Table 2. Compartments and Cellular Components of the Bone
Marrow.
Compartment

Componentsa

Bone Marrow

Myeloid
Erythroid
Megakaryocytes
Adipocytes
Reticular adventitial cells
Macrophages
GranulocytesaLymphocytes are present but cannot be distinguished in H&E sections.

closely associated with blood vessels, sinusoidal endothelial
cells, perivascular cells, and osteoclasts.41 Erythropoiesis, myelopoiesis, and generation of platelets from megakaryocytes
occur within the postnatal marrow and blood cells cross the sinus
endothelium to get into the bloodstream. Lymphocyte progenitors are produced in the marrow and migrate to the thymus andperipheral lymphoid organs. Relatively few mature lymphocytes
and plasma cells return to reside in the marrow. In rodents, EMH
occurs outside of the bone marrow in the spleen and is more
pronounced in mice than in rats. In times of strong demand,
EMH increases, primarily in the spleen.

Histology
Bone marrow consists of hematopoietic islands and cords
enmeshed within a complex network of vascular sinuses supported by stromal cells, reticular fibers, and extracellularmatrix. Vascular sinuses are lined by endothelium. Adventitial reticular cells ensheath sinus endothelium and branch into
the hematopoietic cords along reticular fibers that form thespongiform structural network of the hematopoietic space.42
Hematopoiesis is a compartmentalized process40 that is organized into microniches. Hematopoietic stem cells are localized around vessels and along bone surfaces in association
with osteoclasts. Erythropoiesis is clustered into islets, often
in association with macrophages. Granulopoiesis is more diffusely distributed within hematopoietic cords. Lymphocytes
and monocytes are aggregated near arterial vessels. Pre-T
cells and immature B cells exit the marrow and home to thethymus and secondary lymphoid organs. Megakaryocytes arelocated adjacent to sinus endothelium. Macrophages, mature
B cells, and plasma cells are randomly and singly distributed.
Adipocytes occur in association with adventitial cells surrounding vascular sinuses. Reticular fibers are composed ofvarious types of collagen. Extracellular matrix is composed ofwater, salts, glycosaminoglycans, and glycoproteins. Hematopoiesis is supported and regulated by soluble factors andcognate interactions. Cells differentiate in situ and then crossthe venous sinus endothelium to enter the bloodstream. Platelets enter the bloodstream as they are released from cytoplasmic processes of megakaryocytes that extend into the lumensof venous sinuses. Platelets are variable in size in mice whichis evident on blood smears and flow cytometry.

Sampling and Diagnostic Considerations
Bone marrow for microscopic evaluation in rodents is typicallycollected from sternum, rib, humerus, and/or proximal femur.
Tissue is processed by standard techniques for H&E-stained
formalin-fixed, paraffin-embedded decalcified bone.43Additionally, marrow casts may be collected from long bones andprocessed for histology. Bone marrow smears are routinely
made for cytology. Histopathologic assessment of H&Estained bone marrow tissue sections is qualitative. Identifiable
cellular components of the bone marrow compartment are
given in Table 2.17 General assessment of cellularity (cell density), hematopoietic activity, and myeloid to erythroid (M:E)
ratio and orderly progression of maturation of erythrocytes andgranulocytes can be made. Megakaryocytes and adipocytes areeasily identified. Mature lymphocytes are not easily differentiated from other mononucleated bone marrow cells and therefore are not recommended to be part of the rodent bone marrowevaluation on H&E. Lymphocytes can be identified with IHC.
Plasma cells cannot be accurately quantified in bone marrow
due to low numbers and nonuniform tissue distribution. Visualization of stromal cells and reticular fibers require special
stains. Definitive identification of HSCs and specific immature
stages of erythroid, myeloid, lymphoid, monocytoid, and stromal cells is not routinely possible.40,44 Pigments and abnormalities, such as inflammation, necrosis, and neoplasia, arediscernible. Cytology of Romanowsky stained bone marrowsmears is quantitative and is required for definitive assessmentof hematopoietic cell differentiation and maturation. Flowcytometry may be used to provide additional characterizationof bone marrow subpopulations. Due to inherent variability inbone marrow morphology due to age, strain, sex, environmental, and study conditions, such as, blood collection, evaluationof bone marrow histology requires comparison of treatmentgroups to concurrent controls of the same anatomic site at thesame time point within the same study.
Hematopoietic cellularity of the bone marrow is variable witha relatively wide range of reported values. One study reports
approximately 70% to 80% of the marrow in rats and mice iscomposed of hematopoietic elements and 20% to 30% is composed of adipocytes.45 In a separate study evaluating Fischer rats,
hematopoietic cells varied from 33% to 88% depending upon theage of the rat and the anatomic site evaluated.46 Active hematopoiesis in bone marrow continues throughout the lifespan of therodent. However, hematopoietic cellularity of bone marrow isdependent upon anatomic site, age, sex, and strain of the rodent.
Cellularity is highest in young animals with modest declines withage.40 Comparison to age- and sex-matched controls is therefore
essential. The bone marrow is more cellular in the normal healthy
mouse compared to the rat, making it difficult to differentiate
specific structures, vasculature, and cell types.
Changes in bone marrow should always be interpreted in
the context of clinical pathology/hematology evaluation of

684

Toxicologic Pathology 47(6)peripheral blood. Bone marrow is the source of peripheral
blood cells and therefore the marrow and the circulating blood
are interconnected. Changes to bone marrow should also beinterpreted holistically in context with findings in other organsystems, especially in circumstances of inflammation andneoplasia.

compartments may be confused with hemorrhage where bloodis present outside of endothelial lined spaces. Refer to the
INHAND circulatory system document (see Introduction) for
description of generalized changes to vascular structures applicable to bone marrow.
desNonproliferative Changes

Cellularity, Decreased, Adipocyte (N)Bone marrow

con
des

Angiectasis (N) (Figure 23) Bone marrow

con
enh

Angiectasis

Vessel Dilatation; Sinusoid Dilatation:
or Vessel/Sinusoid Dilatationenh

Atrophy, Adipocyte

Adipocytes, Decreased

Species
Mouse; rat.

Species
Mouse; rat.

Other Terms

Other Terms
Decreased adipocyte cellularity; adipocyte hypocellularity;
hypoplasia; depletion; fat atrophy.

Vascular dilation; vascular dilatation; vascular ectasia.

Pathogenesis/Cell of Origin

Pathogenesis/Cell of Origin

Decreased adipocytes in response to increased metabolic
demand, decreased caloric intake, or crowding/replacement
by increased hematopoietic cells.

Abnormally dilated endothelial-lined vascular spaces may be seen
with severe loss of hematopoietic tissue or associated with inflammation, neoplasia, and vascular or cardiovascular disorders.

Diagnostic Features

Dilatation of bone marrow vessels or sinusoids withblood or serum.
May be diffuse or focal.
Follows vascular patterns throughout the marrow
space.

Diagnostic Features

Medullary adipocytes (adipocytes in bone marrow)decreased.
Medullary hematopoietic cells may appear increased
due to relative decrease in adipocytes.
May be focal or diffuse.

Differential Diagnoses
Serous atrophy of fat

Differential Diagnoses
HemorrhageAbundant mature red blood cells outside of the endothelial-lined vessels.
Increased pigmented macrophages (hemosiderin) suggest chronicity.

HemangiomaWell-circumscribed mass of dilated irregular endothelial-lined spaces.

Comment
Bone marrow angiectasis is characterized by dilated blood orserum-filled vessels/sinusoids that are not increased in number
and have normal structure and well-differentiated endothelial
cells. If severe, the accumulation of blood within vascularAdipocytes and hematopoietic cells both decreased.
Bone marrow contains eosinophilic, seromucinous,
gelatinous, hyaluronic acid-rich material.

Comment
Medullary adipose tissue can be decreased by crowding,obstruction, and/or replacement by increased medullary
hematopoietic tissue. A decrease in the relative proportionof adipocytes in the bone marrow is considered an adaptive
response to an increase in the relative proportion of hematopoietic cells (increased medullary hematopoiesis). Decreased
medullary fat stores may occur with decreased nutritional
status. The overall health status of the animal and its systemic
fat reserves should be taken into consideration when evaluating decreased adipose cellularity as bone marrow fat reserves
are among the last to be mobilized. Decreased adipocyte

Willard-Mack et al.
cellularity should be diagnosed when the primary change is adecrease in adipocytes rather than when adipocytes decrease
as a compensatory response to increased hematopoietic tissue.
Comparison to concurrent control group animals is
recommended.
desCellularity, Decreased, Bone Marrow(Figures 24 and 25) Bone marrow

con
enh

Atrophy (N)Hematopoietic Cells, Decreased(Indicate cell type)Species
Mouse; rat.

Other Terms
Hematopoietic hypocellularity; hypoplasia; aplasia; depletion;
decreased cell numbers.

685



Decreased cellularity.
Necrotic cells and/or necrotic cellular debris present.

Comment
Cell type modifiers (erythroid, myeloid [granulocytic, monocytic], megakaryocytic) should be applied when decreases canbe identified in specific cell populations. Decreased bone marrow cellularity (atrophy) usually occurs diffusely within thebone marrow cavity. Focal or multifocal can be used to characterize the change if it is localized. Atrophy (reduction in size,
wasting), hypoplasia (decreased growth), and depletion (loss ofcells) describe different dynamic processes that all manifest as
fewer than normal numbers of cells in the bone marrow. These
conditions may appear similar histologically, but they havedistinctly different pathogeneses. Use of enhanced terminology
is suggested to avoid unintended or unsubstantiated interpretation of similar appearing clinical syndromes. Age-related
decrease in hematopoietic cells and replacement by adipose
tissue (see Adipocytes, increased) is part of the natural aging
process.47 Comparison of treatment groups to appropriate sex-,
strain-, and age-matched control groups is essential for theassessment of cellularity.

Modifier
Erythroid; myeloid (granulocytic, monocytic); megakaryocytic; NOS.

desDyshematopoiesis (N) Bone marrowcon

Pathogenesis/Cell of Origin
Decreased production or increased destruction of one or morehematopoietic cell lineages can be caused by toxicity, inanition, nutritional deficiencies, irradiation, autoimmune disease,inflammation, neoplasia, infectious agents, genetic defects, andthe normal aging process.

Diagnostic Features












Reduced hematopoietic cellularity.
Reduced area occupied by hematopoietic cells.
Real or apparent relative increase of adipose tissue,
fluid, or dilated bone marrow sinuses relative to hematopoietic cells.
Single or multiple cell lineages may be affected.
An entire cell lineage may be absent.
Maturation may be delayed or arrested at a specific stageof development.
Changes to myeloid/erythroid ratio (M:E ratio) may or
may not be apparent depending upon which cell lineages
are affected.
Distribution may be focal, multifocal, or diffuse.
Decreased cell count of affected cell type(s) may bepresent in peripheral blood.

enh

Dyshematopoiesis

Dyshematopoiesis

Species
Mouse; rat.

Other Terms
Altered hematopoiesis; abnormal maturation; myelodysplasia; dysmyelopoiesis; myeloid dysplasia; dysgranulopoiesis;
granulocytic dysplasia; myelomonocytic dysplasia;
dyserythropoiesis; erythroid dysplasia; erythrodysplasia; redcell dysplasia; thrombodysplasia; dysthrombopoiesis;
dysmegakaryopoiesis.

Modifier
Erythroid; myeloid (granulocytic, monocytic); megakaryocytic; NOS.

Pathogenesis/Cell of Origin
Abnormal or defective differentiation of any of the hematopoietic cell lineages.

Differential Diagnoses

Diagnostic Features

Necrosis

Dyshematopoiesis (General)

686














Toxicologic Pathology 47(6)Failure of normal maturation of erythroid, myeloid, and/or megakaryocytic lineages.
Altered cell and/or nuclear size, morphology, nuclear/cytoplasmic ratio, and/or maturation; asynchronous
nuclear and cytoplasmic development; lack of stages
in maturation series, including early or later stages(maturation arrest).
M:E cell ratio may be altered.
Increased or decreased cellularity may be present.
Increased nonlymphoid immature forms/blasts may bepresent.
When present, immature forms/blasts are 20% of celllineage.
Quantitative assessment best diagnosed by bone marrowsmear evaluation or flow cytometry.
Maturation defect of nonlymphoid hematopoietic
cells may manifest as cytopenia of one or more nonlymphoid hematopoietic cellular lineages in peripheral blood (eg, decreased RBCs, neutrophils, and/or
platelets).
Abnormal cells may be present in peripheral blood.
Nonhematopoietic tissues are not involved.
Diagnostic features generally best assessed by cytology (bone marrow smears) rather than H&E-stained
tissue.

Dyshematopoiesis, Granulocytic

Abnormal nuclear segmentation or asynchrony of chromatin maturation and nuclear segmentation.
Altered granule morphology and/or abnormal cytoplasmic features (eg, size, shape, number, and/or tinctorial quality).
Altered cell and/or nuclear size, morphology, nuclear/cytoplasmic ratio, and/or maturation; asynchronous
nuclear and cytoplasmic development; lack of stages
in maturation series, including early or later stages(maturation arrest).

Dyshematopoiesis, Erythroid

Multiple or satellite nuclei, or nuclear fragmentation
and/or abnormal nuclei shapes.
Abnormal cell size (ie, megaloblasts, abnormal
sideroblasts).
Altered cell and/or nuclear size, morphology, nuclear/cytoplasmic ratio, and/or maturation; asynchronous
nuclear and cytoplasmic development; lack of stages
in maturation series, including early or later stages(maturation arrest).

Dyshematopoiesis, MegakaryocyticAbnormal megakaryocyte size or nuclear morphology,
Asynchronism between nuclear and cytoplasmic
maturation.

Differential Diagnoses
Leukemia (Myeloid, Erythroid, Granulocytic, Megakaryocytic,
Lymphoid)Leukocytosis in the peripheral blood with immatureforms/blasts.
Peripheral blood may contain abnormal megakaryocytes
and/or atypical platelets.
Immature nonlymphoid hematopoietic forms/blasts are>10% to 20% in hematopoietic tissues.
Neoplastic cells are often present in tissues in addition toblood, bone marrow, and spleen. May lead to diffuse
leukemic involvement of tissue.

Comment
Dyshematopoiesis may be used as a general term encompassing abnormalities in one or more hematopoietic cell lineages.
Dyshematopoiesis may be further characterized as abnormal
maturation of specific cell lineages, for example, erythroid,
granulocytic, and/or megakaryocytic lineages. Accurate diagnosis requires knowledge of normal maturation stages of all
lineages. Dyshematopoiesis is characterized by the presence ofprecursors or abnormal cells in the absence of normal maturation. Histological evaluation of bone marrow in decalcified
bone sections stained with H&E is used as a screening assay.
Bone marrow smear evaluation (cytology) is used for quantitative assessment of relative cell numbers and to characterizecell morphology and confirm altered morphology when present.48 Differentiation of dyshematopoiesis and leukemia canoften be made. Dyshematopoiesis may occur de novo ordevelop secondary to xenobiotics, toxicities, and/or irradiation.
Note: Dysplasia is strictly defined as abnormal growth ordevelopment. In the bone marrow, the terms dysplasia andmyelodysplasia have historically been used to describe abnormal development of hematopoietic cells, specifically of myeloid cells. However, the term myelodysplasia is sometimes
used to indicate a preneoplastic condition. While rare, myelodysplasia may be encountered in genetically modified mice. If
this diagnosis needs to be used, it must be made in conjunction
with clinical pathology data.
desFibrosis (N) (Figure 26) Bone marrow

con
enh

Fibrosis
Fibrosis(indicate reticulin, collagen or NOS if appropriate)

Species
Mouse; rat.

Other Terms
Fibroplasia; reticular cell hyperplasia; stromal hyperplasia;
myelophthisis; myelofibrosis; scar formation.

Willard-Mack et al.

Pathogenesis/Cell of Origin
Fibroblasts, fibrocytes, or adventitial reticular cells originating from pluripotential adventitial cells may proliferate in
response to altered cytokine expression by resident cells, toinflammation, to injury, or secondarily to neoplasia resulting in increased collagen or reticulin within the medullary
cavity.

687








Diagnostic Featurescon

Fibrosis and enhFibrosis NOS (Not Otherwise Specified)












enh

Increased extracellular matrix (collagen and/or reticulin)in the medullary cavity.
Increased extracellular matrix may or may not be
accompanied by increased cellular elements (fibroblasts, fibrocytes, adventitial reticular cells) depending
upon chronicity and activity of the process.
Increased cellularity of fibroblasts/fibrocytes.
Fibroblasts/fibrocytes may be morphologically heterogeneous depending on their activity.
Fibroblasts are activated mesenchymal cells.
 Generally have an irregular or elongated cell bodyand an elliptical nucleus having 2 or more nucleoli.
 Synthesize/secrete collagen.
Fibrocytes are mesenchymal cells not engaged in synthesis of extracellular fibers.
 Generally have less eosinophilic cytoplasm with asmaller, more fusiform cell body.
Increased cellularity of adventitial reticular cells.
 Line the adventitial surface of vascular sinuses.
 Have pale cytoplasm, round vesicular nuclei, and asingle nucleolus.
 Synthesize/secrete reticulin.
May be diffuse, multifocal, or focal.

Differential Diagnoses
Fibrous Osteodystrophy























A specific term for increased collagen and reticulin
fibers in the medullary cavity.

Localized increase in the production of osteoblasts.
Well-differentiated localized proliferations of osteoblasts on bone surfaces.
May fill intertrabecular spaces.
Sometimes admixed with focal fibroplasia.

Decreased Cellularity, Bone Marrow

Fibrosis, Collagen

Fibrovascular tissue replacement of bone marrow.
Often accompanied by lesions of the reproductive tract.49
Occurs in mice.
Higher indicence in females than males

Hyperplasia, OsteoblastA specific term for increased reticular fibers in the
medullary cavity.
Increased cellularity of adventitial reticular cells.
Reticular stroma may be more prominent without being
increased.
Reticular fibers are difficult to identify in H&E-stained
tissue.
Reticular fibers stain black with silver stains.
Silver stain is required for definitive diagnosis of reticulin fibrosis.
Reticulin fibrosis may occur independently of, and often
precedes, collagen fibrosis.

Fibrosis of the medullary cavity, especially near theendosteal surface of cortical bone.
Associated with chronic renal disease.
Occurs in rats.

Fibro-Osseous Lesion

Fibrosis, ReticulinenhIncreased reticulin fibers are observed concurrently withincreased collagen.
Fibroblast and adventitial reticular cells may or may not
be increased depending upon chronicity and activity ofthe process.
Diffuse, multifocal, or focal.
Mature collagen stains positively with trichrome
stains.
Reticulin fibers stain positively with silver stains.
Special stains are required for definitive diagnosis ofcollagen fibrosis.

Decreased hematopoietic cellular components may
result in increased prominence of stromal elements.
Should be differentiated from true increase in fibroblastic
cells.

Comment
Bone marrow fibrosis is characterized as increased collagen
and/or reticular fibers with or without proliferation of fibroblasts and adventitial reticular cells. Increased collagen andreticulin are extracellular matrix materials that often, but notalways, occur concurrently with increased fibroblasts and/or
increased adventitial reticular cells, depending upon thechronicity and activity of the fibrotic process. Differentiation of reticulin fibrosis and collagen fibrosis is encouraged
if it would add value to the study. Fibrosis is a componentof chronic inflammation but may also be due to perturbationof cytokine production by resident stromal cells includingtransforming growth factor b, platelet-derived growth factor

688

Toxicologic Pathology 47(6)family, and other factors associated with megakaryocytes
and platelets. 49 Focal fibrosis has been occasionallyobserved in young or aging rats and may be due to injury,
inflammation, or necrosis.50 Fibrosis has been reported inmice in response to administration of recombinant thrombopoietin. Fibroproliferative responses have been associatedwith a variety of conditions (eg, pyruvate kinase deficiency,
gamma radiation, drugs, infectious agents, and malignancies). In humans, reticulin fibrosis is reported to be reversible with resolution of the inciting cause while collagenfibrosis is less likely to be so.51 Abnormal bone metabolism
may also affect the medullary cavity. For a more complete
description of stromal changes associated with altered bonemetabolism, see the INHAND document on Bone (see I.
bone).

Inflammation (N) (Figure 27) Bone marrow
See General Hematolymphoid
Necrosis (N) (Figure 28) Bone marrow
See General HematolymphoiddesSerous Atrophy of Fat (N) (Figure 29)
Bone marrow
con
Serous Atrophy of Fatenh

Serous Atrophy of Fat

Species
Mouse; rat.

desHypersegmentation, Granulocyte(N) Bone marrow

con
enh

Other Terms
Gelatinous transformation.

Hypersegmentation, Granulocyte

Hypersegmentation, Granulocyte

Species
Mouse; rat.

Pathogenesis/Cell of Origin
Reduction of adipocytes associated with cachexia (eg, secondary to neoplasia, endocrinopathies) and advanced severe malnutrition (eg, maldigestion, malabsorption).

Diagnostic Features

Pathogenesis/Cell of Origin

Granulocytes.



Diagnostic Features

Increased numbers of mature granulocytes with nuclear
hypersegmentation.
Hypersegmented granulocytes present in both bone marrow and peripheral blood.

Focal or diffuse depletion of adipocytes and hematopoietic cells.
Replacement of adipose tissue by eosinophilic gelatinous tissue.
Accumulation of extracellular gelatinous substances(hyaluronic acid, mucopolysaccharides) that usually
stain positive with Alcian blue at pH 2.5.

Differential Diagnoses
Cellularity, Decreased, Adipocyte

Differential Diagnoses

Dyshematopoiesis
Abnormal granulocyte precursor cellular morphology
exclusive of nuclear hypersegmentation.



Decrease in adipocytes in the absence of eosinophilicgelatinous tissue.
Relative increase in hematopoietic cells.

Comment
Comment
Granulocyte hypersegmentation in bone marrow is characterized by myeloid cells that have megaloblastic features withgiant metamyelocytes and some hypersegmented mature cells.
In peripheral blood, neutrophil nuclei are hypersegmented withat least 6 nuclear lobes. Similar changes have been reported inassociation with a variety of causes including infectious disease, dietary deficiencies, xenobiotics, and corticosteroids.
Corticosteroids cause retention of granulocytes allowing moretime for maturation.

Serous atrophy of fat is rarely encountered in standard toxicity
studies because humane termination occurs at a predetermined
degree of weight loss. The pathogenesis remains unclear, but isthought to be a basic bioregulatory process that is activated instates of advanced illness often associated with cachexia andweight loss.

Proliferative Changes (Non-Neoplastic)
Hyperplastic changes in all the hematolymphoid organs,including the bone marrow, are generally reactive and are partWillard-Mack et al.
of the normal physiological responses of these organs to acuteand chronic insults or physiologic stimulation. Hyperplasticchanges do not infer preneoplastic or precancerous lesions inthese organs (see Introduction). However, severe or persistent
lymphoid hyperplasia may increase the risk of neoplastic transformation. If there is a concern, clonality studies should beconsidered.

689
rather than when adipocytes increase as a compensatory
response to decreased hematopoietic tissue. Comparison to
concurrent control group animals is recommended.
desCellularity, Increased, Bone Marrow(H) (Figures 31-33) Bone marrow

con
des

Cellularity, Increased, Adipocyte(H) (Figure 30) Bone marrow

con
enh

enh

Hyperplasia, Bone Marrow

Hematopoietic Cells, Increased(indicate cell type(s))

Hyperplasia, Adipocyte

Adipocytes, Increased

Species
Mouse; rat.

Species

Other Terms

Mouse; rat.

Increased hematopoiesis; hematopoietic hypercellularity; panhyperplasia; plasmacytosis; reactive plasma cell hyperplasia;
regeneration.

Other Terms
Increased adipocyte cellularity; increased fat; focal lipomatosis; adipocyte accumulation.

Pathogenesis/Cell of Origin
Adipocytes.

Diagnostic Features

Increased number or cell density of adipocytes within
medullary cavity.
Focal, multifocal, or diffuse.

Modifier
Erythroid; myeloid (granulocytic, monocytic); megakaryocytic; lymphocytic; plasma cell; NOS.

Pathogenesis/Cell of Origin
Proliferation of hematopoietic progenitor cells of one or more
lineages.

Diagnostic Features

Differential Diagnoses
Reduced hematopoietic cellularity.



Focal/Multifocal Atrophy, Hematopoietic CellCellularity, Decreased, Bone MarrowWell-delineated area containing reduced hematopoietic
tissue.

Comment
Rodents generally have less fat and more hematopoietic elements in their marrow cavities compared to other mammals.
Relative fat content of bone marrow varies with species, strain,sex, age, anatomic site, and activity of hematopoietic elements.52 It is generally more physiologically relevant toexpress changes in the relative proportions of adipocytes andhematopoietic cells in terms of increased or decreased hematopoietic cells. Increased adipocyte cellularity should be diagnosed when the primary change is an increase in adipocytesIncreased hematopoietic cellularity involving single ormultiple cell lineages.
Increased area occupied by hematopoietic cells.
Diffuse distribution within the medullary cavity.
Morphology and maturation sequences aresynchronous.
Altered myeloid/erythroid ratio (M:E ratio) may or may
not be apparent depending upon which cell lineages areaffected.
Increased cell count of affected cell type(s) is usually,
but not always, present in peripheral blood depending
upon chronicity and/or peripheral consumption of thecell type.
Extramedullary hematopoiesis may or may not bepresent.
Lymphocytes and monocytes cannot be easily distinguished in routine H&E-stained tissue sections.
Plasma cells are rare and cannot reliably be quantified by histology due to limited sampling of tissue
sections
 Identify plasma cells by IHC for IRF4 and CD138.53
 Quantify plasma cells by flow cytometry.

690

Toxicologic Pathology 47(6)Differential Diagnoses
Hematopoietic Neoplasms









Differentiation of granulocytic hematopoietic hypercellularity from well-differentiated granulocytic leukemia
may be challenging.
Both lymphoid and nonlymphoid organs may be involved.
Abnormal forms of progenitor cells may be present.
Immature forms/blasts increased above 20% of a given
lineage are suggestive of neoplasia.54
Quantitative data from bone marrow smears and flow
cytometry can help differentiate hypercellularity fromhematopoietic neoplasia.
See hematolymphoid neoplasms for additional diagnostic criteria.

Dyshematopoiesis

Lack of maturation.
Lack of normal production.
Need blood smear or bone marrow smear to confirm
diagnosis.

desCellularity, Increased, Macrophage(H) (Figure 34) Bone marrow

con
enh

Hypertrophy/Hyperplasia, Macrophage

Macrophages, Increased

Species
Mouse; rat.

Other Terms
Macrophage accumulation; macrophage infiltrate; macrophage
infiltration; prominent macrophages; histiocytosis; histiocytic
hyperplasia; histiocytic infiltrate; histiocytic aggregates.

Modifier
Tingible body; pigmented; vacuolated; aggregates.

Pathogenesis/Cell of Origin
Comment
Bone marrow cellularity may vary due to species, strain, sex, age,and location (evaluation of both sternal and femoral marrow isrecommended for rodents). Cellularity may be assessed by estimating the ratio of hematopoietic cells to medullary adipocytes.
With increased bone marrow cellularity (hyperplasia), the proportion of hematopoietic cells is increased relative to adipocytes.
Decreased adipocyte cellularity (atrophy) can also result in asimilarly altered ratio and should be differentiated from increased
hematopoietic cells. Mouse bone marrow generally has less fat
and more hematopoietic cells than rat bone marrow; therefore,
the prominent venous sinusoids become compressed as the hematopoietic tissue expands.55 Robust normal proliferation of hematopoietic cells may normally expand beyond the medullary cavity
along perivascular spaces of nutrient blood vessels and must be
differentiated from invasion by neoplastic cells. Multiple
mechanisms may result in increased marrow cellularity. For
example, increased erythroid cellularity may result from administration of exogenous erythropoietin (or potentially from an erythropoietin-producing tumor), from increased endogenous
expression of erythropoietin due to anemia from blood collection,
hemorrhage, or hemolysis or from hypoxia due to chronic heart orlung disease or congenital cyanotic heart defect. Increased myeloid cellularity may result from a variety of factors includingexcessive loss due to hemorrhage or from increased demand forneutrophils or other white blood cells due to infection/inflammation in peripheral tissues. Increased megakaryocyte cellularity
may be due to increased metabolic demand, pregnancy/lactation,
endogenous overexpression, or exogenous administration ofthrombopoietin.56 Plasma cells are a normal component of bonemarrow and may be increased as part of an immune response to an
inflammatory condition, infection, or neoplasia.

Monocyte/macrophages.

Diagnostic Features

Increased abundance and/or size of macrophages within
medullary cavity.
Cytoplasm may or may not contain phagocytized material, pigment, or vacuoles.

Differential Diagnoses
Granuloma

Organized structure with a compact collection of epithelioid macrophages or multinucleated giant cells andother inflammatory cells. May variably include necrosis,
infectious agents, or exogenous materials.
Associated with chronic inflammatory conditions andexposure to xenobiotics.

Cellularity, Increased, Mast Cell

Cells have pale basophilic or eosinophilic cytoplasm
containing abundant basophilic granules that stain metachromatically with Giemsa or toluidine blue stains.
Cytoplasm is not foamy or vacuolated.
Degranulated or immature mast cells may be difficult todifferentiate from macrophages.

Histiocytic SarcomaTumor cells are usually more atypical and pleomorphic
than hyperplastic macrophages.
Multinucleated giant cells often present.

Willard-Mack et al.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace, or destroy normal architecture.
Other tissues may be involved.

Comment
Macrophages may increase in response to demand for phagocytosis or in support of erythropoiesis. Increased macrophage
cellularity (hypertrophy/hyperplasia) often occurs in combination with phagocytosis, pigment storage, vacuolation, andaggregation as macrophages increase to meet the demand driving these processes. The diagnostic terminology for increased
cellularity therefore includes these processes as modifiers toallow the pathologist to construct the most appropriate diagnosis for a particular constellation of features. These findings
can also be diagnosed separately.
des691
Mast Cell Leukemia

Mast cells present in bone marrow and peripheral blood.
Confirm mast cells and rule out basophils with special
stains.
Mast cells are positive for both CAE and c-kit, whereas
basophils are negative for both stains.53

Comment
Mast cells are more prominent in the medullary cavity of the rat
than the mouse.48 Increased mast cells may represent an exaggerated inflammatory response and have been observed in
infection models with or without parasites.

THYMUS
Organization

Cellularity, Increased, Mast Cell
(H) (Figure 35) Bone marrow

con
enh

Hyperplasia, Mast Cell

Mast Cells, Increased

Species
Mouse; rat.

Other Terms

The thymus is a primary lymphoid organ and specialized glandthat is composed of 2 identical lobes connected by an isthmus. In
rats and mice, it is located in the cranial mediastinum in the chest,
adjacent to the cranial part of the heart and dorsal to the sternum.
A thin connective tissue capsule surrounds each lobe and gives
rise to collagenous septa that partially subdivides the lobes into
lobules of variable size and orientation. Each lobule is composedof a central medulla and peripheral cortex. The thymus is composed of cells of hematopoietic and stromal origin including
thymic epithelial cells (TECs), neural crest-derived mesenchymal cells, endothelial cells, and dendritic cells (Table 3).

Increased mast cell cellularity; mast cell infiltrate; mast cell
accumulation; mastocytosis (see comment).

Table 3. Compartments and Cellular Components of the Thymus.
Compartment

Components

Pathogenesis/Cell of Origin

Subcapsular zone

Increased mast cells in bone marrow may be associated with an
inflammatory response, parasitism, or hematologic diseases.

CortexCD4–/CD8–T cells
Epithelial cells
CD4þ/CD8þ T cells
Epithelial cells
Thymic nurse cells
Dendritic cells
Apoptotic cells
Macrophages
Plasma cells
B cells
CD4þ/CD8 T cells
CD4/CD8þ T cells
Epithelial cells
Dendritic cells
Apoptotic cells
Macrophages
Hassall’s corpuscles
B lymphocytes
Myoid cells
Neuroendocrine cells
Mature and immature T cells
B lymphocytes
Plasma cells
CD4þ/CD8þ T cells

Diagnostic Features

Multifocal to diffuse increase of loosely arranged mast
cells in the bone marrow.
Mast cell granules are metachromatic when stained withtoluidine blue or Giemsa.
Mast cells are positive for chloroacetate esterase (CAE)and c-kit (CD117) by IHC staining.

MedullaDifferential Diagnoses
Mast Cell Tumor






Focal nodular increase of mast cells.
Compression and loss of adjacent normal architecture.
Variable degrees of mast cell differentiation.
Often well differentiated in rodents.
See Hematopoietic Neoplasia section for additional
details.

Corticomedullary junction

Epithelial free area(some rat strains only)692

Function
As a primary lymphoid organ, the thymus is a site where T
lymphocytes (T cells) differentiate and mature. T lymphocytes develop from a common lymphoid progenitor in thebone marrow. Those cells that are destined to give rise to T
cells leave the bone marrow and migrate to the thymus where
they divide and mature to produce T cells. During this process, they complete their antigen-independent maturation intofunctional naive T cells. Once mature, the T cells emigratefrom the thymus, join the recirculating pool of lymphocytes,
and begin searching for their cognate antigens in specificcompartments of the secondary lymphoid organs, such as theparacortical regions of lymph nodes and the PALs of thespleen. At this point, T cells have a critical role in the adaptive
immune system, providing an immune response that is highly
specific to a particular pathogen. Immunological memory
occurs after an initial response to a specific pathogen, leading
to an enhanced response to subsequent encounters of thatsame pathogen.

Development
The embryonic development of the thymus and the parathyroid glands is intimately linked. Both develop from the endodermal gut tube and are derived from outpockets of the mostanterior region of the foregut, the pharynx, which is composedof a ventrally located thyroid diverticulum and a series ofpaired transient outpockets of the lateral foregut called thepharyngeal pouches. Each of the pharyngeal pouches of thethird pair of outpockets forms a single epithelial organ primordium surrounded by a mesenchymal capsule. Neural crest
cells migrate into the pharyngeal region and surround the third
pouch during early development and become the mesenchymal cells that eventually form the thymic mesenchymal capsule and become associated with the thymic vasculature. As
the paired primordia detach from the pharynx via apoptosis,
they separate into individual bilateral primordial thymus andparathyroid organs while migrating to their final positions in
the body. Soon after detachment, the parathyroids remain in
the proximity of the thyroid gland. The primordial thymic
lobes migrate caudally into the chest cavity and the 2 lobes
meet at the midline, just above the heart. At this early stage,
the thymus is composed only of TEC and is devoid of lymphocytes. Beginning at around embryonic day 11.5 in themouse, bone marrow–derived lymphocyte progenitor cells are
attracted to the thymus by factors (chemokines) secreted by
the TEC. Lymphocyte progenitor cell immigration occurs at
precise stages of organogenesis in successive discrete waves.
The initial immigration occurs prior to vascularization of thethymus. In mice, the initial entry of the progenitor cells into
the thymic anlage has been shown to be a 2-step process.
Progenitor cells accumulate in the mesenchymal layer atembryonic day 11 and then enter the epithelial cluster onembryonic day 12. Later, they enter via venules at the corticomedullary junction. Lymphocyte progenitor cells migrate

Toxicologic Pathology 47(6)centripetally through the cortex to the subcapsular region
where they proliferate as lymphoblasts. Lymphoblasts mature
into naive T cells as they migrate back through the cortex andinto the medulla. Lymphocyte progenitor cell entry into thethymus from the bone marrow continues in the postnatal
period.

Histology
In the thymus, T cells develop their specific T cell markers,
including the T-cell receptor (TCR), CD3, CD4, CD8, andCD2. T-cell maturation occurs via expression of the TCRCD3 complex and its coreceptors CD4 and CD8 (differentiation antigens). Lymphocyte progenitor cells from the bonemarrow enter the thymus at the corticomedullary junction as
CD3/TCR. At this stage, they begin to express CD2, have notyet begun to rearrange their TCR genes, and have an immature
double negative phenotype (CD4/CD8). They migrate to thesubcapsular zone of the outer cortex and then mature as theymigrate back through the cortex, becoming CD3/TCRþ andCD4/CD8 double positive (CD4þ/CD8þ), and then migrate
to the medulla where they become single positive T cells(CD4þ/CD8, CD4/CD8þ).
As the T cells migrate through the cortex, TCR gene rearrangement occurs and they undergo positive selection. Only
the cells able to recognize antigen in the context of class I orclass II major histocompatibility complex (MHC), expressedby the TEC (thymic nurse cells), will be “positively selected”
to survive. Those that are unable to recognize antigen in thecontext of self-MHC (weakly binding cells) within 3 to 4 days
undergo apoptosis, accounting in part for the apoptotic cells
and tingible body macrophages that are scattered throughout
the cortex in normal control animals. Those T cells that have astrong or medium binding capacity to either MHC class I/II or
peptide molecules will survive. A small degree of negative
selection also occurs in the cortex via exposure to self-antigens, allowing for the removal of autoreactive T cells. To
avoid autoimmunity, those T cells with high affinity are eliminated via apoptosis and those with intermediate affinity
survive.
The T cells that survive then enter the medulla where there
are fewer lymphocytes and relatively more TECs. In the
medulla, T cells undergo further negative selection by interacting with thymic dendritic cells. These cells express transcriptional regulators AIRE and FEZ2 and this allows for thetranscription of a variety of genes related to the expression ofa more complex set of self-antigens than is present in the cortex. As with positive selection, those T cells that don’t survive
during negative selection are eliminated via apoptosis. However, this process is not 100% effective, so some autoreactive T
cells will survive and enter the circulation.
Thymic (Hassall’s) corpuscles are present in the medulla.
These structures consist of one or more central granular cells
surrounded by concentric layers of epithelial cells. In the rat,
they form whorls of flattened cells with central cell debris orconcentrically arranged keratin; in mice, they are less well

Willard-Mack et al.
defined and do not form central keratin. Thymic corpuscles
are remnants of the epithelial tubes that grow out from thethird pharyngeal pouches of the embryo to form the thymus.
They seem to increase in number during times of increased
lymphocyte apoptosis and may have a role in clearing cellular debris.
The TEC are present throughout the thymus and provide a
supporting meshwork for lymphocyte migration. In addition totheir role in the positive and negative selection process, TEC
also secrete hormones such as thymopoietin, thymosin, thymulin, and thymic humoral factor that support T-cell maturationand enhance T-cell function.

Sampling and Diagnostic Considerations
The thymus should be fixed in 10% formalin and thentrimmed along the length of both lobes, to provide standardized longitudinal sections that show all anatomical structures. Cuts should be through the middle of the lobes toallow for evaluation of the largest surface area. The cortex
and medulla should be evaluated separately for both conventional and enhanced histopathology. The cortex:medulla ratio
can be estimated by determining the average of ratios acrossmultiple lobules.
Because the thymus is sensitive to the effects of stressand aging, it is important to differentiate xenobioticinduced thymic decreased cellularity (atrophy) fromstress-related lymphocyte apoptosis and age-related thymic
involution. Because of the effects of aging on the thymus, it
is best to conduct enhanced histopathology on short-term
studies.

Nonproliferative Changes
desApoptosis, Increased, Lymphocyte (N)(Figure 36) Thymus

con
enh

Apoptosis, Increased, Lymphocyte

Apoptosis, Increased, Lymphocyte(indicate compartment and diagnose decreased lymphocytes, decreased area, tingible body macrophages, etc. separately if applicable)

Species
Mouse; rat.

Other Terms
Lymphocyte depletion; atrophy; cell death; single-cell
necrosis693

Pathogenesis/Cell of Origin
Lymphocyte apoptosis may result from direct thymic lymphocyte toxicity or from endogenous factors such as diet or stress(glucocorticoid release).

Diagnostic Features











Single cells or small clusters of cells.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cytoplasm retained in apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis.
Nuclear fragmentation.
Intact cell membrane.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

Differential Diagnoses
Necrosis, Lymphocyte






Necrotic cells are often contiguous, but pattern can be
focal, multifocal, or diffuse.
Cell swelling.
Cell rupture.
Karyolysis, pyknosis, and karyorrhexis.
Inflammation usually present.

Cellularity, Decreased, Lymphocyte (Atrophy)









Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells.
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined by cuboidal to squamous epithelium.

Comment
Apoptosis is a coordinated and often energy-dependent modeof cell death that is considered a vital component of various
normal processes. Apoptosis eliminates activated or autoaggressive immune cells during maturation; therefore, a low
level of lymphocyte apoptosis in the thymus is consideredwithin normal physiological variation. Increased lymphocyte
apoptosis may result from direct thymic lymphocyte toxicity
or from endogenous factors such as diet or stress (glucocorticoid release). Severe ongoing apoptosis results in severe
decreased lymphocyte cellularity (lymphoid atrophy). Necrosis may occur together with apoptosis. While it is preferable to694

Toxicologic Pathology 47(6)identify and record diagnoses of apoptosis and classic necrosis separately, this distinction may not be possible when one
type of cell death histologically obscures the other. Also,
necrotic cell debris can have some similarities to apoptotic
debris, such as pyknosis and karyorrhexis. Apoptosis may
predominate with conversion to a necrotic phenotype, or
necrosis may predominate with scattered apoptosis. In thesecases, it would be appropriate to use both terms together(apoptosis/necrosis) or only diagnose the predominate typeof cell death and discuss the presence of the other type of cell
death in the narrative.34
desCellularity, Decreased, Lymphocyte(Figure 37) Thymus

con
enh

Atrophy (N)Lymphocytes, Decreased(indicate compartments and diagnose apoptosis, tingible
body macrophages, etc, separately if applicable)

Species
Mouse; rat.

Locators
Cortex; medulla.

Other Terms
Lymphocyte depletion; lymphoid depletion; cortical depletion;
lymphoid atrophy.

Pathogenesis/Cell of Origin
Decreased lymphocyte cellularity (atrophy) may result fromchronic direct thymic lymphocyte toxicity or may be related
to endogenous cortisol released in response to stress.



Fat (adipocyte) infiltration is not present.
Finding not present in concurrent controls.

Differential Diagnoses
Necrosis, Lymphocyte






Necrotic cells are often contiguous, but pattern can be
focal, multifocal, or diffuse.
Cell swelling.
Cell rupture.
Karyolysis, pyknosis, and karyorrhexis.
Inflammation usually present.

Apoptosis, Increased, Lymphocyte











Single cells or small clusters of cells.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cytoplasm retained in apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis.
Nuclear fragmentation.
Intact cell membrane.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

Involution, Age-Related









Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined bycuboidal to squamous epithelium.

Diagnostic Features










At the gross and subgross level, the entire organ is small
compared to concurrent controls.
Decreased lymphocyte cellularity in the cortex andmedulla.
Underlying stromal cells may be more prominent.
May have loss of corticomedullary distinction.
May have paler staining cortex due to decreased cellularity and darker staining medulla due to increased
cellularity.
Increased lymphocyte apoptosis and/or tingible bodymacrophages may be present if decreased cellularity(atrophy) is ongoing.
Lymphoid decreased cellularity (atrophy) may be present in other lymphoid organs.

Comment
Treatment-related decreased cellularity is a common finding
in toxicity studies when doses at or above the maximum tolerated dose are used. Treatment-related decreased cellularity
can be a direct effect of immunomodulation or a secondary
effect of stress. It should not be attributed to stress unlessthere are other corroborating findings present, such as marked
body weight loss, cortical hypertrophy in adrenal glands, andeffects in other lymphoid organs, such as reduced lymphoidcellularity and decreased germinal centers in lymph nodes andspleen. In chronic studies, decreased lymphocyte cellularity(atrophy) may overlap with age-related involution and distinguishing them can be challenging. Comparison with

Willard-Mack et al.
concurrent controls may help to make this distinction. A
short-term study evaluated with enhanced histopathology may
be necessary to establish the etiology or mechanism withcertainty. The term atrophy may be used in chronic studies,but atrophy may also be used in short-term studies if thepathogenesis/mechanism of cellular loss is not needed andenhanced histopathology is not performed.

695


Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined by cuboidal to squamous epithelium.

Comment

Mouse; rat.

The corticomedullary ratio is generally determined subjectively
using the average of multiple ratios. The medulla normally occupies about one-third of the lobular volume in the adult rodent, so
the normal ratio of cortex to medulla is approximately 2:1. However, the ratio may vary by species and strain, so comparison
should be made to concurrent controls. The ratio can also varydepending on orientation so a standardized trimming procedure
should be followed. A decrease in the corticomedullary ratio may
be due to either decreased cortical lymphocytes (most commonly)
or increased medullary lymphocytes (or both). Dexamethasone
and some organotin compounds such as dicotyltin dichloride and2-acetyl-4(5)-tetrahydroxybutylimidazole have been associatedwith decreased corticomedullary ratios.57 If both decreased andincreased corticomedullary ratios are present, then the term
“altered corticomedullary ratio” may be used.20,58,59

Other Terms

des

des

Corticomedullary Ratio, Decreased(N) (Figure 38) Thymus
con
Corticomedullary Ratio, Decreasedenh

Corticomedullary Ratio, Decreased(diagnose lymphocyte apoptosis, necrosis, etc, andincreased/decreased area separately if applicable)

Species

Cortical depletion; atrophy; physiological involution; agerelated involution.

Corticomedullary Ratio, Increased(N) (Figure 39) Thymus
con
Corticomedullary Ratio, Increased

Pathogenesis/Cell of Origin

enh

Most commonly results from apoptosis or necrosis in the cortex
resulting in cortical lymphocyte depletion while the medulla
often remains the same in size and cellularity.

Diagnostic Features

Thymus is grossly smaller (decreased size and weight)
than normal or controls.
Loss of immature small lymphocytes from cortex.
 Apoptosis (focal, multifocal, or diffuse) of corticallymphocytes.
 Necrosis (focal, multifocal, or diffuse) of corticallymphocytes.
Medulla is normal in size and cellularity but may appear
relatively larger.

Corticomedullary Ratio, Increased(diagnose lymphocyte apoptosis, necrosis, etc, andincreased/decreased area separately if applicable)

Species
Mouse; rat.

Other Terms
Cortical hyperplasia; medullary depletion; atrophy.

Pathogenesis/Cell of Origin
Regeneration of cortex after insult or treatment-related
increase in cortical lymphocytes or decrease in medullary
lymphocytes, or both.

Differential Diagnoses
Atrophy








Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells (seerelated diagnosis of cellularity, increased, B cell).
Infiltration of adipocytes in connective tissue capsuleand septa.

Diagnostic Features

Cortex larger than normal and/or medulla smaller thannormal.

Differential Diagnoses
LymphomaGrowth may extend beyond thymus, invade mediastinal
fat, or disperse throughout the hematolymphoid systemand to other organs.

696




Toxicologic Pathology 47(6)May present as a focal lesion in the cortex of one lobe or
as diffuse infiltration in one or both thymic lobes withinvolvement of mediastinal lymph nodes.
Possesses a more homogeneous cell population, especiallyof lymphoblasts, compared to the mixture of large andsmall lymphocytes in lymphoid hyperplasia.

Comment
An increase in the corticomedullary ratio may be due to either
increased cortical lymphocytes or decreased medullary lymphocytes (or both). Cyclosporin alters the corticomedullaryratio by increasing lymphocyte cellularity in the medulla sothat it takes on the appearance of the cortex (cortification of
the medulla). The original corticomedullary border is still
located at its original position based on the vasculature.12 If
cortical lymphocytes are increased, lymphoma should be ruledout. If both decreased and increased corticomedullary ratios arepresent within a study, then the term “altered corticomedullaryratio” may be used.20,58,59

Differential Diagnoses
Epidermal Cyst, Squamous

Cyst, Epithelial (N) (Figure 40 and 41)
Thymuscon
enh

Cyst, Epithelial

Epithelial Cysts(indicate compartment if applicable)

Species
Mouse; rat.

Other Terms
Thymopharyngeal duct remnants; thymopharyngeal duct cyst;
epithelial cyst; epidermal cyst.

Adjacent to thymus or within thymic capsule.
Squamous epithelial lining with hair shafts, sebaceous
glands, and keratin.

Hyperplasia, EpithelialdesGenerally arise from dilated tubules (see epithelial hyperplasia).
&
Lined by nonciliated epithelium.
&
Generally are associated with increased epithelial structures (cords and tubules).
&Are found in the medulla.
&
Have cuboidal to columnar epithelium.
&
Vary in size.
In aging mice, cysts with or without ciliated epithelium
may be found within the medulla.
&

Increased epithelial cell component with or without
increased cords and tubules.

Involution, Age-Related









Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells (seecellularity, increased, B cell).
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined bycuboidal to squamous epithelium.

Pathogenesis/Cell of Origin

Comment

Thymic tubular structures or remnants of thymopharyngeal
duct.

Epithelial cysts are a common background finding that canbe congenital or may be associated with age-related physiological involution. Congenital epithelial cysts are derived
from the thymopharyngeal duct which forms during thedevelopment of the thymus from the third pharyngeal pouch.
Congenital cysts are common background findings in rodents
of all ages. Cysts associated with aging are located in the
medulla and are related to epithelial hyperplasia. Epithelial
cysts can also be common in young mice from some mouse
lineages. An increase in epithelial cysts may be related totreatment. At the pathologist’s discretion, all cysts may belumped together under the term “epithelial cysts.” Thymopharyngeal cysts may also be diagnosed separately. Ultimobranchial cysts should not be diagnosed in the thymus; they
are congenital cysts found in the thyroid gland. They arederived from remnants of the ultimobranchial body, an

Diagnostic Features

Cysts may contain homogeneous eosinophilic material
in the lumen.
Cysts may be congenital, age related, or associated withtreatment.
 Congenital cysts arise from the thymopharyngeal
duct.
&
Generally singular with variable size.
&
Lined by ciliated cuboidal epithelium.
&
Generally found in the periphery of the thymus
but may also be found in the medulla.
 Cysts that are not congenital.

Willard-Mack et al.

697

outpocketing of the fourth pharyngeal pouch that combineswith the thyroid diverticulum, giving rise to calcitonin-producing C-cells.50,60,61

Pathogenesis/Cell of Origin

desDiagnostic Features

Ectopic Tissue, Parathyroid (N) (Figure 42)
Thymusconenh

Pathogenesis and cell of origin varies depending on the type oftissue present.



Ectopic Tissue, Parathyroid

Ectopic Tissue, Parathyroid

SpeciesdesEctopic Tissue, Thymus (N) (Figures 43
and 44) Thymus

con

Mouse; rat.
enh

Other Terms

Other ectopic tissues such as skeletal muscle may bepresent within, or adjacent to, the thymus.

Ectopic Tissue, Thymus

Ectopic Tissue, Thymus(indicate location, eg, parathyroid, ectopic tissue, thymus)

Accessory parathyroid tissue.

Pathogenesis/Cell of Origin
Genetic mutation of Hox genes results in aberrant migration of
the parathyroid glands during development.

Diagnostic Features

Small focus of endocrine-like cells in the thymic capsule, connective tissue septa, or isolated foci within thethymic parenchyma.
Can occur in the thymus or dorsolateral to the esophagus
near the larynx.
More common in mice, especially CD-1 mice.

Species
Mouse; rat.

Other Terms
Aberrant thymic tissue; thymic remnant.

Pathogenesis/Cell of Origin
Genetic mutation of Hox genes results in aberrant migration
during development in some cases.

Diagnostic Features

Differential Diagnoses


None.

Comment
The parathyroid glands and the thymus are both derived fromendoderm of the third pharyngeal pouch.62 It has been postulated that ectopic parathyroid tissue may be hormonally active.
Small foci of parathyroid chief cells can be observed normal in
the mouse thymus.63,64
desEctopic Tissue, (specify tissue)(N) Thymus

con
enh

Ectopic Tissue, (specify tissue)Histologically normal ectopic thymus tissue located in
the thyroid and/or parathyroid gland.
The ectopic tissue is predominately cortical tissue.
Pale staining clusters of TEC and/or thymic (Hassell’s)
corpuscles within the foci.

Differential Diagnoses
Simple Lymphoid Aggregates

Aggregates of lymphocytes without structure (ie, without cortex or medulla).
Inflammation, Mononuclear, Thyroid Gland

Presence of interstitial inflammatory lymphocytes
within the thyroid.
May include plasma cells.
May be extension of inflammation in adjacent soft tissues or part of a generalized disease.

Ectopic Tissue, (specify tissue)
Comment

Species
Mouse; rat.

Ectopic thymus (remnants of thymus tissue in the neck region
near the thyroid gland) is common in rodents. Murine thymic

698

Toxicologic Pathology 47(6)tissue remnants can support T-cell differentiation and can
export T cells to the periphery. High incidence has beenreported in BALB/c, CBA/J, and C57BL/6 mice with no elevated incidence of autoimmunity. Prenatal exposure to retinoic
acid has been associated with an increase in “ectopic” thymus.65 No toxicological significance has been attached to thisfinding. It can be a reason for failure of neonatal thymectomy
in immunological studies.50,62,66-70
desHypoplasia (N) (Figures 45 and 46)
Thymusconenh

Genetic Thymic Hypoplasia

Occurs in nude rat or mouse, SCID mouse, Rag1/2 nulls,
and other mutant strains.
 In nude rat, thymus is cystic.
 In SCID, Rag, and NSG mice, thymus consists
predominantly of cytokeratin-positive epithelial
cells.

Aplasia/HypoplasiaCongenital absence of thymus tissue.

Hypoplasia

Comment

Lymphocytes, Decreased

Hypoplasia must be distinguished from thymic decreased cellularity (atrophy) or age-related thymic involution and phenotypes (chemical or genetically engineered models) that result in
reduced/negligible thymic tissue.

(indicate compartments and diagnose decreased area separately if applicable)

Species
Mouse; rat.

Inflammation (N) Thymus
See General Hematolymphoid

Other Terms
Decreased cellularity; aplasia.

desPathogenesis/Cell of Origin

con

Involution, Age-Related (N) (Figures 47-49)
ThymusInvolution, Age-Related

Hypoplasia is a congenital condition characterized by underdevelopment or incomplete development of the organ.

enh

Diagnostic Features

Species

Decreased lymphocytes in the cortex and medulla.
Some thymic tissue present but not fully developed.

Not Applicable

Mouse; rat.

Differential Diagnoses

Other Terms

Atrophy/Involution, Age-Related

Physiological involution; lymphocyte depletion; lymphocyte
atrophy; aging atrophy; atrophy.









Thymus tissue may be absent when severe.
Not present in short-term study with young animals.
Lymphocyte populations decline gradually with age
beginning at puberty.
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined bycuboidal to squamous epithelium.
Increased foci of B lymphocytes and plasma cells

Cellularity, Decreased, Lymphocyte

Decreased lymphocyte cellularity in the cortex and/or
medulla.
Lymphocyte necrosis or apoptosis may be present.
Underlying stromal cells may be more prominent.
May have loss of corticomedullary distinction.

Pathogenesis/Cell of Origin
Lymphocyte populations in the thymus gradually decline withage beginning at puberty.

Diagnostic Features









Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.
Increase in foci of B lymphocytes and plasma cells
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined by

Willard-Mack et al.
cuboidal to squamous epithelium (see related diagnosis
of epithelial hyperplasia).

Differential Diagnoses
Cellularity, Decreased, Lymphocyte (Atrophy)

Loss of lymphocytes is often more prominent incortex.
Treatment-related apoptosis or necrosis of lymphocytes.

Apoptosis, Increased, Lymphocyte











Single cells or small clusters of cells.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cytoplasm retained in apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis.
Nuclear fragmentation.
Intact cell membrane.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

699
des

Loss Of Corticomedullary Distinction(N) (Figure 50) Thymus

con
enh

Loss Of Corticomedullary Distinction

Loss Of Corticomedullary Distinction(diagnose lymphocyte apoptosis, necrosis, etc. andincreased/decreased area separately if applicable)

Species
Mouse; rat.

Other Terms
Atrophy; hyperplasia.

Pathogenesis/Cell of Origin
Loss of lymphocytes may occur after toxins, irradiation, andviral infection or as a congenital change in immunodeficient
mice that results in loss of corticomedullary distinction in
adult mice.

Necrosis, Lymphocyte






Necrotic cells are often contiguous, but pattern can be
focal, multifocal, or diffuse.
Cell swelling.
Cell rupture.
Karyolysis, pyknosis, and karyorrhexis.
Inflammation usually present.

Diagnostic Features

Decreased cortical and medullary lymphocytes.
Little or no distinction between cortical and medullarycellularity/cell density.
Thymus size and weight grossly decreased.

Differential Diagnoses
Comment
Age-related involution is characterized by a gradual decline
in lymphocyte populations beginning at puberty and continuing with age in association with increased circulating levelsof sex steroids. There are some species, strain, and sex differences in the evolution of age-related thymus involution.
Lymphocyte populations also decline with age in other lymphoid organs. Age-related thymic involution may share similar features with chronic experimentally induced thymic
lesions, such as a reduction in thymus weight and histological depletion of cortical lymphocytes. Since age-related involution can be a confounding factor in chronic studies, careful
evaluation of dose relationship including concurrent controls
and examination of other lymphoid organs is essential.
Sometimes it may be necessary to conduct a short-term study
to distinguish the 2 diagnoses. Enhanced histopathology is
not recommended for diagnosis of age-related involution inchronic studies since there is generally no need to characterize the finding or explain its mechanism of action. Agerelated decline may be reversible; hormonal changes canreconstitute/restore the thymus morphologically and possibly
functionally.

Lymphoma

Early lymphoma may present as diffuse infiltration inboth thymic lobes with loss of corticomedullary
distinction.
Growth may extend beyond compartments.
Cell population is more homogeneous, especially of
lymphoblasts.
Mitotic figures are generally evident.

Comment
There is normally a clear demarcation between the cortex andmedulla which can become obscured by changes in the lymphocyte populations of these compartments. Histologically,
there may be a loss of cortical and medullary lymphocytes witha subsequent decrease in compartment areas. Depending on
cause and severity, other lesions may be present (see Hyperplasia, Epithelial, and Cellularity, Increased, B Cell). The
development of neoplasia may also result in loss of corticomedullary distinction. Rebound regeneration may be seen after thewithdrawal of suppression and is not a preneoplastic lesion.

700

Toxicologic Pathology 47(6)Increased medullary lymphocytes can be induced by compounds that inhibit lymphocyte migration.71




des



Necrosis, Lymphocyte (N) (Figures 51 and 52)
Thymusconenh

Increase in foci of B lymphocytes and plasma cells.
Infiltration of adipocytes in connective tissue capsuleand septa.
Prominent epithelial cells in the medullary region that
may form cords, ribbons, tubules, or cysts lined bycuboidal to squamous epithelium.

Necrosis, Lymphocyte

Necrosis, Lymphocyte(indicate compartment and diagnose decreased lymphocytes, decreased area, pigment, etc, separately if applicable)

Species
Mouse; rat.

Other Terms
Necrotic cell death; oncotic necrosis; lymphocyte depletion.

Pathogenesis/Cell of Origin
Necrosis can be seen in areas of thymic infarction or as a direct
treatment-related effect.

Comment
Lymphocyte necrosis is considered to be the result of a toxic
process where the cell is a passive victim and follows anenergy-independent mode of cell death. Necrosis in the thymus is generally classic necrosis rather than single-cell necrosis. Necrotic cell injury is mediated by 3 main, potentiallyoverlapping, mechanisms: interference with the energy supply of the cell, direct damage to DNA, and direct damage tocell membranes. If both necrosis and apoptosis are present,
necrosis may predominate with scattered apoptosis or apoptosis may predominate with conversion to a necrotic phenotype. In such cases, necrosis and apoptosis may be diagnosed
separately or may be diagnosed together as a single entity(apoptosis/necrosis or apoptosis/single cell necrosis). Alternatively, the predominant type of cell death can be diagnosed
and the presence of the other type of cell death can be discussed in the narrative.34

Diagnostic Features






Necrotic cells are often contiguous but pattern can be
focal, multifocal, or diffuse.
Cell swelling.
Cell rupture.
Karyolysis, pyknosis, and karyorrhexis.
Inflammation usually present.

Differential Diagnoses
Apoptosis, Increased, Lymphocyte











Single cells or small clusters of cells.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cytoplasm retained in apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis.
Nuclear fragmentation.
Intact cell membrane.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

Age-Related Involution






Decrease in overall size/weight of thymus.
Decrease in cortical lymphocytes.
Thinning and irregularity of the cortex.
Variable loss of corticomedullary demarcation.
Increase in perivascular spaces.

Tingible Body Macrophage, Increased(N) (Figures 53 and 54) Thymus
See General Hematolymphoid

Proliferative Changes (Non-Neoplastic)
Hyperplastic changes in all the hematolymphoid organs, including the thymus, are generally reactive and are part of the normal
physiological responses of these organs to acute and chronic
insults or physiologic stimulation. Hyperplastic changes do notinfer preneoplastic or precancerous lesions in these organs (seeIntroduction). However, severe or persistent lymphoid hyperplasia may increase the risk of neoplastic transformation. If
there is a concern, clonality studies should be considered.
desCellularity, Increased, Epithelial Cell
(H) (Figures 55 and 56) Thymus

con
enh

Hyperplasia, Epithelial

Epithelial Cells, Increased, Medulla

Species
Mouse; rat.

Other Terms
Hyperplasia, epithelial tubules, and cords.

Willard-Mack et al.

Pathogenesis/Cell of Origin

701Epithelial component of the thymus; increased cords andtubules most likely derived from branchial remnants.

Lined by ciliated cuboidal epithelium containing variable amounts of homogenous eosinophilic material.

Comment
Diagnostic Features
One or more of these epithelial cell components are increased:






Epithelial cords and tubules.
 Tubules.
&
Gland-like structures composed of cuboidal tocolumnar cells.
&
May be partially cystic and filled with a markedly
eosinophilic colloid-like material (see Epithelial
Cysts).
&
Lined by nonciliated epithelial cells (subseptal
TEC).
&
May respond to hormonal stimulus in older
females.
 Squamous metaplasia may be a feature.
 Localized in thymic medulla.
 Frequently located at the mid ventral portion of thethymus.
 Focal or diffuse.
 Pleomorphism may be considerable.
Epithelial pseudofollicles.
Invaginations of superficial epithelial cells into overlying septal connective tissue.
Thymic (Hassall’s) corpuscles.
 Increased number and size.
 Increased calcification.
 May occur independently from other epithelialchanges.
 See diagnosis of thymic (Hassall’s) corpuscles,
increased for additional information.

Differential Diagnoses
Thymoma, Benign







Epithelial component is neoplastic.
Lymphocytes may or may not be neoplastic.
Percentage of lymphocytes can vary.
May have a focal expansile mass of tubules with orwithout intertubular stroma, central areas of squamous
epithelium, and/or keratin formation.
May have nodules of keratinized squamous epithelium.

Thymoma, MalignantMay resemble a poorly differentiated carcinoma withsheets of pleomorphic epithelioid cells and scatteredpoorly formed tubules.

Cysts, EpithelialThymopharyngeal duct remnants forming cysts.

A variety of factors (neural, endocrine, growth factors, cytokines,
and chemokines) modulate the proliferative and secretory activityof medullary TEC. For example, TECs are stimulated by estrogenand inhibited by testosterone. Age-associated changes in the thymus can result in a disturbance of thymocyte–TEC interactions,
resulting in increased proliferative and secretory activity of various epithelial subsets at different time points. There may be a
relatively high incidence in some strains of rats, such as the Wistar
and BN/Bi rat. In mice, the incidence is relatively high in somestrains and rare in others. In mice, epithelial and lymphoid hyperplasia may be present simultaneously. In aged Wistar Hannoverrats, a strain prone to developing thymomas, a focal or multifocal
lobular hyperplasia has been described which displays a medullary zone and an enlarged and distorted cortical zone. This hyperplasia resembles a thymoma with lobular pattern but shows little
or no compression or nodular growth pattern. The size is smalleror equivalent to the transverse axis of normal thymus.9,72-79
desCellularity, Increased, Lymphocyte(H) (Figures 57-59) Thymus

con
enh

Hyperplasia, Lymphoid

Lymphocytes, Increased(indicate compartment, increased area if applicable, etc)

Species
Mouse; rat.

Other Terms
Diffuse hyperplasia; atypical hyperplasia; focal hyperplasia;
nodular hyperplasia.

Modifier
T cell; B cell.

Pathogenesis/Cell of Origin
Lymphocytes; may be treatment related or associated with agerelated involution (medullary B-cell hyperplasia).

Diagnostic Features
Cellularity, Increased, T Cell

Increased small, hyperchromatic, mostly uniform
lymphocytes.
May be present unilaterally (one lobe) or bilaterally(both lobes).

702








Toxicologic Pathology 47(6)May be present in cortex or medulla.
May be nodular, focal, or diffuse.
May diffusely affect one or both compartments.
Normal delineation of compartments is retained.
No obvious proliferation of epithelial cells.
Distinct foci of proliferating subcapsular thymocytes
may be present.
Positive for CD3, CD4 and/or CD8 by IHC.

Cellularity, Increased, B Cell











Increased small, mostly uniform lymphocytes.
Lymphocytes may diffusely infiltrate the medulla orform follicles or follicle-like nodules with or without
germinal centers.
No extension beyond the thymic capsule.
Loss of normal corticomedullary demarcation.
Cortex generally shows patchy atrophic changes.
Epithelial and lymphoid hyperplasia may be present
together, more so in rats than mice.
In mice, positive for CD45R/B220, CD79b, PAX5,
CD79acy by IHC (CD19 works in flow cytometry but
does not work in formalin-fixed paraffin-embedded tissue. CD20 does not work in mice).
In rats, positive for CD45R/B220, CD79b CD45 RA,
and KiB1R by IHC.

if the identity of the lymphocyte population is known with certainty. Diffusely increased lymphocyte cellularity can be established only by comparison with age-matched controls. Increased
cellularity may be induced by growth factors or cytokines. B-cell
hyperplasia in the medulla is a common component of agerelated involution in both rats and mice (female mice may showa higher incidence than males). In some strains of mice (ie,CD1), this change may be prolific with marked expansion of
the medullary area. In old rats, the thymus occasionally is muchlarger (hyperplastic or persistent) than would be expected given
the age of the animal. The architecture of both lobes is relatively
normal. Atypical lymphocyte hyperplasia has been diagnosed insome studies where there is a confirmed chemical or geneticallyinduced progression from thymic T-cell lymphocyte hyperplasia
to thymic lymphoma. The diagnostic term “atypical lymphocyte
hyperplasia” has been used for a preneoplastic lesion in male andfemale haploinsufficient p16(Ink4a)/p19(Arf) mice and p53(þ/) mice treated with phenolphthalein (NTP 2007, ntp.niehs.
nih.gov/testing/types/altmodels/reports/gmm12/index.html, lastaccessed August, 23, 2016). This diagnosis should be used at
the discretion of the pathologist, depending on the study requirements.58,80-86
desEpithelium-Free Areas, Increased (N)(Figures 60 and 61) Thymus

con

Differential Diagnoses
LymphomaGrowth may extend beyond compartments, invade mediastinal fat, or disperse throughout the hematolymphoidsystem and to other organs.
May present as a focal lesion in the cortex of one lobe or
as diffuse infiltration in one or both thymic lobes with orwithout involvement of mediastinal lymph nodes.
Usually lymphoblastic type when only the thymus is
involved.
Possesses a more homogeneous cell population, especially of lymphoblasts, compared to the mixture of large
and small lymphocytes in lymphoid hyperplasia.

enh

Epithelium- Free Areas, Increased

Lymphocytes, T Cell, Epithelium-Free
Areas, Increased

Species
Rat.

Other Terms
Epithelium-free zone

Pathogenesis/Cell of Origin
CD4þ/CD8þ T lymphocytes.

Atypical Hyperplasia






Preneoplastic precursor in treated B6C3F1 mice and p53
knockout mice.
May be unilateral or bilateral.
Diffuse change with loss of corticomedullary distinction.
Sheets of large atypical lymphocytes and fewer admixed
small lymphocytes.
Heterogeneous cell population with a variable mitotic
index that fails to extend beyond the thymic capsule.

Comment
Increased lymphocyte cellularity (hyperplasia) may be moredefinitively diagnosed as B- or T-cell lymphocyte hyperplasia

Diagnostic Features






Discrete cortical areas of densely packed lymphocytes.
 Higher than normal density of small T lymphocytes.
 No epithelial component.
 No obvious vascularization.
 Extend from capsular surface to deep cortex and often
border the medulla (as seen with serial sections).
Populated by CD4þ/CD8þ T lymphocytes and fewer
TCR a/b and CD5þ/ cells.
High rate of proliferation assessed by Bromodeoxyuridine (BrDU).
Apoptotic cells and tingible body macrophages may bepresent.

Willard-Mack et al.


Various macrophage types present (large and round toslightly dendritic or small and dendritic).
May be difficult to discern with H&E.
 Negative for cytokeratin and laminin (no epithelial
component).
 Negative for MHC class II except for single cells.

Differential Diagnoses
Cellularity, Increased, LymphocyteIncreased amount of small, mostly uniform lymphocytes.
Epithelial cells intermixed, visualized with cytokeratin
or laminin.

Comment
The function of epithelium-free areas (EFAs) is unknown. They
may represent an alternate intrathymic pathway for T lymphocytes to move from the cortex to the corticomedullary region andmedulla while avoiding cortical stromal elements and thus avoiding positive/negative selection. They may also serve as reservoirsof lymphocytes. These areas are present in some strains of rats.
The occurrence and extent varies between rat strains and by age,with more abundant EFAs in younger rats. There is no apparent
difference between sexes. If an increase or decrease in the EFAs issuspected, IHC can be done to more fully characterize thechanges. This finding is not a common change.87
desThymic Corpuscles, Increased (N) (Figure 62)
ThymusconenhThymic Corpuscles, Hyperplasia

Thymic Corpuscles, Increased(indicate medulla)Species
Mouse; rat.

Locators
Medulla.

Other Terms
Increased Hassall’s bodies; increased Hassall’s corpuscles.

Pathogenesis/Cell of Origin
Type VI epithelial reticular cells.

Diagnostic Features

Corpuscle consists of concentric layers of flattened eosinophilic epithelial cells.

703




Cellular debris and/or keratin may be present in center ofcorpuscle.
Located in medulla.
Inapparent in some mouse strains.

Differential Diagnoses


None.

Comment
The function of thymic (Hassall’s) corpuscles is currently
unclear, but they have been proposed to act in the removal of
apoptotic thymocytes and the maturation of developing thymocytes. In rodent studies with a high degree of lymphocyte apoptosis, increased thymic corpuscles with central cell debris may bepresent. In some mouse strains, thymic corpuscles are inapparent
unless there is increased apoptosis. Many studies suggest thatthese structures are active in antigen expression, cell signaling,transcription, and metabolism mediated by cytokines or growth
factor receptors. In humans, they are a potent source of thymic
stromal lymphopoietin, a cytokine that directs the maturation ofdendritic cells in vitro and increases the ability of dendritic cells toconvert naive thymocytes to a Foxp3þ regulatory T-cell lineage.

SPLEEN
Organization
The spleen is a highly vascular organ that has 2 grossly visible
components, the red pulp and the white pulp. The splenic vasculature is complex and must be fully understood to appreciatenormal structure and function as well as pathologic changes.
Unlike other organs, medium-sized splenic arteries and veins
do not routinely run side by side. Arterial vessels are locatedpredominantly in the white pulp and venous vessels are locatedin the red pulp. The afferent splenic artery enters along the hilum
and gives off central arteries which become ensheathed by white
pulp. Each central artery and its accompanying white pulp
sheath bifurcates repeatedly while descending from the hilus
toward the parietal surface. The central arteries give off arterioles and capillaries which traverse the white pulp and either
terminate at the periphery of the white pulp or pass out of thewhite pulp and terminate in the red pulp. Many of these bloodvessels are open-ended and release their blood into the reticularmeshworks of the MZ and red pulp. In the red pulp, venous
vessels coalesce into trabecular veins that empty into the splenicvein which exits the spleen along the hilum. Trabeculae arecontinuous with the splenic capsule; both are composed offibroelastic tissue and smooth muscle.88,89
The white pulp is subdivided into morphologically identifiable compartments with distinct cell populations. The central
arteries are surrounded by PALS populated primarily by T cells.
Primary follicles populated by B cells are located along the
PALS. A primary follicle becomes a secondary follicle when
it forms a germinal center and a germinal center is considered
a compartment in its own right. The MZ is a distinct layer

704

Toxicologic Pathology 47(6)Table 4. Compartments and Cellular Components of the Spleen.
Compartment

Components

Periarteriolar
Dominant T lymphocyte area
lymphoid sheath- T lymphocytes- B lymphocytes- Fibroblastic reticular cells (FRCs)- Interdigitating dendritic cells- White pulp macrophages
Follicle
Dominant B lymphocyte area
- B lymphocytes- Follicular dendritic cells (FDCs)
Germinal center
Dominant B lymphocyte area
- B lymphocytes in several stages ofdevelopment and maturation- T lymphocytes- Follicular dendritic cells (FDCs)- Tingible body macrophages
Marginal Zone
- Marginal zone B cells- Marginal zone macrophages (MZMs)- Marginal metallophilic macrophages (MMMs)- Fibroblastic reticular cells (FRCs)- T lymphocytes
Red pulp- Red pulp macrophages- Red blood cells- Hematopoietic cells- Lymphocytes- Plasma cells- Fibroblastic reticular cells (FRCs)

surrounding the PALS and follicles that is interposed between
these compartments and the red pulp. The MZ is sometimes
considered a third area along with the white pulp and red pulp.
However, for the purposes of this document, the MZ is considered part of the white pulp.90,91 The large white pulp compartment is therefore composed of 4 smaller compartments, thePALS, follicles, germinal centers, and MZ, which may be evaluated together as a single large compartment (white pulp) or
evaluated as separate smaller compartments using enhanced histopathology.88 Table 4 indicates the various compartments andcellular components of the spleen.

Function
The white pulp compartments serve as part of both the adaptive
and the innate arms of the immune system. In mice and rats, thewhite pulp is composed of T-cell zones (the PALS) and 2
different B-cell zones (the follicles and the MZ). The B cells
associated with the follicle and the MZ belong to the B-2 celllineage. The T- and B-cell zones of the PALS and follicle,
respectively, are involved with T-cell-dependent adaptive
immunity. In addition to B-2 cells, the MZ also has specificpopulations of macrophages and thus it is involved with bothinnate T cell-independent and adaptive T-cell-dependent
immunity. The red pulp serves as a filter for the blood and is
an important site of innate immunity by virtue of its populationof red pulp macrophages. These macrophages clear aged anddefective red blood cells and pathogens and store andrecirculate iron. The splenic red pulp is also a normal site of
low levels of hematopoiesis in the rat and mouse.92-94

Development
In both the mouse and the rat, the spleen first develops as acomposite of splenopancreatic mesenchyme around embryonicday (E) 10.5 to 11 and separates from the pancreas around E12.5
to 13. Erythroblasts and F4/80 positive monocytes/macrophages
are first detected in the fetal spleen around E12 and lymphoidprogenitor cells are evident around E12.5 to 13.5.95,96 Hematopoiesis occurs in the spleen around E14.5 before there ishematopoietic activity in the fetal bone marrow. Compartmentalization of the spleen into MZ, follicle, and PALS does notdevelop until after birth. Although T lymphocytes are localizedaround central arterioles at birth, MZ and PALS do not begin todevelop until postnatal day (PND) 5 and PND 7, respectively. In
the mouse, MZ and PALS are not fully developed until 3 to 4
weeks of age and mature follicles form around 6 weeks of age.97
Leukocytes are the most abundant cell population in the spleen
during the first 4 to 5 days after birth. At PND 6, a massive
accumulation of nucleated immature erythroid cells takes place.
This erythroid population remains the most dominant populationin the neonatal spleen up to 3 weeks of age.98

Histology
Blood released from capillary and arterial terminations deposit
lymphocytes into the open circulation of the red pulp and MZ.
Lymphocytes actively make their way back into the white pulp
where they are directed to specific compartments by FRCs.2
These stromal cells and their reticular fibers form a reticularmeshwork that subdivides the white pulp into compartments.
Fibroblastic reticular cells secrete specific chemokines that
attract T and B cells to their respective compartments. Chemokines CXCL19 and CCL21 produced by stromal cells in the
T-cell zone are crucial for attraction and retention of T cells andinterdigitating dendritic cells (IDCs). The T-cell zone is composed of CD4þ and CD8þ T cells (CD4 > CD8), IDCs, andmigrating B cells. In the T-cell zone, T cells search for theircognate antigens by interacting with IDCs and migrating antigen-presenting B cells. In follicular B-cell zones, follicles arecomposed primarily of naive B cells, a few follicular dendritic
cells (FDCs) and a few CD4þ T cells. Chemokine CXCL13 is
produced by the CD21þ/CD35þ FDC and stromal cells of thefollicular B zone. This chemokine is required for B cells to
migrate to the B-cell follicles. Larger B cells are found in thecenter of the primary follicles and are surrounded by smaller
lymphocytes. This difference in lymphocyte size is difficult toappreciate by H&E. B cells interact with FDC in their search fortheir cognate antigen. Follicular dendritic cells may collect soluble antigens from the FRC conduit system and present them to B
cells.99 Positive identification of a cognate antigen initiates primary follicular cell differentiation, clonal expansion, and development of a germinal center (secondary follicle). Germinal
centers are composed of centroblasts and centrocytes with

Willard-Mack et al.
subsequent differentiation into plasma cells and memory B cells
and the production of T-cell-dependent humoral antibody. Upon
plasma cell differentiation, the plasma cells migrate from thefollicle through the MZ into the red pulp where they reside, often
in close association with veins which carry their antibodies to thesystemic circulation. Some long-lived plasma cells migrate to
the bone marrow where they reside for months.89
The reticular meshwork of the MZ is populated predominantly
by specific subsets of B cells and macrophages. Marginal zone B
cells express IgM, CD45R/B220, CD1dhi, and PAX5 but are
negative for IgD. Marginal zone macrophages (MZMs) express
CD209bþ (SIGN-R1) and marginal metallophilic macrophages(MMMs) line the marginal sinus and express CD169. Thesemacrophages clear particulate pathogens and endogenous apoptotic debris from blood as it passes through the MZ and marginal
sinuses. Loss of MZMs and MMMs will lead to MZ B-cell loss
and impaired trapping of particulate antigens.91-93 Commonly,with MZ B-cell hyperplasia, germinal center hyperplasia is concurrently present. Marginal zones generally do not fully develop
in mice until 3 to 4 weeks of age. The MZ is more prominent andlarger in rats than in most mouse strains. Generally, the MZ inmice is 3 to 5 cells wide, but the width varies with strain and age.
Marginal zone B cells and MZMs decrease in number as miceage. Mice 18 to 24 months old have fewer MZ B cells and MZMs
than mice <6 months old.100 In aged mice, MMMs diffuse into thewhite pulp follicle.100
The red pulp is composed of a large number of venous
sinusoids (rat) or venules (mouse) interspersed with pulp
cords (cords of Billroth). The reticular meshwork in the pulp
cords is populated by red pulp macrophages, hematopoietic
cells, and blood cells, predominantly red blood cells that areundergoing filtration. Blood released from capillary and arterial terminations enter the open circulation of the spleen. Bloodcells move slowly through interstices of the MZ and/or the
splenic cords of the red pulp (cords of Billroth) and thenreturn to the closed circulation by passing through interendothelial slits in the walls of venous sinuses (rats) or venules(mice). The rodent splenic red pulp is a normal site of extramedullary hematopoiesis and generally contains low numbersof myeloid, erythroid, and megakaryocytic cells. In reactive
and neoplastic conditions, one or more of these lineages may
increase in volume substantially causing enlargement of thespleen. The mouse spleen is also reported to store half thebody’s monocytes.101 In addition to macrophages, the red
pulp cords also contain scattered lymphoid cells consisting
predominantly of CD8þ T cells and occasional plasmablasts
and plasma cells. The sinusoids (venules) predominantly contain red blood cells. Their endothelial cells express both phagocytic and vascular antigens, but unlike human sinus
endothelial cells, they do not express CD8. Pigments (hemosiderin, ceroid, lipofuscin, and melanin) may be present inboth the cord macrophages and the phagocytic sinusoidal
endothelial cells.
The splenic capsule is composed of fibrous tissue, elastic
fibers, and a small amount of smooth muscle with a thin overlying layer of mesothelium.

705

Sampling and Diagnostic Considerations
A single cross section of the spleen is generally used for routine
histopathological assessment and is adequate for an evaluationof the red pulp and assessment of splenic enlargement. However, a splenic cross section may not provide sufficient white
pulp to render an adequate diagnosis. A longitudinal section of
the spleen will generally have an adequate amount of white
pulp on which to base a diagnosis. Due to branching of the
PALS as they course from hilar to parietal areas, the size of the
PALS will vary with the depth of the longitudinal section.102
Therefore, it is important to routinely sample cross sections
and/or longitudinal sections consistently.102,103
The white pulp compartments, red pulp, and cellular components of the spleen may react individually or collectively inresponse to insults to cells or tissue. Optimal interpretation of aspleen is best done by reviewing the whole tissue section at low
magnification in order to observe the anatomic compartments andto compare the compartments in treated tissue with those observedin normal or control tissue.103 While clear diagnoses may beevident at low magnification for normal tissues, hyperplasticchanges, and neoplasia, some lesions will require evaluation ofcytological detail at high magnification. Proliferative splenic
lesions can involve the white and/or the red pulp and it can sometimes be difficult to differentiate reactive proliferations from lymphoma and/or leukemia. In making the differentiation between
reactive and neoplastic proliferations, it is essential to interpret thechanges in the spleen in conjunction with clinical history (age,sex, drug treatment, etc), clinical pathological data, and lesions inother tissues (systemic inflammatory lesions, tumor, etc.).
Immunohistochemistry can be helpful in the interpretation ofhistological changes in the spleen. Each compartment of thespleen has unique antigen expression patterns that can be used
to compare expression patterns in control and treated tissues.21

Nonproliferative Changes
Aplasia/Hypoplasia (N) Spleen
See General Hematolymphoid
White Pulp
desApoptosis, Increased, Lymphocyte(N) Spleen

con
enh

Apoptosis, Increased, Lymphocyte

Increased Apoptosis, Lymphocyte(indicate compartment and diagnose decreased lymphocytes, decreased area, tingible body macrophages, etc, separately if applicable)

Species
Mouse; rat.

706

Toxicologic Pathology 47(6)Other Terms

Species

Lymphocyte depletion; atrophy; single-cell necrosis.

Mouse; rat.

Pathogenesis/Cell of Origin

Other Terms

Lymphocyte death by apoptotic cell death pathway.

Lymphoid depletion; lymphoid degeneration; decreased
cellularity.

Diagnostic Features










Single cells or small clusters of cells affected.
Small, dark hyperchromatic cells.
Apoptotic bodies.
Cell shrinkage and convolution.
Pyknosis and karyorrhexis, nuclear fragmentation.
Intact cell membranes.
Cytoplasm retained in apoptotic bodies.
Increase in tingible body macrophages containing apoptotic bodies.
Inflammation usually not present.

Differential Diagnoses
Necrosis, lymphocyteCell swelling.
Cell rupture.
Karyolysis, pyknosis, karyorrhexis.
Inflammation may be present.

Modifier
Lymphoid, lymphocyte.

Pathogenesis/Cell of Origin
Decreased lymphocytes as a result of apoptosis, necrosis,
decreased lymphopoiesis (reduced, defective, or deficient),or redistribution in response to stress, aging, toxicity, or
genetics.

Diagnostic FeaturesComment
Apoptosis is a coordinated and often energy-dependent modeof cell death that is considered a vital component of various
normal processes. 34 Increased lymphocyte apoptosis may
result from direct lymphocyte toxicity or from endogenous
factors such as diet or stress (glucocorticoid release). Apoptosis
may occur together with necrosis. While it is preferable toidentify and record diagnoses of apoptosis and classic necrosis
separately, this distinction may not be possible to make histologically when one type of cell death obscures the other. Also,
necrotic cell debris can have some similarities to apoptotic
debris, such as pyknosis and karyorrhexis. Apoptosis may predominate, with conversion to a necrotic phenotype, or necrosis
may predominate with scattered apoptosis. In these cases, itwould be appropriate to use both terms as a single entity (apoptosis/necrosis) or to only diagnose the predominate type of cell
death and discuss the presence of the other type of cell death in
the narrative.
desCellularity, Decreased, White Pulp
(N) (Figures 63-67) Spleen

con
enh

Atrophy, White Pulp

Lymphocytes, Decreased(Indicate compartment and diagnose decrease in area if
applicable)

Decreased cellularity and/or area of one or more lymphoid compartment(s).
Lymphoid necrosis and/or apoptosis may be present.
Tingible body macrophages containing apoptotic bodies
may be present.
Hyaline material may be present in regressing germinal
centers.
 Eosinophilic proteinaceous material.
 Negative for amyloid, may be positive for IgM.
 Other term—paramyloid.

Differential Diagnoses
Aplasia/HypoplasiaCongenital disorder.
Lymphoid tissue is completely absent.
History helpful in differentiating atrophy fromhypoplasia.
Refer to General Terms for additional information.

Comment
When using conventional terminology, the modifiers “lymphoid”
or “white pulp” can be used to specify that decreased cellularity(atrophy) is limited to the white pulp. The absence of a modifier
implies that the entire spleen is atrophic and that the white pulp
and red pulp are similarly affected. Alternatively, the modifier“diffuse” can be used to specify that white pulp and red pulp areboth atrophic. Immunosuppressive drugs may cause decreased
cellularity of specific compartment(s) of the spleen. Other causesof decreased cellularity include aging, cachexia, poor nutrition,
toxins, chemotherapy, autoimmunity, irradiation, or viral infections. Age-related atrophy may be species and strain related.
Atrophic effects can be influenced by whether the spleen is in a
resting or activated state at the time a test material is administered.

Willard-Mack et al.
Decreased lymphoid cellularity in the spleen can be a secondary
effect of lymphocyte toxicity in the thymus due to decreased
release of thymic lymphocytes into the circulation. Decreased
lymphocyte cellularity via thymus depletion takes at least 3 weeks
to manifest in the spleen.

Necrosis, Lymphocyte (N) Spleen

707
HemangiosarcomaSee General Hematolymphoid

Tingible Body Macrophage, Increased(N) Spleen

Cyst

See General HematolymphoidRed Pulp

desAngiectasis (N) (Figure 68) Spleen

con
enh

Angiectasis

Vessel Dilatation; Sinusoid Dilatation or
Vessel/Sinusoid Dilatation(indicate compartment and diagnose increased area if
applicable)

No clear border.
Tumor cells are plump and form interconnecting vascular channels.
Tumor cells may or may not be invasive into adjacenttissues.
Tumor cell cytology is more pleomorphic and hyperchromatic and mitotic figures are present.
Tumor cells are CD34 positive.

Multiple blood-filled or thrombi-filled cystic cavities ofvariable size in the red pulp.
Cystic cavities lacking an endothelial lining.
Peliosis—may be used as other term.

Comment
Hemangiomas and hemangiosarcomas (angiomas and angiosarcomas in humans) often arise in the red pulp from, or associated with, angiectasis. Unless there is a nodular or infiltrative
pattern of growth or clear atypia of endothelial cells, it can bedifficult to differentiate angiectasis from a vascular neoplasm.
Irregular blood-filled spaces can occur as a handling artifact
during necropsy procedures or from trauma.

Species
Mouse; rat.

Other Terms

desCellularity, Decreased, Red Pulp
(N) (Figure 69) Spleen

con

Dilatation, vascular.
enh

Atrophy, Red Pulp

Pathogenesis/Cell of Origin(Indicate Cell Type), Decreased,
Red Pulp

Endothelium of splenic veins, sinusoids, arteries, or arterioles.

Diagnose decrease in area separately if applicable.

Diagnostic Features








Dilatation of arteries, veins, or sinusoids of the spleen.
Often only the red pulp sinusoids are involved.
Focal, multifocal, or diffuse.
May be associated with thrombosis.
Endothelial cells are typically small, flat, and spaced
wide apart.
Endothelial cells of the spleen are CD34 negative.
Mitotic figures are not present.

Differential Diagnoses

Species
Mouse; rat.

Other Terms
Decreased size.

Pathogenesis/Cell of Origin
Any or all of the red pulp cell types may be decreased due tophysiological effects (ie, aging, decreased body weight),
changes in cell production/removal or toxicity.

HemangiomaTumor cells are plump and form vascular structures
which appear as new blood spaces.
May grow in a nodular pattern.
Tumor cells are CD34 positive.

Diagnostic Features

Decrease in one or more hematopoietic cell types (erythroid, myeloid, megakaryocytic, lymphoid, and/or
macrophages) compared to controls.

708



Toxicologic Pathology 47(6)Fibroblastic reticular cell meshwork and venous sinuses
may also be proportionally decreased.
Spleen capsule is not crenated or corrugated.

Differential Diagnoses

Differential Diagnoses
Leukemia, Erythroid
Contraction






Erythrocytes are decreased in red pulp sinuses and cords.
Fibroblastic reticular cell meshwork is contracted andmore prominent.
Splenic capsule may be crenated or corrugated.
History of cardiovascular insufficiency (hypotension,
hemorrhage, moribundity) or exsanguination in theabsence of barbiturate euthanasia.

Comment
Decreased cellularity (atrophy) of the red pulp may beobserved in rats showing marked chemical-induced lesions,fasting, or other factors affecting weight gain.
desCongestion (N) (Figure 70) Spleen

con
enh

Congestion

Red Blood Cells, Increased(indicate red pulp and diagnose increased area if applicable)

Idiopathic ErythrocytosisMouse; rat.

Increased hematocrit and hemoglobin.

Lack of ExsanguinationCan be seen in animals found dead.

Comment
Congestion commonly occurs with barbiturate euthanasia due
to smooth muscle relaxation. It can also be associated with bothacute cardiac failure (acute congestion) and chronic cardiac
failure (chronic passive congestion). Less commonly inrodents, chronic congestion can occur secondary to vascularabnormalities in the liver and portal vein obstruction. Vasoactive drugs such as histamine, bradykinin, and prostaglandins
increase storage of blood in the mouse spleen.104
desContraction (N) (Figure 71) Spleen

con

SpeciesTumor cells (primarily nucleated immature erythroid
precursors) present in blood (intravascular) and/or red
pulp sinusoids.
Tumor cells may be present in other tissues.

enhContraction

Contraction(indicate red pulp and diagnose decreased area if applicable)

Other Terms
Hyperemia; chronic passive congestion.

Species
Mouse; rat.

Pathogenesis/Cell of Origin
Increased erythrocytes in red pulp cords and sinusoids due toactive congestion (hyperemia, infection, inflammation, hypertension, increased clearance activity) or chronic passive congestion (venous obstruction, cardiac decompensation, chronic
passive hyperemia).

Diagnostic Features







Enlargement of the red pulp; grossly, microscopically,and by increased splenic weight.
Abundant erythrocytes within dilated red pulp sinusoids
and pulp cords.
Usually diffuse but may be focal.
Acute—dilated sinusoids.
 May involve arteries and veins in trabeculae.
 May involve MZ.
Chronic—red pulp fibrosis of the pulp cords may occur
resulting in sinusoidal dilatation.

Pathogenesis/Cell of Origin
Smooth muscle contraction stimulated by epinephrine release,drop in blood pressure, hypoxia, and so on.

Diagnostic Features










Erythrocytes in red pulp are decreased.
Venous sinuses may be open and prominent or collapsed
and difficult to distinguish, depending on kinetics ofcontraction process.
Fibroblastic reticular cell meshwork contracted andmore prominent.
Splenic capsule may be crenated or corrugated.
History of cardiovascular insufficiency (hypotension,
hemorrhage, moribundity) or exsanguination withoutbarbiturate euthanasia.
Spleen section has decreased area compared to controls.
Correlates with gross finding of small spleen.

Willard-Mack et al.

709

Differential Diagnoses

Comment

Cellularity, Decreased, Red Pulp

Ectopic spleen tissue is a rare finding in rodents and may becongenital or acquired. It may develop as a result of fusion failureof the splenic anlage during embryogenesis and may be acquired
as a consequence of splenectomy or traumatic injury.

Hematopoietic cell type (s) decreased.
Fibroblastic reticular cell meshwork and venous
sinuses may also be decreased, but they lackprominence.
Capsule generally not crenated or corrugated.

desErythrophagocytosis (N) (Figure 74) Spleen

conComment
The splenic capsule and trabeculae contain smooth muscle
capable of rapid contraction to expel blood from the spleen.
Fibroblastic reticular cells are considered to be myofibroblasts and play a role in splenic contraction in rats andmice.7,105 Rodent spleens have less smooth muscle and less
red pulp than some large animal species, so they tend to vary
less in gross appearance when contracted. Contraction can be
stimulated by drugs that lower blood pressure, acute blood
loss, hypoxia, and so on. This is a physiologic diagnosis that
may be used to correlate with macroscopic observations ofsmall spleen. Splenic contraction in an animal found dead orin the absence of barbiturate euthanasia is suggestive of cardiovascular insufficiency.

enh

Erythrophagocytosis

Erythrophagocytosis(indicate red pulp and diagnose increased macrophages if
applicable)

Species
Mouse; rat.

Other Terms
Phagocytosis; hemosiderosis; increased pigment; pigmented
macrophages; hemophagocytic syndrome.

Pathogenesis/Cell of Origin
Phagocytosis of erythrocytes by red pulp macrophages.

desEctopic Tissue, Spleen (N) (Figures 72 and 73)
Spleenconenh

Diagnostic FeaturesEctopic Tissue, Spleen

Ectopic Tissue, SpleenSpeciesMouse; rat.

Other Terms
Accesory spleen, ectopic spleen; spleen nodule; supernumery
spleen; splenule; splenunculus; daughter spleen.

Pathogenesis/Cell of Origin
Normal splenic tissue in an abnormal location

Diagnostic Features

One or more dark red to black nodules 1 mm in diameter.
Embedded in the mesentery, mesenteric attachment of
the spleen, pancreas, or gonads.
Have all or some components of normal spleen (capsule,trabeculae, red pulp, and/or white pulp).

Differential Diagnoses
Neoplasm of the pancreas, gonadRed pulp macrophages contain phagocytized intact orfragmented erythrocytes with or without nuclei or erythrocyte ghosts.
Often associated with increased pigmented macrophages containing hemosiderin.
May be associated with red pulp congestion if acute.
May be associated with increased red pulp macrophages
if chronic.
Hemosiderin pigment positive for Prussian blue.
History of infection or autoimmune disorder.

Differential Diagnoses
Vacuolation, Macrophages

Due to a variety of causes, including interleukin (IL) 4
exposure.

Histiocytic Sarcoma

Typical tumor cell morphology is seen.
Erythrophagocytosis and/or hematopoiesis may beobserved (Magali 2003)
Presence of histiocytic sarcoma with similar cell morphology in other tissues.

Comment
Erythrophagocytosis is a normal phenomenon in the red pulp of
the spleen.106 The spleen’s open vascular system provides a

710

Toxicologic Pathology 47(6)filter for removing senescent, parasitized, and otherwise alterederythrocytes in a physiological manner. Erythrophagocytosis
occurs primarily by resident macrophages located in the pulp
cords and venous sinuses. Fibroblastic reticular cells and cells
lining the sinuses (littoral cells) may also be phagocytic. Physiological erythrophagocytosis is often difficult to appreciate
on routine examination of the spleen. As a result of physiological erythrophagocytosis, iron is recycled by the macrophages
resulting in accumulation of intracellular iron pigment (hemosiderin). In young adult rats, hemosiderin is more prominent infemales than in males. Blood collection may also influence theamount of hemosiderin stored in macrophages. Splenic erythrophagocytosis can be stimulated by IL-4 exposure which
also causes hepatic erythrophagocytosis. Extensive macrophage activation, which can occur with anemia, infection,
immunodeficiency, neoplasia, and so on, may lead to hemophagocytosis (engulfing of erythrocytes, leukocytes, and platelets) by the splenic macrophages. Morphologically, there issplenic macrophage hyperplasia with the macrophages constipated with engulfed erythrocytes and/or other hematopoietic
cells. Unlike physiologic erythrophagocytosis, hemophagocytosis is often fatal.
desFibrosis (N) (Figures 75 and 76) Spleen

con
enh

Fibrosis

Fibrosis(indicate compartment and distribution ie, focal, multifocal,
or diffuse)May contain mature fat cells (lipomatosis or fatty change).
A silver stain and trichrome stains are helpful for theassessment of reticular or collagen fiber content andstructure of sinuses within the lesion.
Osseous metaplasia may occur in fibrotic areas.

Differential Diagnoses
Fibroma/Fibrosarcoma

Fibroblasts and bands of dense mature collagen are themajor components.
Fibrosarcoma often has a fascicular growth patternwhich may or may not have a “herring bone” pattern.
Tumor cell nuclei are plump and have a distinct
nucleolus.

Fibrosarcoma, PleomorphicContains abundant collagen and storiform or cartwheel
growth pattern.
Bizarre multinucleated neoplastic cells are present in thepleomorphic type.

Histiocytic Sarcoma


Cytoplasm is often more abundant than in stromal
hyperplasia.
Multinucleated cells may be present, but they are not ofa bizarre type.
Contains no prominent fibers and no mature fat cells.
Multiple foci present within the spleen and there may besimultaneous occurrence in other organs.

Species
Mouse; rat.

Comment

Other Terms

Chronic passive congestion and thrombosis of the venous sinusoids can lead to fibrosis of the red pulp with narrowing of the
red pulp sinusoids. The presence of excessive nucleated cells inthe sinusoids, such as those of large granular lymphocyte(LGL) leukemia of the F344 rat, can result in sludging of blood
leading to ischemia and subsequent fibrosis.
Capsular fibrosis is typically focal and usually occurs as areparative process following inflammation or an injury such as
an accidental needle puncture during intraperitoneal injection.
It may also occur as a secondary process related to inflammatory, toxic, or neoplastic lesions of the spleen. Pigment may be
associated with the fibrosis and is usually secondary to hemorrhage related to the primary event. Fibrosis can be induced by avariety of chemicals (aniline compounds, azobenzene, o-toluidine hydrochloride, 4,40 -sulfonyldianiline, D & C Red No. 9).
Spontaneous splenic fibrosis is very rare.

Reticular cell hyperplasia; stromal cell hyperplasia.

Pathogenesis/Cell of Origin
Stromal cells (of various cell types but usually fibroblasts),
most often of the red pulp, also of the capsule.

Diagnostic Features









Focal, multifocal, or diffuse.
Generally localized in the red pulp but may occur incapsule and/or white pulp.
Expansive, larger lesions may enclose atrophic white pulp.
Not well circumscribed.
Consists of eosinophilic fibroblasts/fibrocytes with oval
nuclei with small nucleoli and mature collagen fibers.
Collagen fibers may be markedly increased.
In denser fibrotic foci, sinuses are partly replaced bynarrow capillaries.
Mitotic figures are rare.

Pigment, Macrophage (N) (Figure 77)
SpleenSee General Hematolymphoid

Willard-Mack et al.

711

Vacuolation, Macrophage (N) Spleen

Differential Diagnoses

See General Hematolymphoid

Granuloma

Proliferative Changes (Non-Neoplastic)
Hyperplastic changes in all the hematolymphoid organs,including the spleen, are generally reactive and are part of thenormal physiological responses of these organs to acute andchronic insults or physiologic stimulation. Hyperplasticchanges do not infer preneoplastic or precancerous lesions inthese organs (see Introduction). However, severe or persistent
lymphoid hyperplasia may increase the risk of neoplastic transformation. If there is a concern, clonality studies should beconsidered.

White Pulp
desAggregates, Macrophage, Increased(N) (Figure 78) Spleen

con
enh

Aggregates, Macrophage, Increased

Macrophage Aggregates, Increased(indicate compartment and increased area if applicable)

SpeciesOrganized structure with a compact collection of epithelioid macrophages and other inflammatory cells which
may include multinucleated giant cells.
Multifocal or diffuse lesions that may be variably associated with necrosis, other inflammatory cells, infectious agents, or foreign materials.
Associated with inflammatory conditions and exposure
to xenobiotics.

Increased Cellularity, Mast Cells
Cells have pale basophilic or eosinophilic cytoplasm
containing abundant basophilic granules that stain metachromatically with Giemsa or toluidine blue stains.
Cytoplasm is not foamy or vacuolated.
Degranulated or immature mast cells may be difficult todifferentiate from macrophages.

Histiocytic SarcomaTumor cells may be larger and are often more atypicaland pleomorphic than hyperplastic macrophages.
Multinucleated giant cells often present.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace, or destroy normal architecture.
Other tissues may be involved.

Mouse; rat.

Locators
White pulp; red pulp.

Other Terms
Macrophage hyperplasia; histiocytic aggregates; histiocytic
hyperplasia; histiocytic granuloma; granulomatous
inflammation.

Modifier
Vacuolated; pigmented.

Comment
Macrophages form aggregates when they cannot completelydegrade microorganisms or ingested macromolecules, including some vehicles or test materials. Phagocytized test article
may have a specific identifiable morphological character.
Macrophage aggregates can be found anywhere in the spleen
but occur most commonly in the PALS. Some red pulp macrophages form small aggregates, known as ellipsoids or periarterial macrophage sheaths (PAMS), which are centered aroundarterial terminations in the red pulp. They are small and inconspicuous in the rodent spleen but may enlarge in response tostimulus for increased clearance of cells or particulates fromthe blood.

Pathogenesis/Cell of Origin
Monocyte/macrophages.

desDiagnostic Features

con








Adherent macrophages clustered together to form variably sized aggregates.
Cell borders may be distinct or may appear syncytial.
Macrophages may or may not contain pigment.
Hemosiderin often increased if present.
Normal cellular elements are not displaced.
Most commonly located in the PALS.

Cellularity, Increased, Plasma Cell, White
Pulp (H) (Figure 79) SpleenenhHyperplasia, Plasma Cell

Plasma Cells, Increased(indicate compartment)

Species
Mouse; rat.

712

Toxicologic Pathology 47(6)Other Terms
Plasmacytosis.

a common aging change in the rodent spleen but is not wellrecognized unless immunohistochemical stains for plasma
cell markers, heavy chain Igs, or kappa (k) light chains are
performed.

Pathogenesis/Cell of Origin
In response to antigen, clonal expansion in germinal centers
gives rise to antibody-secreting plasma cells.

Diagnostic Features










Increased plasma cells in the white pulp.
Cytoplasm is abundant and stains purple with routine
H&E stains.
Eccentric nuclei and prominent Golgi apparatus.
Mott cells with Russell bodies may be present.
May be associated with an acute or chronic disease process in the spleen or any other tissue, including infectious etiology or neoplasm.
Often occurs with blood-borne infections, systemic
inflammatory conditions, and autoimmune disorders.
Germinal centers may be hypertrophic/hyperplastic.
Mitotic figures are absent.

desCellularity, Increased, White Pulp
(H) (Figures 80-82) Spleen

con
enh

Hyperplasia, White Pulp

Lymphocytes, Increased(indicate compartment and diagnose increase in area if
applicable)

Species
Mouse; rat.

Locators
White pulp.

Other Terms
Differential Diagnoses
Plasmacytic Lymphoma










Cells may have anaplastic/plasmablastic features in lesswell-differentiated plasmacytic lymphomas.
May be difficult to differentiate a well-differentiatedtumor from plasma cell hyperplasia based on morphologic features.
Ki67 or other proliferation markers can help differentiate because plasma cell hyperplasia does not have uniform Ki67 immunoreactivity.
Mitotic figures may be present but are rare in well-differentiated tumors.
Tumor cells may be mature plasma cells, immature cellswith an obvious plasmacytoid differentiation, or a mixture of mature and immature plasma cells.
Formation of eosinophilic intracytoplasmic crystal-likestructures considered diagnostic for plasmacytic lymphomas, although rarely seen.

Comment
Increased plasma cell cellularity (hyperplasia) is a common
response of the spleen to acute and chronic inflammatory
stimulation. Plasma cells are the mature end-stage cell of aprocess involving antigen recognition and the production of
mature and memory B cells in the follicle germinal centers or
MZs. The resulting plasma cells are commonly located in the
red pulp, especially around draining veins, but they may also
be seen in the PALS and along the interface between the
PALS and the MZ. Increased plasma cell cellularity is also

Nodular hyperplasia; follicular hyperplasia; MZ hyperplasia;
increased cellularity; reactive lymph node.

Modifier
Lymphoid, lymphocyte.

Pathogenesis/Cell of Origin
Increased lymphocytes in one or more lymphoid compartments due to antigenic stimulation, other immunomodulatory mechanisms, redistribution, or aging changes; may
include dendritic cell and macrophage hyperplasia to varying degrees.

Diagnostic Features









Increased cellularity and/or area of one or more lymphoid compartment(s).
Lymphoid compartments may be differentially affected.
Increased cellularity may involve lymphoid and/or antigen-presenting cells.
Lymphoid cells can include mixtures of lymphoblasts,
immunoblasts, medium-sized lymphocytes, mature lymphocytes, and/or plasma cells.
Antigen-presenting cells can include dendritic cells, B
cells, and macrophages.
In rare cases, lymphoid hyperplasia can be due primarily
to increased dendritic cells or macrophages.
May be associated with an acute or chronic disease process in the spleen or any other tissue, including infectious etiology or neoplasm.

Willard-Mack et al.


May be a consequence of lymphoid regeneration after
exposure to a toxin or from exposure to an immunomodulatory chemical.
Often occurs with blood-borne infections, systemic
inflammatory conditions, and autoimmune disorders.

Differential Diagnoses
Normal Aging Process

Can vary with strain or line of rat or mouse.
Compare with controls.

Lymphoma

Neoplastic cellular morphology of a uniform or pleomorphic population of lymphocytes.
 Early or late stages of differentiation.
 Focal, multifocal, or diffuse.
Normal or atypical mitotic figures may be prominent.
Effacement of white pulp architecture with or without
red pulp involvement.
Presence of lymphoma in other tissues.

Marginal Zone LymphomaMay be difficult to distinguish from MZ hyperplasia.
Germinal center hyperplasia occurs more commonlywith MZ hyperplasia than with MZ lymphoma.
Marked red pulp involvement with MZ lymphoma.
IHC for Pax5, k light chain, IgM, or Ki 67 is often
helpful in elucidating the degree of red pulp involvement and differentiating MZ lymphoma from MZ
hyperplasia.

Pleomorphic/Follicular Lymphoma






If present, germinal cells have no polarity and minimal
to no mantle zone.
Germinal centers may be adjacent to each other and may
fuse together.
Multinucleated cells may be present.
Blastic cells have prominent nucleoli.
IHC for IgG1, IgG2a, and IgG2b may help establish
clonality.107

Lymphoproliferative Disorder:103,108

Polyclonal lymphoproliferation.
Seen in genetic mutant mice.
Difficult to diagnose without knowing genetic
background.

Comment
When using descriptive or conventional terminology, the modifiers “lymphocyte,” “lymphoid,” or “white pulp” can be used to
specify that the increased cellularity (hyperplasia) is limited to thewhite pulp. The absence of a modifier or the use of the term713
“diffuse” implies that cellularity is increased in both the white
pulp and the red pulp. An increase in size and/or cellularity of theentire white pulp or in any of its compartments may be diagnosed
as lymphoid hyperplasia. Increased lymphoid cellularity (lymphoid hyperplasia) may be focal, multifocal, or diffuse (ie, involving all white pulp areas in a section) and may also include
increases in white pulp dendritic cells and/or white pulp macrophages. Increased plasma cell cellularity is generally diagnosed
separately (see Cellularity, Increased, Plasma Cell).
Increased white pulp cellularity may be a response to immunomodulatory drugs or to antigens, infectious agents, toxins
and infected, ulcerated, and/or necrotic tissues or tumors. Lymphoid hyperplasia may be indistinguishable from early stagesof lymphoma. Lymphoma should be diagnosed conservatively,
preferably with overwhelming evidence of neoplasia, especially involvement of other tissues.
Increased cellularity is generally a reactive response thatinvolves the production of additional cells and/or changes inthe trafficking of existing cells, either at the site of the response
or in a distant location. Lymphocyte proliferation and redistribution often occur together and may be morphologically indistinguishable, so the term “lymphoid hyperplasia” is understoodto encompass both processes in the spleen.
It is important to be familiar with the normal variation in
lymphoid tissue within different sections of a spleen andbetween spleens of control animals, especially in very young
and aging control mice and rats. Strain differences should also
be considered. The MZ does not fully develop in the mouse
until 3 to 4 weeks of age and its size (thickness, ie, the numberof cell layers) varies among mouse strains and is often thin. In
contrast, the MZ is very wide in most strains of rats. The
spleen is larger and denser in aging rodents than in young
animals. White pulp is oriented around arborizing central
arteries and can vary in appearance depending on where thetissue section is taken relative to the arterial tree. The spleen istriangular in cross section, so careful attention should begiven to standardizing the depth of longitudinal sections as
the size and arrangement of white pulp structures vary withdepth of sectioning, resulting in artifactual differences in sizeand cellularity.

Red Pulp
desCellularity, Increased, Adipocyte(N) (Figure 83) Spleen

con
enh

Hyperplasia, Adipocyte

Adipocytes, Increased(indicate compartment and distribution and diagnoseincrease in area if applicable)

Species
Mouse; rat.

714

Toxicologic Pathology 47(6)Other Terms

Pathogenesis/Cell of Origin

Lipomatosis; lipidosis; fatty infiltration; fatty or lipid metaplasia; fatty replacement; fatty change.

Monocyte/macrophages.

Pathogenesis/Cell of Origin
Probably derived from local connective tissue, other stromal
tissue, adipocytes, or splenic pluripotent stem cells.

Diagnostic Features

Lipid-containing cells in the red pulp.
May be focal, multifocal, or diffuse.
May be associated with collagen deposition.

Differential Diagnoses
Lipoma

Nodular space occupying lesion composed of mature
differentiated adipocytes.

Diagnostic Features









Increased abundance and/or size of macrophages insinusoids and/or cords of red pulp.
Macrophages are generally individualized and have distinct cell borders.
Cytoplasm may or may not contain phagocytized material, pigment (commonly hemosiderin), or vacuoles.
Macrophages containing phagocytized apoptotic bodies
are known as tingible body macrophages (see General
section for additional information).
May be focal, multifocal, or diffuse.
May also occur in white pulp.
Dendritic cells in the white pulp may be increased in size
and/or number.

Differential Diagnoses
Aggregates, Macrophage, Increased

Comment
Increased adipocyte cellularity is a rare lesion in the spleenof control mice and rats. It has been reported in rats
exposed to aniline dyes in association with splenic
fibrosis.109,110






Discrete clusters of adherent macrophages.
Lack organized structure or encapsulation.
Focal or multifocal.
Macrophages may transform into epithelioid cells.

Granuloma
desCellularity, Increased, Macrophage(N) (Figure 84) Spleen

con
enh

Hypertrophy/Hyperplasia, Macrophage

Macrophages, Increased(indicate compartment and diagnose increase in area if
applicable)

Organized structure with a compact collection of epithelioid macrophages and other inflammatory cells which
may include multinucleated giant cells.
Multifocal or diffuse lesions that may be variably associated with necrosis, other inflammatory cells, infectious agents, or injected materials.
Associated with inflammatory conditions and exposure
to xenobiotics.

Cellularity, Increased, Mast Cell

Species
Mouse; rat.



Locators
Red pulp; white pulp.



Other Terms
Increased macrophage cellularity; macrophage accumulation;
macrophage infiltrate; macrophage infiltration; prominentmacrophages; histiocytosis; histiocytic hyperplasia; histiocytic
infiltrate; histiocytic aggregates.

Modifier
Tingible body; pigmented; vacuolated; aggregates.

Cells have pale basophilic or eosinophilic cytoplasm
containing abundant basophilic granules that stain
metachromatically with Giemsa or toluidine blue
stains.
Cytoplasm is not foamy or vacuolated.
Degranulated or immature mast cells may be difficult todifferentiate from macrophages.

Histiocytic SarcomaTumor cells are usually larger and more atypical andpleomorphic than hyperplastic macrophages.
Multinucleated giant cells often present.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace or destroy normal architecture.
Other tissues may be involved.

Willard-Mack et al.

715

Comment

Differential Diagnoses

Increased macrophage cellularity (hypertrophy/hyperplasia)
occurs most commonly in the red pulp where the majority ofthe splenic macrophages are located, but it may also occur in thewhite pulp, including the MZ. In the red pulp, resident macrophages clear the blood of aged and damaged cells (especially
erythrocytes) and particulates such as microorganisms.
Increased macrophage cellularity may occur diffusely as partof a reactive response to a variety of conditions, such as infectious diseases, immunological status, erythrocyte breakdown,
metabolism of xenobiotics, or distant neoplasia. Focal collections of macrophages, known as ellipsoids or PAMS , are clusters of macrophages centered around arterial terminations in the
red pulp that may enlarge in response to stimulus for increased
clearance. In rodents, these macrophage collections are normallysmall and inconspicuous. Increased macrophage cellularity often
occurs in combination with phagocytosis, pigment storage,
vacuolation, or aggregation as macrophages increase to meet thedemand driving these processes. The diagnostic terminology
therefore includes these processes as modifiers to allow thepathologist to construct the most appropriate diagnosis for aparticular constellation of features. These findings can also bediagnosed separately.

Cellularity, Increased, Macrophage

desCellularity, Increased, Mast Cell
(H) (Figure 85) Spleen

con
enh



Cells do not have pale blue cytoplasm or basophilic
granules.

Tumor, Mast Cell, Benign/Tumor, Mast Cell, MalignantNeoplastic cytology and/or malignant growth pattern.

Comment
Increased mast cell cellularity (hyperplasia) is uncommon in
the rodent spleen.
desCellularity, Increased, Mesothelial Cell
(H) (Figure 86) Spleen

con
enh

Hyperplasia, Mesothelial Cell

Mesothelial Cells, Increased(indicate compartment and distribution and diagnoseincreased area if applicable)

Species
Mouse; rat.

Hyperplasia, Mast Cell
Other Terms

Mast Cells, Increased

Hyperplasia; capsule.

(indicate compartment and diagnose increase in area if
applicable)

Diagnostic FeaturesSpecies
Mouse; rat.

Other Terms
Mastocytosis (see comment).

Diagnostic Features







Mast cell infiltrates in red pulp.
 No alteration or destruction of the splenicarchitecture.
 Focal, multifocal, or diffuse.
Cells in the same stage of differentiation, usually welldifferentiated.
Large cells with abundant pale blue or vacuolated
cytoplasm.
Abundant dark blue cytoplasmic granules.
Cytoplasmic granules stain metachromatically with
Giemsa or toluidine blue stain.






Increased mesothelial cells on the surface of thespleen.
Often associated with inflammation including fibrosis.
May be focal, multifocal, or diffuse.
Often associated with other peritoneal or splenic lesions
including inflammation and fibrosis.
May be seen with tumors metastatic to the peritoneal
cavity.

Differential Diagnoses
Mesothelioma, Malignant
Mesothelial tumor cells have characteristics of neoplastic cells and may be found on peritoneal surfaces ofother organs in addition to the spleen.

Comment
Increased mesothelial cell cellularity (mesothelial hyperplasia)
may be seen with chronic peritonitis, ascites, peritoneal tumor
metastases, and splenic inflammation.

716
des

Toxicologic Pathology 47(6)Cellularity, Increased, Plasma Cell,
Red Pulp (H) (Figure 87) Spleen

con
enh

Hyperplasia, Plasma Cell

Plasma Cells, Increased

Tumor cells may be mature plasma cells, immature cellswith an obvious plasmacytoid differentiation, or a mixture of mature and immature plasma cells.
Formation of eosinophilic intracytoplasmic crystal-likestructures considered diagnostic for plasmacytic lymphomas, although rarely seen.

(indicate compartment)

Comment
Species
Mouse; rat.

Other Terms
Plasmacytosis.

Pathogenesis/Cell of Origin
In response to antigen, clonal expansion in germinal centers
gives rise to antibody-secreting plasma cells.

Increased plasma cell cellularity (hyperplasia) is a common
response of the spleen to acute and chronic inflammatory
stimulation. Plasma cells are the mature end-stage cell of aprocess involving antigen recognition and the production of
mature and memory B cells in the follicle germinal centers
or MZs. The resulting plasma cells are commonly located in
the red pulp, especially around draining veins, but they may
also be seen in the PALS and along the interface between
the PALS and MZ. Plasmacytosis is also a common aging
change in the rodent spleen but is not well recognized
unless immunohistochemical stains for plasma cell markers,
heavy chain Igs, or k light chains are performed.

Diagnostic Features














Increased plasma cells in the red pulp.
May be arrayed around trabecular veins.
May see intermixed with other hematopoietic cell types
as part of extramedullary hematopoiesis or as pure populations of plasma cells, depending on the nature of theinciting cause.
Cytoplasm is abundant and stains purple with routine
hematoxylin and eosin stains.
Eccentric nuclei and prominent Golgi apparatus.
Mott cells with Russell bodies may be present.
May be associated with an acute or chronic disease process in the spleen or any other tissue, including infectious etiology or neoplasm.
Often occurs with blood-borne infections, systemic inflammatory conditions, and autoimmune
disorders.
Germinal centers may be hypertrophic/hyperplastic.
Mitotic figures are absent.

desCellularity, Increased, Stromal Cell
(H) (Figure 88) Spleen

con
enh

Hyperplasia, Stromal Cell

Stromal Cells, Increased(indicate compartment and distribution, ie, focal, multifocal or diffuse, and diagnose increase in area if
applicable)

Species
Mouse; rat.

Other Terms
Reticular cell hyperplasia; FRC hyperplasia.

Differential Diagnoses

Pathogenesis/Cell of Origin

Plasmacytic Lymphoma

Presumed to be increased FRCs.








Cells may have anaplastic plasmablastic features in lesswell-differentiated plasmacytic lymphomas.
May be difficult to differentiate a well-differentiatedtumor from plasma cell hyperplasia based on morphologic features alone.
Ki67 or other proliferation markers can help differentiate because plasma cell hyperplasia does not have uniform Ki67 immunoreactivity.
Mitotic figures may be present but are rare in well-differentiated tumors.

Diagnostic Features

Focal proliferation of stromal cells in the red pulp.
Cells have pale cytoplasm, round to oval vesicular
nuclei, and a single nucleolus.
Reticulin (fiber) staining is present but collagen is notseen or is not prominent.
 Fibroblastic reticular cells are positive for smoothmuscle actin, desmin, and podoplanin.2
 Reticular fibers are positive with silver stains.

Willard-Mack et al.

Differential Diagnoses

717


Focal Fibrosis

Localized proliferation of fibroblasts with collagendeposition.

Focal LipomatosisWell-demarcated foci of adipocytes in the red pulp.

Cellularity, Increased, Macrophage
Immunohistochemistry for F4/80 may be necessary todifferentiate stromal cell hyperplasia from increased
macrophage cellularity.

Comment
Induced stromal cell hyperplasia of the red pulp has beenreported after long-term treatment of rats with aromatic
amines. This lesion is milder than fibrosis and may be a precursor of fibrosis.

Differential Diagnoses
Leukemia (Myeloid, Erythroid, or Megakaryocytic)






Extramedullary Hematopoiesis, Increased(N) (Figures 89-92) Spleen

con

enh

Tumor cells are often all at one stage of differentiation
especially in less well-differentiated tumors.
Other organs are often involved with the same neoplastic cells as in the spleen.
Some leukemias present with minimal tissue involvement but with a high white blood cell count and numerous immature forms/blasts in the blood.
Mitosis, apoptosis, and necrosis may be present.

LymphomadesHematopoietic cells of 3 lineages (myeloid, erythroid,and megakaryocytic) may be present.
 Proportions of cell types vary depending on the
etiology of increased hematopoiesis.
 Any of the lineages may be predominant.
 Each lineage is often represented by cells in various
stages of differentiation.



Cell morphology is different from that of myeloid, erythroid, or megakaryocytic cells.
May be difficult to distinguish from undifferentiatedmyeloid leukemias, IHC would help.

Extramedullary Hematopoiesis,
Increased

Extramedullary Hematopoiesis,
Increased(indicate red pulp and diagnose increase in area if
applicable)

Species
Mouse; rat.

Locators
Red pulp.

Other Terms
Hematopoietic hyperplasia; red pulp hyperplasia; myeloid
hyperplasia; erythroid hyperplasia; megakaryocytic hyperplasia; hematopoietic cell proliferation; myeloid metaplasia.

Comment
Increased EMH is the commonly used term for diffuse hyperplasia of the red pulp. Increased EMH is usually diffuse but
can also occur as a focal change. Extramedullary hematopoiesis consists of one or more of the 3 hematopoietic celllineages, that is, erythroid precursors, myeloid precursors, andmegakaryocytes. In rodents, particularly in mice, the spleenshares hematopoietic function with the bone marrow to varying degrees throughout the animal’s life. Although somedegree of EMH is normally present in the rodent spleen, it
can increase in response to hematotoxic insult, anemia, bonemarrow suppression, inflammation, tumors elsewhere in the
body and pregnancy. Depending on the underlining initiator(anemia, inflammation, etc), one of the 3 cell lineages may
predominate, and depending on the dominant cellular lineage,
the hyperplasia maybe referred as erythroid hyperplasia, myeloid (granulocyte) hyperplasia, or megakaryocyte hyperplasia. Hyperplasia may be related to mouse or rat strain, sex,
and age.

Pathogenesis/Cell of Origin
Hematopoietic cells normally present in the red pulp or thathave migrated there from the bone marrow.

desHyperplasia, Nodular (H) Spleen

con

Diagnostic Features

Increased size/area of the red pulp.
Increased hematopoietic cells in the red pulp.

enhHyperplasia, Nodular

Hyperplasia, Nodular(indicate red pulp)

718

Toxicologic Pathology 47(6)Species
Mouse; rat.

Other Terms
Splenoma; hamartoma; malformation; focal red pulp
hyperplasia.

Pathogenesis/Cell of Origin
Red pulp of the spleen.

Diagnostic Features








Focal, solitary occurrence in the red pulp.
Nodule protrudes from the spleen.
Well-demarcated, distinct border.
Consists of normal-appearing red pulp (sinusoids, hematopoietic foci, hemosiderin).
No white pulp elements/lymphoid tissue included in the
nodule.
Expansile growth with focal expansion of the capsule.
Often grossly visible.

Differential Diagnoses
HemangiomaTumor cells are plump and form vascular structures,
which appear as blood spaces with varying amounts ofconnective tissue.
Consists predominately of endothelial-lined blood vessels with mature erythrocytes and few or no other hematopoietic cell lineages.

Hemangiosarcoma






Lack sharp, distinct border.
Blood-filled vascular channels associated with varying
amounts of connective tissue.
Cellular atypia of endothelia.
Solid areas with fibrosarcomatous appearance may bepresent.
Consists predominately of endothelial-lined blood vessels with mature erythrocytes and few or no other hematopoietic cell lineages.

Cellularity, Increased, Stromal Cells

Fibrostromal tissue.
No distinct border.
Reduced or absent sinuses replaced by capillaries.
Cellular pleomorphism may be present.

Comment
Nodular hyperplasia is a rare lesion in rats, possibly causedby splenic trauma and probably not a true hamartoma.

Focal hyperplasia of the red pulp has been reported in
F344 rats.

LYMPH NODE
Organization
The lymph nodes are part of an integrated network of lymph
nodes and lymphatics which communicates with the body’s
sites of contact with the outside world (the skin and the mucosae) and with the internal organs such as liver and kidneys.
Some lymph nodes also drain other lymph nodes.

Function
Lymph nodes conduct antigen surveillance on tissue fluids(lymph), mount cellular, and antibody responses against antigens and filter the lymph.105 Adaptive immune responses are
initiated in the lymphoid compartments where recirculating
lymphocytes are brought together with antigen-presenting cells
bearing antigens from the site of contact with the outside world
or xenobiotic application (for example, skin, mucosae). Lymphocytes that encounter their cognate antigens undergo clonal
proliferation in germinal centers (B cells) and the paracortex (Tcells). Activated B cells give rise to plasma cells that produce
antigen-specific antibodies and memory B cells that will augment the immune response to future encounters with the antigens. Activated T cells produce a variety of effector T
lymphocytes. Innate immune responses occur in the sinuses
where macrophages identify, phagocytize, and eliminate pathogens, foreign material, and cell debris.

Development
Lymph nodes are present at birth and develop upon antigenic
stimulation.

Histology
Lymph nodes have several lymphoid compartments and a filtration compartment (the sinuses). The cellular components ofeach compartment are outlined in Table 5.
The lymph node stroma consists of a reticular meshwork
composed of FRCs and reticular fibers. The reticular meshwork
subdivides the lymph node into several functional compartments4 and provides a scaffold for lymphocyte migration andsegregation within the node.1,2,5,6 Fibroblastic reticular cells provide surfaces for adherence and produce trophic factors (chemokines) that direct lymphocyte movement (trafficking by meansof haptotaxis) to B-cell and T-cell regions. Lymphocytes enter
the lymphoid tissue via HEVs in the paracortex and interfollicular cortex and migrate to the follicles (B cells) and paracortex(T cells) where they survey antigens displayed on antigen-presenting cells. If they do not find their cognate antigens, they exit
into the sinuses and pass out of the node via the efferent lymphatics. Interdigitating dendritic cells (IDC) collect antigen fromthe skin, mucosal surfaces, and organs, enter lymphatics, and are

Willard-Mack et al.

719

Table 5. Compartments and Cellular Components of Lymph Nodes.
Compartment

Components

Follicle

Dominant B lymphocyte area
-Primary follicles contain small
resting naive B cells-Follicular dendritic cells (FDCs)

Germinal center-B lymphocytes in several stages ofdevelopment and maturation-T lymphocytes-Follicular dendritic cells (FDCs)-Macrophages
Interfollicular cortex and Dominant T lymphocyte areas
Paracortex-T lymphocytes-B lymphocytes-Fibroblastic reticular cells (FRCs)-Interdigitating dendritic cells (IDCs)-High endothelial venules (HEVs)-Macrophages
Medullary cords-Lymphocytes-Plasma cells-Fibroplastic reticular cells (FRCs)-Macrophages
Sinuses (subcapsular,-Fibroblastic reticular cells (FRCs)
transverse, paracortical,-Macrophages
medullary)transported in the lymph to the local draining lymph node.
Lymph also transports soluble antigens, inflammatory mediators, cell debris, inflammatory cells, erythrocytes, and microorganisms. Upon reaching the subcapsular sinus, IDCs settle on thesinus floor and migrate into the lymphoid tissue. Inflammatory
mediators and soluble antigens (<70 kD) are picked up by reticular fibers in the meshwork which form a system of conduitsthat carry these substances deep into the lymphoid tissue.4,5Inflammatory mediators stimulate HEVs to upregulate recruitment of recirculating lymphocytes which can cause increased
lymphocyte cellularity, particularly around the HEVs in the peripheral paracortical and interfollicular areas. Soluble antigens
are sampled from the conduit system by antigen-presenting cells,
including follicular dendritic cells (FDCs) in the follicles. Whena lymphocyte encounters its cognate antigen on an antigen-presenting cell, it is stimulated to remain in the node and undergoclonal proliferation.
Antigenic stimulation of B cells leads to germinal center
development within primary follicles. The trapping of antigen
by FDCs and the migration of B lymphoblasts into the follicle
are regarded as the first events in germinal center formation.
Small recirculating B cells in the primary follicles are pushedaside by the developing germinal center and form the follicular
mantle that surrounds it. The germinal center with its surrounding mantle is referred to as a secondary follicle. In an H&Estained section, the mantle of the secondary follicle stains
densely basophilic because of the high number of small B cells.
The germinal center is paler because of the presence oflymphoblasts and FDCs. The antigen-stimulated B blasts proliferate exponentially and alter phenotypically, for example, bylosing their surface Ig (sIg). In this state, they are called centroblasts. Centroblasts migrate apically and mature into nondividing centrocytes that again express sIg. Centrocytes with highaffinity for antigen express the cell survival gene bcl-2 andsurvive. Centrocytes with low or no affinity for antigen do not
express bcl-2 and are lost by apoptosis. At this stage, the darkzone (centroblasts) and light zone (surviving centrocytes) of thegerminal center become apparent.111 Antibody isotype switching occurs in the apical light zone, most probably with help of
CD4þ T lymphocytes. The presence of a dark and light zone
depends on the phase of activation and also on the plane ofsection. The presence of tingible body macrophages reflects
apoptosis of the centrocytes.112 Mature B cells leave the germinal center either as plasma cells or as B memory cells. Plasma
cells can accumulate in the medullary cords and secrete antibodies into the lymph or they can leave the lymph node. B memory
cells reside in the mantle zone. Germinal centers appear within a
few days after antigen administration. In lymph nodes whichhave low background germinal center activity, germinal center
reactions last about 3 weeks after antigen administration. The
presence, size, and number of germinal centers are valuable
indicators of immune activation. The location of germinal centers in the lymph node can provide information on the amount ofactivation. A very active lymph node may have germinal centersin the paracortex and even in the medullary cords.105
Antigenic stimulation of T cells leads to enlargement of thecentral paracortical areas. T cells no not produce a distinctive
structure like a germinal center and are less well characterized.
Paracortical cellularity varies depending on the relative proportions of small basophilic recirculating lymphocytes, larger proliferating lymphoblasts, pale IDCs, and macrophages.
Lymph is cleared of particulates, cells, and cell debris byresident macrophages (also known as sinus histiocytes)
located in the reticular meshwork of the sinuses. Macrophages
may contain pigment derived from erythrocytes (hemosiderin) or tattoo pigment. Sinuses vary in appearance depending on the numbers and types of blood cells passing throughthem. Lymphocytes on their way out of the lymph node arepresent in variable numbers in the sinuses. Draining erythrocytes, neutrophils, eosinophils, and mast cells may be seen
depending on activity in the drainage area. The sizes of
medullary cords and medullary sinuses are inversely related.
Sinuses narrow as medullary cords expand with plasma cells
and sinuses become wider when cellularity in medullary cords
decreases.

Sampling and Diagnostic Considerations
The lack of grossly visible features and the small size of lymph
nodes often make standardized midsagittal sections difficult toobtain. The complexity of normal nodal architecture, variation
in lymphocyte numbers, and variation in plane of section allcontribute to the difficulty in evaluating lymph nodes. Fibroblastic reticular cells and lymphocyte subpopulations are not720
easily visualized in routine H&E-stained sections. The microscopic appearance of a lymph node can vary widely dependingon the relative activity of surveillance/antigen presentation,
lymphopoiesis, and filtration functions, each of which expands
a different compartment within the lymph node. The names to
identify the different lymph nodes are not standardized and canpresent difficulty in the communication of effects. Harmonization of lymph node names has been proposed for the rat113 andthe mouse.114
Mesenteric lymph nodes and/or respiratory tract draining
lymph nodes are generally sampled in toxicity studies, alongwith a quiescent peripheral lymph node that does not drain amucosal surface, typically the axillary or popliteal lymph
node. The use of peripheral lymph nodes in the evaluationof the immune system has been questioned115 but their use
has been suitable for evaluating immune stimulation. For evaluation of an immune-modulatory effect, it is of utmost
importance to have concurrent controls for comparison.
Descriptive or enhanced nomenclature is recommended for
subacute (2-4 week) and subchronic (13 week) studies as a
descriptive approach is the most useful in evaluating andcharacterizing immune modulatory effects.13,15 For carcinogenicity studies and for special studies including those intransgenic animals, conventional interpretative nomenclature
is suitable.
Knowledge about the area(s) drained by the lymph nodes isneeded to interpret their morphology.
For evaluation of treatment-related effects in lymph nodes,
it is important to realize that although they are located at manysites throughout the body, they are part of one integrated
system together with the bone marrow, thymus, spleen and
MALT.116

Toxicologic Pathology 47(6)Other Terms
Lymphocytolysis; increased tingible body macrophages;
increased cell death.

Pathogenesis/Cell of Origin
Lymphocyte death by apoptotic cell death pathway.

Diagnostic Features







Apoptotic lymphocytes in various stages of shrinkage,
nuclear condensation, and fragmentation.
Apoptotic bodies free in tissue.
Increased tingible body macrophages (macrophages
with phagocytized apoptotic bodies).
Pale tingible body macrophages scattered amongbasophilic lymphocytes create “starry sky”
appearance.
Often results in a reduction in cellularity of one or more
lymphoid compartments (atrophy).

Differential Diagnoses
Necrosis, lymphoid






Nonproliferative Changes

Morphological appearance may include ghost cells or
coagulation necrosis of multiple cells leading to areas
without cellular detail.
Inflammatory response present.
Increased macrophages contain necrotic debris, but not
apoptotic bodies.
Macrophages often contain pigment or other phagocytosed material.

NormalCortex, Paracortex, and Medullary Cords
Aplasia/Hypoplasia (N) Lymph nodeLow level of apoptosis may be seen as part of normal
lymphocyte development, for example, in active germinal centers.

See General Hematolymphoid

Comment
desApoptosis, Increased, Lymphocyte(N) (Figures 93 and 94) Lymph node

con
enh

Apoptosis, Increased, Lymphocyte

Apoptosis, Increased, Lymphocyte(indicate compartment and diagnoses decreased lymphocytes, decreased area, tingible body macrophages, etc, separately if applicable)

Species
Mouse; rat.

Apoptosis is a coordinated and often energy-dependent modeof cell death that is considered a vital component of various
normal processes. Apoptosis eliminates activated or autoaggressive immune cells during maturation; therefore, a low
level of lymphocyte apoptosis is considered normal. Lymphocyte apoptosis can be increased above background levels by
viruses, obstruction (of blood, lymph, etc), radiation, cytotoxic drugs, corticosteroids, and endogenous cortisol (stress).
Increased apoptosis can also be seen in some tumors, such as
lymphoma. Increased lymphocyte apoptosis may result fromdirect nodal lymphocyte toxicity or may result from endogenous factors such as diet or stress (glucocorticoid release). In
coordination with macrophages, apoptotic cells are cleared

Willard-Mack et al.
quickly and efficiently. Apoptotic lymphocytes, tingible bodymacrophages, and/or decreased lymphoid cellularity are variably present, depending on the duration of the process.
Ongoing and severe apoptosis results in lymph nodedecreased cellularity (atrophy). Differentiation of necrosis
from apoptosis and other types of cell death can be difficult.
There may be a mixture of cell death types (eg, apoptotic cells
may occur as a bystander effect with necrotic foci) and necrotic and apoptotic cell debris share some common features,such as pyknosis and karyorrhexis. While it is preferable toidentify and record diagnoses of apoptosis and classic necrosis separately, this distinction may not be possible when theseprocesses occur together. This may be because one type ofcell death histologically obscures the other. Also, necrotic celldebris can have some similarities to apoptotic debris, such as
pyknosis and karyorrhexis, or vice versa. Apoptosis may predominate, with conversion to a necrotic phenotype, or necrosis may predominate with scattered apoptosis. In these cases,
it would be appropriate to use both terms or only indicate thepredominate type of cell death and discuss the presence of theother type of cell death in the narrative.34

desCellularity, Decreased, Lymphocyte(N) (Figures 95 and 96) Lymph node

con
enh

Atrophy, Lymphoid

Lymphocytes, Decreased(indicate compartment)

Species
Mouse; rat.

Other Terms
Lymphoid depletion; lymphocyte depletion.

Modifier
Lymphoid; lymphocyte

Pathogenesis/Cell of Origin
Change in lymphocyte kinetics—increase in destruction or
decrease in production (intranodal or systemic) and/or decrease
in recruitment or retention (intranodal).

Diagnostic Features

Decreased lymphocytes.
Decreased size and/or cellularity of one or more lymphoid compartments.
Decreased size and/or number (involution) of germinal
centers.

721







Increased apoptosis and/or increased tingible bodymacrophages.
 Indicative of increased lymphocyte destruction.
 May affect one or more compartments.
Distribution.
 Changes may be local involving one lymph node orsystemic involving all lymph nodes.
 Other lymphoid organs may be affected, especiallythymus.
Decreased prominence of HEVs.
 Suggestive of decreased recruitment.
Decreased lymphocytes in sinuses.
 Suggestive of decreased lymphocyte flux throughsinuses.

Differential Diagnoses
Aplasia/HypoplasiaComplete lack of development of a lymphoid organ.
Absence of tissue or organ.

Comment
The etiology of decreased lymphocyte cellularity (atrophy) is
potentially complex. Decreased cellularity in the lymph node
can be the direct result of atrophy (increased cell death ordecreased cell production) in the lymph node. In some cases,
however, it may be the result of decreased cellularity (atrophy) in the bone marrow or thymus or may reflect changes inlymphocyte trafficking and distribution patterns or may be
due to effects on FRCs. The conventional term “atrophy” may
be used in chronic studies or in cases where lymphocyte
destruction is known to have occurred in the lymph node
(evidence of apoptosis or necrosis is present). The descriptive
term “cellularity, decreased” or the enhanced term “decreased
lymphocytes” are preferred for short-term studies because
they are purely descriptive and do not imply a mechanism forthe reduction in lymphocytes or suggest where the reduction
occurred. Decreased lymphoid cellularity can occur withadvancing age, physiological stress, cachexia, toxins, chemotherapy, immunosuppressive drugs, irradiation, viral
infections, and interference with lymphocyte trafficking. The
extent of age-related atrophy is species and strain related. The
extent of induced atrophy is dependent on dose, duration ofadministration/exposure, and whether the lymph node was inan activated or resting state during exposure. The diagnosis ofatrophy depends heavily on the history and the anatomic
lymph node examined, namely either continuously activatedlymph nodes draining mucosal surfaces, such as the mesenteric or mandibular lymph nodes, or resting lymph nodes,
such as the popliteal lymph nodes. Atrophy of the paracortex
and medullary cords is easily recognized as decreased cellularity, whereas atrophy in follicles often presents as a decreasein size without prominent decreased cellularity. Lack of

722

Toxicologic Pathology 47(6)germinal center development (in activated lymph nodes) may
accompany decreased cellularity. Necrosis and/or apoptosis
may result in atrophy, so necrotic and/or apoptotic cells may
be seen in atrophic lymph nodes depending on the stage of theprocess. If systemic immunomodulatory effects are present,
generally all the lymphatic organs (lymph node, spleen, thymus) may show a similar effect reflecting the “one system but
multiple sites” integrated interactions among theseorgans.13,116

Necrosis (N) Lymph nodeOften associated with lymph node decreased cellularity/
atrophy, especially of the medullary cords.
Medullary sinuses are most commonly affected.

Differential Diagnoses
Lymphangiectasis

Dilation of afferent or efferent lymphatics.
Sinuses unaffected.

Edema

See General HematolymphoidIncrease in interstitial fluid not restricted to sinuses.

Pigment, Macrophage (N) Lymph node
See General Hematolymphoid

Comment

Tingible Body Macrophage, Increased(N) Lymph nodeSinus dilatation is common in aged rodents. It may be of undetermined etiology or due to inflammatory lesions, edema, or
tumors in adjacent tissues. It is more common in the mesentericand mediastinal lymph nodes. When medullary cords decreasein diameter due to lymphoid depletion, medullary sinuses may
secondarily increase in actual or apparent size.

See General Hematolymphoid

Inflammation (N) Lymph node
See General HematolymphoiddesSinuses and Lymphatics

Erythrocytes, Intrasinusoidal (N)(Figures 98 and 99) Lymph node

con
des

Dilatation, Sinus (N) (Figure 97) Lymph node

con
enh

Dilatation, Sinus

Dilatation, Sinus(indicate affected sinus, ie, transverse, medullary, etc, if
applicable)enh

Erythrocytes, Intrasinusoidal

Erythrocytes, Intrasinusoidal(indicate affected sinus, ie, transverse, medullary, etc, if
applicable)

Species
Mouse; rat.

Species
Mouse; rat.

Other Terms
Sinus ectasia; sinus dilation; widened sinuses; cystic dilation;
cystic degeneration; lymphatic cysts; lymphangiectasia.

Pathogenesis/Cell of Origin
Diffuse or focal dilatation of subcapsular, paracortical, or
medullary sinuses.

Diagnostic Features

Focal or diffuse sinus enlargement.
Enlarged sinuses are often filled with an eosinophilicstaining material, presumably lymph, containing lymphocytes and other cells.

Other Terms
Sinus erythrocytosis/erythrophagocytosis; draining erythrocytes, sinusoidal hemorrhage.

Pathogenesis/Cell of Origin
Erythrocytes are transported to lymph node sinuses via lymphatic drainage from extranodal site of hemorrhage.

Diagnostic Features

Erythrocytes present in sinuses, either individualized orin aggregates.
Erythrocytes rosetted around and/or phagocytized bysinus macrophages.
Sinus macrophages contain hemosiderin pigment if
exposure to draining erythrocytes is chronic or
ongoing.

Willard-Mack et al.

723

Differential Diagnoses

Other Terms

Hemorrhage

Lymphatic dilation; lymphatic cysts; lymphatic ectasia.



True intranodal hemorrhage is rare.
Erythrocytes are present in lymphoid compartment(s)instead of, or in addition to, the sinuses.
Localized lesion in the absence of other causes.

Vascular Ectasia

Dilation of congested blood vessels.
Absence of free blood cells in sinuses and lymphoidtissue.

HemangiomaProliferation of endothelial cells and vascular structures
forming a discrete, nodule-like lesion.
Hemal lymph nodes (rats).

Comment
Intrasinusoidal erythrocytes generally originate from sites ofextranodal hemorrhage and are carried to the lymph node in thedraining lymph and are therefore not a true biological lesion per
se. They are often focally located in the sinuses that were receiving lymph from hemorrhagic area(s) of the drainage field. Intrasinusoidal erythrocytes are commonly seen as a perimortem or
postmortem artifact secondary to terminal blood collection or
necropsy manipulations, especially in mediastinal and bronchial
lymph nodes. They may also be found in mesenteric and, to alesser extent, mandibular lymph nodes. Acute hemorrhage in thelung is common with euthanasia and drainage is often observed inbronchial lymph nodes, especially in animals euthanized with
CO2.50,117 Hemosiderin-containing sinus macrophages (pigmented macrophages) are indicative of antemortem hemorrhage. If
notable accumulations of erythrocytes are due to manipulations at
necropsy or in-life handling (eg, injection in the draining area),
the incidence is expected to be distributed about equally among
the groups, including controls. The association with handling canbe discussed in the pathology narrative if deemed necessary tocorrelate with a gross lesion. Erythrocytes adherent to sinus
macrophages (rosette formation) without erythrophagocytosis
may be a sign of deficient phagocytosis, as found in studies withorganotins.118 Intranodal hemorrhage originating from nodal
blood vessels is rare and can be distinguished by large numbersof free erythrocytes intermingled with lymphocytes in the lymphoid compartment(s).
desLymphangiectasis (N) Lymph node

con
enh

Lymphangiectasis

Lymphatics, Dilated

Species
Mouse; rat.

Pathogenesis/Cell of Origin
Lymphatics in soft tissue surrounding lymph nodes.

Diagnostic Features

Dilatation of any afferent or efferent lymphatic
vessel(s) in connective tissue adjacent to lymph
nodes.
Lymphatics may be cystic, especially in nude athymic rodents, but are also seen in carcinogenicity
studies.
Can be filled with acellular proteinaceous material.

Differential Diagnoses
Vascular DilationDilation of blood vessels, characterized by the presenceof erythrocytes in existing dilated vessels.

Edema


Increased interstitial fluid not associated with avessel.

Comment
Dilatation of lymphatic vessels is usually due to blockage oflymphatics by disease processes such as tumors or by inflammatory lesions.

Pigment, Macrophage (N) Lymph node
See General Hematolymphoid

Vacuolation, Macrophage (N) Lymph node
See General Hematolymphoid

Proliferative Changes (Non-Neoplastic)
Hyperplastic changes in all the hematolymphoid organs,including the lymph nodes, are generally reactive and are partof the normal physiological responses of these organs to acuteand chronic insults or physiologic stimulation. Hyperplasticchanges do not infer preneoplastic or precancerous lesions inthese organs (see Introduction). However, severe or persistent
lymphoid hyperplasia may increase the risk of neoplastictransformation. If there is a concern, clonality studies should
be considered.

724

Toxicologic Pathology 47(6)Cortex, paracortex, and medullary cords
desAggregates, Increased, Macrophage(N) (Figure 100) Lymph node

con
enh



Increased Cellularity, Mast CellAggregates, Increased, Macrophage

Aggregates, Increased, Macrophage(indicate compartment and diagnose increase in area if
applicable)

Species

Associated with inflammatory conditions and exposure
to xenobiotics.



Cells have pale basophilic or eosinophilic cytoplasm
containing abundant basophilic granules that stain
metachromatically with Giemsa or toluidine blue
stains.
Cytoplasm is not foamy or vacuolated.
Degranulated or immature mast cells may be difficult todifferentiate from macrophages.

Histiocytic Sarcoma

Mouse; rat.



Other Terms



Macrophage hyperplasia; histiocytic aggregates; histiocytic
hyperplasia; histiocytic granuloma; granulomatous inflammation; Potter’s lesion.35

Tumor cells may be larger and are often more atypicaland pleomorphic than hyperplastic macrophages.
Multinucleated giant cells often present.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace, or destroy normal architecture.
Other tissues may be involved.

Comment
Modifier
Pigmented; vacuolated.

Pathogenesis/Cell of Origin
Monocyte/macrophages.

Diagnostic Features







Adherent macrophages clustered together to form variably sized aggregates.
Cell borders may be distinct or may appear syncytial.
Macrophages may or may not contain pigment.
Hemosiderin often increased if present.
Normal cellular elements are not displaced.
Most commonly located in medullary cords andparacortex.

Differential Diagnoses
Increased Cellularity, Macrophage, IntrasinusoidalIncreased abundance and/or size of macrophages in one
or more sinuses.
Macrophages are generally individualized and have distinct cell borders.

Inflammation, GranulomaOrganized structure with a compact collection of epithelioid macrophages and other inflammatory cells which
may include multinucleated giant cells.
Multifocal or diffuse lesions that may be variably associated with necrosis, other inflammatory cells, infectious agents, or injected materials.

Macrophages accumulate and form aggregates when they cannot
completely degrade ingested macromolecules or microorganisms. Small scattered macrophage aggregates, often containing
pigment, may be seen as a background finding.117 Aggregates canform in any of the lymphoid compartments, but they are mostcommonly located in the medullary cords and paracortex. Specific distribution patterns in the same node within a dose groupmay be consistent with a treatment-related effect. Phagocytized
test article may have a specific identifiable morphological character. Some vehicles and dietary antigens can cause increased
macrophage aggregates. Corn oil used in gavage studies will frequently result in small aggregates, commonly called“microgranulomas,” within the mesenteric lymph node. If macrophage aggregates are increased in the lymphoid compartments,
sinusoidal macrophages may also be increased and should generally be diagnosed separately (see cellularity, increased, macrophage, intrasinusoidal). Enlarged lymph nodes containing sheets
and bands of fusiform macrophages in the cortex and medulla(Potter’s lesion) have been reported in NZB, CD1 mice, andoccasionally other mouse strains.35,117 This change has been associated with viral infection and is considered to be non-neoplastic.
desCellularity, Increased, Interdigitating
Dendritic Cell (H) (Figure 101) Lymph node

con

enh

Hyperplasia/Hypertrophy, Interdigitating
Dendritic Cell

Interdigitating Dendritic Cells,
increased(indicate compartment and diagnose increase in area if
applicable)

Willard-Mack et al.

Species
Mouse; rat.

Other Terms
Cellularity, increased, nonlymphoid; hyperplasia, nonlymphoid

Pathogenesis/Cell of Origin
Increased immigration of IDC to paracortex.

Diagnostic Features









Expansion of paracortical area.
Increased number of IDCs.
IDCs initially accumulate around the periphery of
the paracortex but may infiltrate the entire
compartment.
Cells are larger than lymphocytes with irregularly contoured nuclei and pale cytoplasm.
Lymphocytes may be decreased in number or celldensity.
Positive for S-100, variably and weakly positive forCD68 and CD45.
May be associated with inflammation or antigen in thedrained tissues.

Differential Diagnoses
Aggregates, Increased, Macrophage







Adherent macrophages clustered together to form variably sized aggregates.
Cell borders may be distinct or may appear syncytial.
Macrophages may or may not contain pigment.
Hemosiderin often increased if present.
Normal cellular elements are not displaced.
Most commonly located in medullary cords andparacortex.

Other types of inflammatory response in paracortex:

Neutrophils and/or other inflammatory cells present.

Comment
The IDC is a mature form of a tissue dendritic cell withincreased antigen-presenting capabilities.119 Interdigitating
dendritic cells originate from bone marrow and the immature cells are distributed in peripheral tissues including skinand mucosal epithelia.120 They collect and process antigens
in the peripheral tissues and then migrate via afferent lymphatics to the draining lymph nodes where they are distributed in the paracortical area as mature IDCs.121 Increased
IDC cellularity (hyperplasia) is thus a reactive change in
response to the presence of antigens in the organs and725
tissues drained by the lymph node and is not considered apreneoplastic lesion. IDC hyperplasia has been observed inathymic nude mice and rats as a compensatory response to
T-cell deficiency. The IDCs in superficial lymph nodes arederived from the Langerhans cells of the skin. Increased
Langerhans cells, IDCs, and paracortical hyperplasia havebeen observed in contact dermatopathic lymphadenopathy inhumans. Similar paracortical hyperplasia due to increased
IDCs has been observed in the superficial lymph nodes ina mutant strain of hairless rats.122 In routine studies without
special stains, the diagnosis of “cellularity, increased, nonlymphoid, paracortex” or “hyperplasia, nonlymphoid” canbe used for this finding.
desCellularity, Increased, Lymphocyte(H) (Figure 102) Lymph node

con
enh

Hyperplasia, Lymphoid

Lymphocytes, Increased(indicate compartment and diagnose increase in area if
applicable)

Species
Mouse; rat.

Other Terms
Lymphocyte proliferation; lymph node activation; reactive
lymph node; follicular hyperplasia; increased follicularnumber.

Modifier
Lymphoid; lymphocyte

Pathogenesis/Cell of Origin
Change in lymphocyte kinetics—increase in production (intranodal or systemic) due to antigenic stimulation, increase inrecruitment/retention (intranodal), and/or decrease indestruction.

Diagnostic Features







Increased lymphocytes.
Increased size and/or cellularity of one or more lymphoid compartments compared to concurrent controls
exposed to the same environmental conditions.
Increased size of paracortical units.
Increased size and/or number of germinal centers.
 Indicative of chronic antigenic stimulation.
Distribution.
 Changes may be local involving one lymph node orsystemic involving multiple lymph nodes.

726




Toxicologic Pathology 47(6) Other lymphoid organs may be affected.

Increased prominence of HEVs.
 Suggestive of increased lymphocyte recruitment.
Increased lymphocytes in sinuses.
 Suggestive of increased lymphocyte flux throughsinuses.



Medullary sinuses may be collapsed by enlarged medullary cords and paracortex may be partially or completely
replaced by plasma cells.
Often observed as a reactive change in mandibularlymph nodes in chronic studies.
Not a systemic lesion.

Differential Diagnoses

Differential Diagnoses

Lymphoma

LymphomaDistortion of nodal architecture.
Starry sky appearance with tingible body macrophages.
Generally systemic with multi-organ involvement.
No obvious association with an inflammatory lesion.

Plasmacytic LymphomaComment
Increased lymphocyte cellularity (lymphoid hyperplasia) can
occur under a variety of circumstances which can be either
specific, that is, antigen driven by viruses or bacteria, or nonspecific, for example, driven by pollutants, particulates, tissue
damage, or drainage of inflammation from a distant site. The
etiology of lymphoid hyperplasia is potentially complex, so theuse of this interpretive diagnostic term should be considered
carefully. Increased lymphocyte cellularity in the lymph node
can be the direct result of hyperplasia (increased cell production and/or decreased cell death) in the lymph node. In somecases, however, it may reflect changes in lymphocyte trafficking and distribution patterns.
desCellularity, Increased, Plasma Cell (H)(Figures 103 and 104) Lymph node

con
enh




Undifferentiated blasts rather than fully mature plasma
cells.
Systemic lesion.
Affected tissues have distorted architecture.

Comment
Increased plasma cells are part of an inflammatory response tochronic antigenic stimulation that originates in a lymph node’s
drainage field, such as an infected catheter or pododermatitis.
They are often seen in the mandibular lymph nodes in chronic
studies. Affected lymph nodes may be macroscopicallyenlarged. Since mature plasma cells are fully differentiated, they
are inherently not proliferative123 and do not express Ki67.
desCellularity, Increased, Stromal Cell
(H) Lymph nodeHyperplasia, Plasma Cellcon

Plasma Cells, Increasedenh(indicate compartment and diagnose increase in area if
applicable)

Generally consists of lymphocyte populations without
mature nuclear chromatin features.

Hyperplasia, Stromal Cell

Stromal Cells, Increased(indicate compartment and diagnose increase in area if
applicable)

SpeciesSpecies

Mouse; rat.

Mouse; rat.

Other Terms

Other Terms

Plasmacytosis.

Fibrous hyperplasia; fibrosis fibroplasia; FRC hyperplasia.

Pathogenesis/Cell of Origin

Pathogenesis/Cell of Origin

Plasma cells, B lymphocytes.

Proliferation of FRCs.

Diagnostic Features

Diagnostic FeaturesMedullary cords are expanded by well-differentiatedplasma cells.



FRCs have large irregularly oval nuclei and pale cytoplasm and secrete reticular fibers.

Willard-Mack et al.


Focal to multifocal change, most prominent in theparacortex.
FRCs are positive for cytokeratins 8 and 18 by IHC124
Reticular fibers are positive for Gomori’s reticulin silver
stain.
Definitive diagnosis may also be made by EM or IHC
for desmin, podoplanin or smooth muscle actin.

727

Other Terms
Angiomatosis; vascular transformation.

Modifier
Lymphatic (if vessels contain only proteinaceous fluid and noerythrocytes)

Differential Diagnoses

Pathogenesis/Cell of Origin

Inflammation, Granulomatous

Endothelial cell (both lymphatic and blood vessel origin).

Presence of other inflammatory cells and possibly multinucleated giant cells.

Diagnostic Features


Fibroplasia/FibrosisPresence of fibrous tissue and mature collagen.
Collagen is positive for trichrome stains.

Histiocytic Sarcoma
Systemic neoplasm that often involves nonlymphoidorgans.
Neoplastic proliferation of polymorphic cells with eosinophilic cytoplasm, often with multinucleated giant cells.
IHC for macrophage markers may be required for adefinitive diagnosis.

Hemangioma/Lymphangioma and Hyperplasia, AngiomatousMay look fibrotic with relatively few vascular spaces.
Focal lesion.
Vascular endothelia are often enlarged or slightly
pleomorphic.
IHC for endothelial cell markers may be required for adefinitive diagnosis.

Comment
Definitive differentiation from fibrosis may require special
stains, IHC, or electron microscopy. Fibrosis may be diagnosedin the absence of a definitive diagnosis. Reported cases ofincreased FRC cellularity (hyperplasia) in the lymph node oflaboratory animals are rare.124-127
desHyperplasia, Angiomatous (H) (Figure 105)
Lymph nodecon
Hyperplasia, Angiomatous
enh

Increased/Dilated, Blood Vessels(indicate compartment and diagnose increase in area if
applicable)

Species
Mouse; rat.






Increased numbers and/or size of blood vessels in thecortex, paracortex, and/or medullary cords or adjacent
perinodal connective tissue.
Cortex and medullary cords most commonly affected.
Focal or diffuse proliferation of (blood) vessels which
are often dilated.
Dilated blood vessels filled with blood/erythrocytes
which may be thrombosed.
In early stages, proliferating vessels may be filled with aproteinaceous fluid without the presence of erythrocytes
suggesting lymphatic origin.

Differential Diagnoses
Erythrocytes, Intrasinusoidal


Erythrocytes located in the sinuses, not within vessels
lined by endothelium.

CongestionDilation of existing blood vessels of normal diameter,that is, size is not increased.

Lymphangiectasis

Dilation of existing lymphatics.
Devoid of erythrocytes.

Angioma and Angiosarcoma (Hemangioma andHemangiosarcoma)






Increased number of vascular spaces.
Endothelial proliferation forming a nodular mass.
May extend out into the perinodal connective tissue.
Neoplastic vascular spaces contain RBCs.
Neoplastic cells with cytoplasmic microcapillaryfeatures.

Comment
Angiomatous hyperplasia is seen as a common aging lesion,
especially in the mesenteric lymph nodes of B6C3F1 mice andin some strains of rats such as the Wistar strain. The lesion

728

Toxicologic Pathology 47(6)appears to start in vessels in the medullary cords and adjacenthilar tissue and is thought to be caused by the occlusion ofefferent lymphatics. Larger lesions invariably contain erythrocytes. Angiomatous hyperplasia is generally not regarded aspreneoplastic to hemangiomas and hemangiosarcomas,
although there appears to be a progression. It has some morphological resemblance angiomatosis, a rare condition inhumans.128-131

Sinuses and Lymphatics
desCellularity, Increased, Macrophage,
Intrasinusoidal (N) (Figure 108) Lymph node

con

enh
des

Hypertrophy/Hyperplasia, High
Endothelial Venules (HEVs)(N) (Figures 106 and 107) Lymph node

con

enh

Hypertrophy/Hyperplasia, High
Endothelial Venules (HEVs)

High Endothelial Venules, Increased orHigh Endothelial Venules, Hypertrophy(indicate compartment)

Hypertrophy/Hyperplasia, Macrophage,
Intrasinusoidal

Intrasinusoidal Macrophages, Increased
OR Intrasinusoidal Macrophages,
Hypertrophy(indicate compartment and diagnose increase in area if
applicable)

Species
Mouse; rat.

Other Terms

Mouse; rat.

Sinus histiocytosis; histiocytic hyperplasia; histiocytic infiltrate; histiocytic aggregates; macrophage accumulation;
macrophage infiltrate; macrophage infiltration; prominentmacrophages.

Pathogenesis/Cell of Origin

Modifier

Species

Endothelial cells of high endothelial venules in lymph nodes.

Diagnostic FeaturesHypertrophy of the normal “high” vascular endothelium.
Increased lymphocyte migration across vessel walls.
High endothelial venules are often dilated or increasedin cross-sectional area.
Hyperplasia (increased numbers of HEVs) may occur.

Differential Diagnoses
Atrophy, lymphoid, paracortex
High endothelial venules may appear relatively more
prominent when the surrounding paracortex isatrophic.
High endothelial venule cells are not hypertrophic.

Pigmented; vacuolated.

Pathogenesis/Cell of Origin
Monocyte/macrophages.

Diagnostic Features

Increased abundance and/or size of macrophages in one
or more sinuses.
Macrophages are generally individualized and have distinct cell borders.
Cytoplasm may or may not contain phagocytized material, pigment, or vacuoles.
Macrophages tend to accumulate in subcapsular sinus
first, then in transverse sinuses, then in medullary sinuses.

Differential Diagnoses
Aggregates, Macrophage, Increased

Comment
Hypertrophy/hyperplasia of high endothelial venules occurs
in response to immune stimulation, specifically to cytokines
delivered to the HEVs via the FRC conduit system. Hypertrophy of the endothelial cells increases lymphocyte recruitment across the HEV wall and is usually associated withincreased cellularity of one or more compartments of thelymph node. It can also be induced by xenobiotics such as
hexachlorobenzene.118,132






Multiple discrete clusters of adherent macrophages.
Lack organized structure or encapsulation.
Focal or multifocal.
Generally not found in the sinuses.

Granuloma

Organized structure with a compact collection of epithelioid macrophages and other inflammatory cells which
may include multinucleated giant cells.

Willard-Mack et al.
Multifocal or diffuse lesions that may be variably associated with necrosis, other inflammatory cells, infectious agents, or injected materials.
Associated with inflammatory conditions and exposure
to xenobiotics.

Cellularity, Increased, Mast Cell

Cells have pale basophilic or eosinophilic cytoplasm
containing abundant basophilic granules that stain
metachromatically with Giemsa or toluidine blue
stains.
Cytoplasm is not foamy or vacuolated.
Degranulated or immature mast cells may be difficult todifferentiate from macrophages.

Histiocytic SarcomaTumor cells may be larger and are often more atypicaland pleomorphic than hyperplastic macrophages.
Multinucleated giant cells often present.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace, or destroy normal architecture.
Other tissues may be involved.

729
des

Cellularity, Increased, Mast Cell
(H) (Figures 109 and 110) Lymph node

con
enh

Hyperplasia, Mast Cell

Mast Cells, Increased(indicate compartment and diagnose increase in area if
applicable)

Species
Mouse; rat.

Other Terms
Mast cell accumulation; mastocytosis.

Pathogenesis/Cell of Origin
Mast cell proliferation, immigration, or redistribution within
the sinuses.

Diagnostic FeaturesComment
Macrophages residing in the sinuses are a distinct population ofphagocytic cells that filter lymph and they are functionallydifferent from macrophages located in the lymphoid compartments.117 Note that the diagnosis is restricted to an increase in
intrasinusoidal macrophages (hypertrophy/hyperplasia) without aggregate formation. An increase in intrasinusoidal macrophages (also known as sinus histiocytes) may be due to either
an increase in resident macrophages or an influx of macrophages that traffic to the lymph node via afferent lymph orblood.117,123 Sinus histiocytosis was a common term used forincreased macrophages in the subcapsular, transverse, paracortical, and/or medullary sinuses. Specific patterns in the samenode within a dose group may be consistent with a treatmentrelated effect. Note that the number of macrophages within thesinuses can vary with the node and plane of section, variables
which should be considered in the evaluation of this finding.
Increased cellularity of intrasinusoidal macrophages (hypertrophy/hyperplasia) is often indicative of increased clearance ofparticulates from lymph. Sinus macrophages commonly phagocytize draining erythrocytes and tattoo pigment and thenstore the pigments as hemosiderin or carbon, respectively.
These conditions may be diagnosed separately (erythrophagocytosis, pigmented macrophages), or may be included in the
diagnosis of intrasinusoidal macrophage hypertrophy/hyperplasia as a modifier (pigmented) or simply noted as a commentin the data. The diagnostic terminology includes these processes as modifiers to allow the pathologist to construct themost appropriate diagnosis for a particular constellation offeatures.









Increased mature mast cells within sinuses without
nodule formation.
No loss/distortion of normal architecture or compressionof adjacent tissues.
Mitotic figures are not present.
Mast cells are uniform, round or polygonal, mediumsized well differentiated, and noncohesive.
Nuclei are uniformly round but may be obscured bycytoplasmic granules.
Cytoplasm is abundant, granular, and slightly to heavily
basophilic.
Cytoplasmic granules may or may not be visible withhematoxylin and eosin depending on the type of fixation.
Cytoplasmic granules are metachromatic and generally
are positive for Giemsa, toluidine blue, or other metachromatic stains.

Differential Diagnoses
Mast Cell Tumor, Benign

A single, solitary, compact (dense) mast cell aggregate
or nodule.
Compression of adjacent tissue.

Mast Cell Tumor, MalignantCompact solitary nodule, local sarcomatous growth, or
sheet-like accumulation(s) of round, spindle-shaped, or
immature mast cells.
Cytoplasm is often hypogranular, but may have typicalbasophilic granules.
May have atypical bilobed or polylobed nuclei.

730








Toxicologic Pathology 47(6)Located in the lymphoid tissue of the paracortex andmedullary cords (not exclusively in the sinuses).
Eosinophils may be associated with the mast cells.
Destructive growth pattern, may be locally infiltrative.
Multiple organs may be involved.
No bone marrow involvement.
No clear inflammatory stimulus.
Considered malignant.

Mast Cell LeukemiaAtypical mast cells are present in the bone marrow and/or peripheral blood.
Mast cell accumulations with sheet-like or leukemic
pattern present in one or more hematolymphoid organs.

Comment
Mast cell numbers can vary according to location and species
and strain of animal. The etiology and pathogenesis are often
unknown. Mast cells in the peripheral tissues can migrate to thelymph node sinuses in response to certain hypersensitivity
conditions.123
desFibrosis (N) Lymph node

con
enh

Fibrosis

Fibrosis

Species
Mouse; rat.

Other Terms
Chronic inflammation.

Pathogenesis/Cell of Origin
Fibroblasts reactive to a local stimulus.

Diagnostic Features

Fibroblasts with oval to spindle-shaped nuclei andeosinophilic extracellular matrix (collagen) whenmature.
Increase in connective tissue and collagen with distortion of normal architecture/outline.
Often restricted to the capsule but may extend into subjacent node with loss of normal cells and architecture.

Differential Diagnoses
Plane of Sectioning of Normal TissueTangential section of capsule.
Absence of fibroplasia, reactive cells.

Comment
Fibrosis restricted to the capsule/capsular surface of a lymph
node is a sequel to inflammation or necrosis which may belocalized to the node or secondary to local inflammation suchas peritonitis.

MUCOSA-ASSOCIATED LYMPHOID
TISSUE
Organization
The mucosal immune system is organized into (a) lymph nodes
draining the mucosae, (b) the more or less organized tissues
associated with the mucosal epithelium, and (c) mucosal single
cells. Mucosal single cells include intraepithelial lymphocytes(IELs), macrophages, and dendritic cells within the mucosal
epithelium and lamina propria lymphocytes (LPLs), macrophages, and dendritic cells in the mucosal lamina propria. Organized tissues associated with the intestinal mucosa are thePeyer’s patches (PP), colonic and rectal lymphoid aggregates,
cryptopatches (CPs) and isolated lymphoid follicles (ILFs).
Rodents do not have an appendix, but they have several variably
developed lymphocytic aggregates associated with thececum.133 The organized tissues associated with the respiratory
mucosa are NALT (nose/nasopharynx-associated), LDALT(lacrimal duct-associated), LALT (larynx-associated) and BALT
(bronchus-associated). BALT is normally present in (moststrains of) rats but needs to be triggered to become apparent inmost strains of mice (inducible or iBALT).134 In both rats andmice, BALT/iBALT are observed at a fixed location, namely atthe bifurcations of bronchi and bronchioles. Although PP are
already present at birth, their number increases after birth andcan vary during life depending on antigenic stimulus. Thus, PP
are also inducible as well as constitutive. Therefore, the distinction between inducible and constitutive MALT is not strict.
Less well known in rodents is CALT/EALT (conjunctiva/
eye-associated). Organized tissue associated with the urogenital mucosa has not been found in rodents, which suggests thatthe urogenital mucosae are served by MALT elsewhere,
namely in the intestinal and/or respiratory tract.135 Nonlymphoid organs and tissues like mucosal glands are involved in
the mucosal immune response as effector sites.136 The mucosal
immune system is connected to the systemic immune system
via the mucosa-draining lymph nodes.
The abovementioned list of MALT is probably not complete
as more MALT tissues will likely be identified in rodent mucosae in the future due to increased sampling or different planesof sectioning. Therefore, it is important to keep in mind that
aggregates of mononuclear/lymphocytic cells in the mucosae
could be MALT rather than cell infiltrates responding to an
inflammatory stimulus.

Definition and Function of MALT
Several definitions are used for mucosa-associated lymphoidtissues, or MALT. Some definitions include the IELs and LPLs

Willard-Mack et al.
and the mucosa-draining lymph nodes. Here, the definition of
MALT is restricted to the mucosa-associated, more or lessorganized tissues operating as immune-inductive or immune
response-generating sites,136 that is, sites where naive immune
cells are triggered by antigens and where memory cells are
generated. Therefore, MALT-like tissues in the salivary glands
and elsewhere are not defined as MALT, because they are
immune-effector, that is, involved in the effectuation of theimmune response, rather than immune-inductive sites.137 It
needs to be investigated if LDALT (lacrimal duct-associated)
lymphoid tissue is really an immune-inductive site. However,the distinction between inductive and effector sites is not absolute, which really reflects the high plasticity of immune tissues
and organs in general. Isolated lymphoid follicles and CPs act
as precursors of PP. Both IELs and LPLs are considered to beeffector cells.
The mucosal immune system plays a decisive role in therelationship of the body itself with the enormous load of
micro-organisms inhabiting the body (the microbiome). The
immune system in the intestines needs to maintain a balance
between tolerance of the microbiome and the uptake of nutrients on the one hand and the exclusion of pathogens on theother hand.
In humans, certain B-cell lymphomas in gut, salivary
glands, and elsewhere, including kidneys, are being diagnosed“MALT lymphoma” or “Maltoma”. There is often a backgroundof chronic inflammation, either infective or of autoimmune
character.138 The term Maltoma or MALT lymphoma is linked
to small B cells in the mantle zone of germinal centers. Since
germinal centers can arise in MALT as well as in TLSs, the term
MALT lymphoma is somewhat confusing.

731
Table 6. Compartments and Cellular Components of MALT.a
Compartment

Components

Follicle-associatedepithelium (FAE)- Microfold (M) epithelial cells within theepithelium- (scarcity or absence of goblet cells)- (dendrites of) dendritic cells- Macrophages- Lymphocytes

Subepithelial dome(SED) Syn. Dome-

Follicle

MantleGerminal center

Interfollicular area
Syn. Parafolliculararea-

Dendritic cells
Macrophages
T lymphocytes
Reticular cells
Dominant B lymphocyte area
Primary follicles contain small restingvirgin B cells
Follicular dendritic cells (FDCs)
Small resting B cells like in primary follicle
B lymphocytes in several stages ofdevelopment and maturation
T lymphocytes
Follicular dendritic cells (FDCs)
Tingible body macrophages
Dominant T-lymphocyte area
T lymphocytes
B lymphocytes
Fibroblastic reticular cells (FRCs)
Dendritic cells
High endothelial venules (HEV)

Abbreviation: MALT, mucosa-associated lymphoid tissues. a Adapted from
Kuper et al 2017. Compartmentalization is based on Peyer’s patches and
NALT. Isolated lymphoid follicles (ILFs) and cryptopatches do not show suchdegree of organization.140

Sampling and Diagnostic Issues
Development
Most MALT is present at birth and develops rapidly thereafter.90 Age-related functional decline of the mucosal immune
response has been described, but light microscopic age-relatedchanges in MALT of rodents have not been reported. Therefore, age-related involution is not described below as a separate
diagnosis; comparison with concurrent controls is needed todecide whether or not age-related changes have occurred in astudy. Abnormal MALT development has been reported in
GEM.139

Histology
The compartments of MALT are outlined in Table 6.140 In
general, MALT is structured like lymph nodes, but MALT
lacks afferent lymphatics. Instead, its epithelium (follicle-associated epithelium or FAE) and associated dendritic cells serveas porte d’entre´e of antigens.90,141-143 Species-dependent differences in the percentage of T and B cells in MALT have been
described by Haley.133

Some guidance for sampling MALT may be helpful because
sampling can influence the results. NALT sampled in situ
within nasal cross sections can reveal prominent changes in
NALT. However, the detection of more subtle effects requires
dissected and longitudinally-embedded NALT, but this preparation damages nasal tissue (see144 mice;145 rat;146 mice andrats). Peyer’s patches in the small intestine may not all react
similarly to xenobiotics, thus standardized selection for microscopy is needed.147 In rats, the number of grossly visible PP in
the 40 cm of ileum just proximal to the ileocecal junction canbe counted as a general estimate, although it remains to beinvestigated if the distal PPs are sufficiently sensitive. An alternative method is the use of “Swiss rolls” for microscopy, allowing examination of a considerable portion of the small
intestines.148 The use of “Swiss rolls” also allows examinationof solitary ILFs and CPs. Examination of CPs can be facilitatedby horizontal sectioning in a plane perpendicular to the villous
axis.149 When single lymphocytes in the epithelium or lamina
propria are the focus of investigation, these cell populations canbe isolated according to methods described by Sheridan andLefrancois.150

732

Toxicologic Pathology 47(6)As mentioned under “Organization,” aggregates of mononuclear/lymphocytic cells at the mucosae could be MALT rather
than being cell infiltrates due to an inflammatory stimulus, especially when they are observed consistently at a certain location.
Structures such as ILFs and CPs in the intestines cannot be
distinguished from TLSs without the use of IHC for specificlymphocyte subpopulations like innate lymphocytes. As a practical approach, TLSs in the gastrointestinal and respiratory tracts
are only diagnosed when they are present outside the mucosa.
Mucosa-associated lymphoid tissue is often not reported
independently, but instead is recorded as part of the respiratory
tract, nasal passages (NALT), lung (BALT), or intestines/
gastrointestinal tract. Toxicity reports on PP and NALT are
infrequent and reports on ILFs and CPs are essentially nonexistent.136,147,151 This could be due to sampling and diagnostic
issues or because MALT is highly resistant to xenobiotic insults.

Nonproliferative Changes
Aplasia/Hypoplasia (N) MALT
See General Hematolymphoid

Apoptosis, Increased, Lymphocyte(N) MALT

Decreased germinal center development may accompany decreased cellularity or may be the most prominent
feature.

Differential Diagnoses
Aplasia/HypoplasiaIncomplete or arrested development of one or more/all
compartments.
Stroma may appear collapsed or prominent.
See General section for additional information.

Comment
Decreased lymphocyte cellularity (atrophy) can result fromdecreased lymphocyte and/or macrophage recruitment, direct
immunotoxicity (may be accompanied by necrosis/apoptosis),decreased stimulation (activation) due to diminished antigenic
presentation (eg, by housing under SPF conditions), or stressand other endocrine disruption-related mechanisms. In the caseof PPs, the diagnosis of “decreased cellularity” depends heavily on the plane of section and on the location in the small
intestines.147,152 BALT is not normally seen histologically inmost mouse strains unless there is antigenic stimulation (inducible BALT or iBALT),139 so decreased cellularity is seldom
an appropriate diagnosis in murine BALT.

See General HematolymphoiddesdesCellularity, Decreased, Lymphocyte(N) (Figures 111 and 112) MALT

con
enh

Degeneration, Follicle-Associated
Epithelium (N) (Figure 113) MALTcon

Atrophy, Lymphoid

Lymphocytes, Decreased

enh

Degeneration, Follicle-Associated
Epithelium

Follicle-Associated Epithelium, Decreased(indicate compartment)

SpeciesSpecies

Mouse; rat.

Mouse; rat.

Other Terms
Other Terms

Lymphoepithelial degeneration.

Lymphoid depletion.

Pathogenesis/Cell of Origin
Modifier

M cells and ciliated respiratory epithelium.

Lymphoid; lymphocyte

Diagnostic Features
Pathogenesis/Cell of Origin
Decreased cellularity of lymphocytes.

Diagnostic Features

Decrease in size and/or cell density of individual PPs orother MALT (see comment) in a specific compartment.
Lymphoid necrosis or apoptosis.



Loss of cilia (ciliated respiratory epithelium).
Flattening and thinning of epithelial cells.

Differential Diagnoses
Necrosis

Pyknosis or karyorrhexis of nuclei.
Cytoplasmic eosinophilia.

Willard-Mack et al.
Erosion/Ulcer

Loss of follicle-associated epithelium (erosion).
Loss of epithelium and underlying basement membrane(ulceration).

Metaplasia
Change in epithelial cell types present, usually with amixture of cell types in transition area.

733
Igs). The subepithelial dome appears to be a particularly sensitive area of the Peyer’s patch; fibrosis (scarring) and mineralization have also been observed in this area.

Inflammation (N) MALT
See General HematolymphoiddesLymphangiectasis (N) (Figure 116) MALT

con

Comment
Epithelial degeneration of ciliated respiratory epithelium andM (microfold) cells in NALT can be caused by exposure toxenobiotics or by an inflammatory condition such as rhinitis.112,146 Degeneration of follicle-associated epithelium mayalter uptake of antigens and thus may lead to changes in theunderlying lymphoid tissue, such as atrophy or decreased orincreased germinal center development. Follicle-associatedepithelial alterations have not yet been reported for other
MALT locations.

enh

Lymphangiectasis

Lymphatics, Dilated(indicate compartment)

Species
Mouse; rat.

Other Terms
Dilatation of lymphatics, lymphatic ectasia.

desHyaline Material (N) (Figures 114 and 115)
MALT

con
enh

Pathogenesis/Cell of Origin

Hyaline Material

Lymphatic vessels.

Hyaline Material

Diagnostic Features(indicate compartment)

Species
Mouse; rat.

Other Terms
Eosinophilic material; cysts; hyaline change; hyalinization;
paramyloid.

Pathogenesis/Cell of Origin
Deposition of eosinophilic material.

Diagnostic Features






Material may be associated with macrophages.
Predilection for the PP dome.
May be positive for Ig, amyloid, or other protein.
Positive for P component, protein AA, AL, and FAP
with IHC.
Positive with Congo Red stain (green birefringence) todemonstrate amyloid.

Dilatation of the efferent lymphatics of MALT.

Differential Diagnoses
Angiectasis
Vessels may contain red blood cells.

NeoplasiaLymphangioma, hemangioma, or hemangiosarcoma
Extremely rare.
Plump endothelial cells, mitotic figures.

Comment
Dilatation of lymphatic vessels may be due to blockage oflymphatic outflow by diseases or to increased demand(increase in interstitial fluid), for example, in (activated) lymphoid tissue associated with the lacrimal duct.112

Mineralization (N) MALT
See General Hematolymphoid

Comment
Specific stains may help with identification of material (IHC
staining with antibodies against the different (sub)classes of

Necrosis (N) MALT
See General Hematolymphoid734

Toxicologic Pathology 47(6)Pigment, Macrophage (N) MALT

See General Hematolymphoid

Associated with inflammatory conditions and exposure
to xenobiotics.

Histiocytic Sarcoma

Tingible Body Macrophage, Increased(N) MALT
See General Hematolymphoid

Proliferative Changes (Non-Neoplastic)
Hyperplastic changes in all the hematolymphoid organs, including MALT, are generally reactive and are part of the normal
physiological responses of these organs to acute and chronic
insults or physiologic stimulation. Hyperplastic changes do notinfer preneoplastic or precancerous lesions in these organs (seeIntroduction). However, severe or persistent lymphoid hyperplasia may increase the risk of neoplastic transformation. If there isa concern, clonality studies should be considered.
desAggregates, Macrophage (N) MALT

con
enh






Comment
Macrophages form aggregates when they cannot completelydegrade microorganisms or ingested macromolecules, including some vehicles or test materials. Phagocytized test article
may have a specific identifiable morphological character.
desCellularity, Increased, Lymphocyte(H) (Figures 117 and 118 ) MALT

Aggregates, Macrophage

con

Aggregates, Macrophageenh(indicate compartment)

Tumor cells are usually larger and more atypical andpleomorphic than hyperplastic macrophages.
Multinucleated giant cells often present.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace, or destroy normal architecture.
Other tissues may be involved.

Hyperplasia, Lymphocyte

Lymphocyte, Increased(indicate compartment)

Species
Mouse; rat.

Species
Mouse; rat.

Other Terms
Granulomatous inflammation; histiocytic granuloma.

Pathogenesis/Cell of Origin
Monocyte/macrophages.

Diagnostic Features






Adherent macrophages clustered together to form variably sized aggregates.
Cell borders may be distinct or may appear syncytial.
Macrophages may or may not contain pigment.
Hemosiderin often increased if present.
Normal cellular elements are not displaced.

Other Terms
Lymphocyte proliferation; lymphocyte infiltration; germinal
center stimulation; lymphoid accumulation.

Pathogenesis/Cell of Origin
Lymphocytes.

Diagnostic Features

Differential Diagnoses
Inflammation, GranulomaOrganized structure with a compact collection of epithelioid macrophages and other inflammatory cells which
may include multinucleated giant cells.
Multifocal or diffuse lesions that may be variably associated with necrosis, other inflammatory cells, infectious agents, or injected materials.



Increased mucosal lymphoid tissue.
 Increased cellularity.
 Increased area.
 Increased size/number of germinal centers.
&
Increased tingible body macrophages.
&
Germinal centers may coalesce and form bizarre
shapes surrounded by thin mantle zones.
Increased macrophages in lymphoid tissue.
 Accumulation rather than aggregation.
 Diffuse or focal.
Efferent lymphatics are filled with lymphocytes.
High endothelial venule hypertrophy/hyperplasia.
 High endothelial venules prominent in interfollicular tissue.

Willard-Mack et al.

 High endothelial venules may be increased in number.
 Increased lymphocyte traffic across HEV walls.

735



Differential Diagnoses
Preneoplastic Lymphocyte Proliferation/Lymphoma
Normal MALT architecture is disturbed.
Lymphocytes invade the epithelial structures.

Comment
Local proliferation and influx of lymphocytes and/or increase in
macrophages occur in response to antigens or nonspecificimmune-stimulating compounds. Within a few days after stimulation, primary or resting follicles develop into secondary follicles with a germinal center. Germinal centers may last about 3
weeks after antigen administration. Increased germinal centers
indicate increased activation of MALT. Generally, the PP closest
to the cecum has the largest follicles with prominent germinal
centers and distinct interfollicular areas, whereas those closer tothe stomach are small and often without germinal centers.147
Germinal centers in NALT are relatively uncommon.153
desCellularity, Increased, Macrophage(N) MALTenh

Differential Diagnoses
Aggregates, Macrophage

Multiple discrete clusters of adherent macrophages.
Lack organized structure or encapsulation.
Focal or multifocal.
Macrophages may transform into epithelioid cells.

Granuloma

Organized structure with a compact collection of epithelioid macrophages and other inflammatory cells which
may include multinucleated giant cells.
Multifocal or diffuse lesions that may be variably associated with necrosis, other inflammatory cells, infectious agents, or injected materials.
Associated with inflammatory conditions and exposure
to xenobiotics.

Histiocytic SarcomaHypertrophy/Hyperplasia, Macrophage

Macrophages, Increased or
Macrophages, Hypertrophycon

Cytoplasm may or may not contain phagocytized material, pigment, or vacuoles.
Can be diffuse or focal.

Tumor cells are usually larger and more atypical andpleomorphic than hyperplastic macrophages.
Multinucleated giant cells often present.
Nodular or coalescing sheets of neoplastic macrophages
efface, displace, or destroy normal architecture.
Other tissues may be involved.

(indicate compartment)

Comment
Species
Mouse; rat.

Other Terms
Macrophage accumulation; macrophage infiltrate; macrophage
infiltration; prominent macrophages; histiocytosis; histiocytic
hyperplasia; histiocytic infiltrate; histiocytic aggregates.

Modifier
Tingible body; pigmented; vacuolated; aggregates.

Pathogenesis/Cell of Origin

Increased macrophage cellularity (hypertrophy/hyperplasia)
in MALT may be a reactive response to a variety of conditions such as infectious diseases, immunological status, erythrocyte breakdown, metabolism of xenobiotics, or distantneoplasia. Macrophages often increase in combination withphagocytosis, pigment storage, or vacuolation, so these processes are included as modifiers to allow the pathologist to
construct the most appropriate diagnosis for a particular constellation of features. These findings can also be diagnosed
separately.
desHyperplasia, Follicle-Associated
Epithelium (H) MALT

con

Monocyte/macrophages.

Diagnostic Features

Increased abundance and/or size of macrophages in lymphoid tissue.
Macrophages are generally individualized and have distinct cell borders.

enhHyperplasia, Follicle-Associated
Epithelium

Follicle-Associated Epithelium,
Increased

Species
Mouse; rat.

736

Toxicologic Pathology 47(6)Other Terms

Other Terms

Lymphoepithelial hyperplasia.

Lymphoepithelial goblet cell hyperplasia.

Pathogenesis/Cell of Origin

Pathogenesis/Cell of Origin

Follicle-associated epithelium (M cells intermingled withrespiratory cells).

Goblet cells.

Diagnostic Features
Diagnostic Features

Thickened epithelium as a result of an increase in number of respiratory and/or M cells.
Often in association with hyperplasia of the surrounding
respiratory epithelium.
Epithelial hyperplasia may include proliferation of atypical, pleomorphic, or undifferentiated cells.

Increased goblet cells.
Undulation of the epithelium by increased number ofgoblet cells.

Differential Diagnoses
None.

Differential Diagnoses

Comment

Metaplasia, Squamous, Follicle-Associated Epithelium

Normal follicle-associated epithelium contains a low numberof goblet cells compared to the surrounding respiratory epithelium. Often, goblet cells are absent in the plane of section.
Hyperplasia of goblet cells may present as an increase in thenumber of goblet cells or even as the presence of a few goblet
cells in the plane of section. In more severe cases, the epithelium may undulate, due to clustering of goblets cells.

Hyperplasia of respiratory cells with a reduction in oreven complete absence of M cells.

Comment
Hyperplasia of the follicle-associated epithelium may diminish
or facilitate uptake of antigens, and thus may lead to inactivationor activation of underlying lymphoid tissue, seen as decreased
cellularity (atrophy) with or without decreased germinal center
development or increased cellularity (hyperplasia) withincreased germinal center development. Follicle-associatedepithelial hyperplasia has so far been reported for NALT, but
not for PP. Exposure to xenobiotics or an inflammatory condition (such as rhinitis in the case of NALT) can cause these kindof epithelial alterations.112,146 Distinction between M cells andrespiratory cells is difficult in H&E-stained sections, unless M
cells are numerous, have clustered together, and/or haveenwrapped few or numerous lymphocytes (lymphocytes in cytoplasmic “pockets” of the M cell)154 In that case, M cells can beseen as a cluster of nonciliated cells amid ciliated epithelium in
NALT.155 In contrast, hyperplasia of respiratory cells can potentially lead to a decrease in the number of M cells. In addition,
granulocytes may occasionally be present in FAE.
desHyperplasia, Goblet Cell,
Follicle-Associated Epithelium (H) MALT

con

enh

Hyperplasia, Goblet Cell,
Follicle-Associated Epithelium

Follicle-Associated Epithelium, Goblet
Cell, Increased

Species
Mouse; rat.

desHypertrophy/Hyperplasia, High
Endothelial Venule (HEV)(N) (Figure 119) MALT

con

enh

Hypertrophy/Hyperplasia, High
Endothelial Venule (HEV)High Endothelial Venules, Increased
or High Endothelial Venules, Hypertrophy(indicate compartment)

Species
Mouse; rat.

Pathogenesis/Cell of Origin
Endothelium of HEVs.

Diagnostic Features






Prominent HEVs have an increased number of crosssections in the MALT due to hyperplasia and (often
associated) hypertrophy of the endothelium.
Increased height of endothelium and/or increased vascular elements.
Increased lymphocyte migration across vessel walls.
Associated MALT activation/lymphocyte hyperplasia
occurs.

Willard-Mack et al.

Comment
High endothelial venule hypertrophy/hyperplasia occurs in
response to immune stimulation, specifically to cytokines
delivered to the HEVs via the FRC conduit system. Hypertrophy of the endothelial cells increases lymphocyte recruitment
across the HEV wall and is usually associated with increased
cellularity of the lymphoid tissue. It is not known if HEV
hypertrophy/hyperplasia is always part of activation of MALT,
that is, lymphocyte hyperplasia and germinal center development, but it is observed upon antigenic stimulation in lymph
nodes and PP.156-158
des737
inactivation or activation of underlying lymphoid tissue, seen
as atrophy with or without decreased germinal center development or increased cellularity and increased germinal center
development. Follicle-associated epithelial metaplasia has so far
been reported for NALT, but not for PP. Exposure to xenobiotics
or an inflammatory condition (such rhinitis in the case of NALT)
can cause these kinds of epithelial alterations.112,146

OTHER LYMPHOID TISSUES
Tertiary Lymphoid Structures
Organization

Metaplasia, Squamous, Follicle-Associated
Epithelium (H) MALT

con

enh

Metaplasia, Squamous, Follicle-Associated
Epithelium

Follicle-Associated Epithelium, Metaplasia,
Squamous

Species
Mouse; rat.

Other Terms
Lymphoepithelial squamous metaplasia.

Pathogenesis/Cell of Origin
Follicle-associated epithelium.

Diagnostic Features






Replacement of the follicle-associated epithelium bysquamous epithelium.
Several layers of stratified epithelial cells, with flattening of the more superficial cells.
Ciliated epithelial cells are absent.
Surface cells may contain keratohyaline granules or may
be keratinized.
Desquamation of surface cells may occur.

Differential Diagnoses
Degeneration/Regeneration

Usually follows acute injury.
Cells are only one or two layers thick.
No horizontal layering of flattened superficial cells.

Under chronic inflammatory conditions, lymphocyte-specificmicrodomains can be formed in nonlymphoid organs like salivary glands, liver, pancreas, thyroids, joints, and kidneys.159
The liver has a hematopoietic function during embryonicdevelopment which is lost postnatally, but the liver can host
TLS in adult life.160 The lymphocyte-specific microdomains
are defined as TLSs and can arise as a result of autoimmune
responses (as for example in rheumatoid arthritis, Sjögren disease and Hashimoto thyroiditis in humans), graft rejection,
atherosclerosis (humans and ApoE knockout mouse), microbial
infection, and neoplasia.161

Function
Tertiary lymphoid structures function like lymph nodes or
MALT. Their precise role in disease progression is still
unknown. Their presence in specific areas suggest that theyfacilitate local antigen presentation and immune responses andlikely play a role in epitope spreading by cross-reactivity of
immune cells and antibodies with endogenous host cellularantigens. Formation of well-developed TLSs with germinal
centers are often associated with increased severity of (autoimmune) disease and the local production of autoantibodies,
but TLSs can have an immune-protective role as well, especially in resolving chronic inflammation. The latter has been
observed in an atherosclerosis model in the APOE knockout
mouse.162 Tertiary lymphoid structures are often indicators ofgood prognosis in patients with cancer.163

Development
Tertiary lymphoid structures are not present at birth and do notdevelop at a predestined location (anlage), although their formation is driven by many of the same cytokines which drive thedevelopment and maintenance of lymph nodes and MALT.161
Tertiary lymphoid structures do not have specific developmental windows or anatomic locations, but instead arise in inflamedtissues.161

Comment

Histology

Squamous metaplasia of the follicle-associated epithelium is
expected to alter uptake of antigens and thus may lead to

Tertiary lymphoid structures contain B and T lymphocytes,
follicular and non-FDC, FRCs, and HEVs and may contain738
efferent lymphatic vessels.164-169 Like MALT, they may not
be supplied by afferent lymphatics and they are not encapsulated. The B and T lymphocytes are organized in similar
fashion to lymph nodes and MALT, with T- and B-celldominant areas including follicle-like compartments withgerminal centers. Lymphoid cell movement into andthrough TLS appears similar to that seen in the lymph nodeand is regulated by the cytokines permitting cells to enter
via HEVs, traffic along the FRC meshwork and exit via
lymphatic sinuses. Tertiary lymphoid structures can progress into a destructive inflammation and lose this lymphoidorgan-like organization.168

Sampling and Diagnostic Considerations
Tertiary lymphoid structures are not reported independently but are instead regarded as chance findings in nonlymphoid organs. Lymphoid follicles with germinal centersthat are located in nonlymphoid organs can be designatedas TLS. Without follicle formation (and in the absence ofdistinct HEVs), it is impossible to distinguish TLSs fromlymphocyte infiltrates in H&E-stained sections unless histochemistry/IHC reveals a lymphoid tissue organization
with dendritic cells, FRCs, some (indistinct) HEV, andlymphatics.
The distinction between TLS and MALT in the gastrointestinal tract and conducting airways can be complicated. Inducible BALT (iBALT) is sometimes considered to be TLS134,170
because of its dependence on inflammatory signaling, but,unlike TLS, it has predestined locations (at the bifurcation ofbrochi/bronchioles). Likewise, ILFs and CPs in the gastrointestinal tract resemble TLSs in their apparent dependence on
inflammatory stimuli. This document considers all types ofmucosal lymphoid accumulations to belong to MALT.
Increases in, or remarkable presence of, iBALT, ILFs, and CPs
or comparable structures are diagnosed according to“Proliferative changes, non-neoplastic” in the MALT section.
Lymphoid follicles outside typical MALT locations are diagnosed TLS.
desTertiary Lymphoid Structures (TLSs)(N) (Figure 120) Other lymphoid tissues

con
enh

Toxicologic Pathology 47(6)Pathogenesis/Cell of Origin
Precise pathogenesis is unknown. Under chronic or unresolved
inflammatory conditions, lymphocyte-specific microdomains
can form in inflamed nonlymphoid organs or tissues.

Diagnostic Features







Differential Diagnoses
Inducible BALT (IBALT) in the Lungs and ILFs in theIntestines

iBALT: Located between bronchus/bronchioles andartery and often in association with the airway
epithelium.
ILF: Located in the mucosa in association with theintestinal epithelium.

Increased SALCs in the Body Cavities

Located in the serosa and adipose tissue depots of theabdominal and thoracic cavities.
Follicles (with germinal centers) absent or rare.

Lymphocyte Inflammatory Cell Infiltrates


Infiltrates lack organization, especially follicle formation and presence of HEVs.
May include granulocytes or other types of inflammatory cells.

LymphomaTertiary Lymphoid Structures (TLSs)

Tertiary Lymphoid Structures (TLSs)

Ectopic, that is, in nonlymphoid organs such as salivary
glands, thyroid, liver, pancreas, kidney, or outside
mucosa in the gastrointestinal and respiratory tracts.
Uncommon in brain and skin.
Follicle formation, preferably with some germinal center development.
Presence of distinct HEVs.
Presence of inflammatory condition involving the organof concern.

Presence of neoplastic lymphocyte accumulations
elsewhere.
Involvement of primary and/or secondary lymphoidorgans.
Absence of an inflammatory stimulus.

Species
Mouse; rat.

Other Terms
Tertiary lymphoid organs (TLOs); tertiary lymphoid tissues;
lymphoid follicles; germinal centers; lymph node-like structures; ectopic lymphoid structures; lymphoid neogenesis.

Comment
Immunohistochemical staining for follicular and non-FDC,
FRCs, HEVs and other structural elements can help in the
diagnosis of TLS in case a distinct follicle formation is absent,
because it can reveal the lymphoid organ-like organization of
the lymphocyte accumulation(s).

Willard-Mack et al.

Serosa-Associated Lymphoid Clusters
Organization
Serosa-associated lymphoid clusters include fat-associatedlymphoid clusters (FALCs) and milky spots (MSs), also known
as Kampmeier’s foci, which are considered to be identicalstructures that differ only in location.171 Serosa-associatedlymphoid clusters172 are tiny white (“milky”) lymphoid focipresent in the serosa and the adipose tissue depots of the peritoneal, pleural, and pericardial cavities.

Function
The presence of SALCs in the serosal surfaces in the peritoneal
and thoracic cavities points to a role as first-line defense in thecavities. During intraperitoneal inflammation, they increase
greatly in number and size in the omentum. They may beobserved in increased numbers following intraperitoneal injection and may function as “the MALT of the serosa.” Based on
the abundant presence of innate lymphoid cells (ILC and B1 B
cells) and innate B1 B cells, the clusters probably form a special
link between innate and adaptive immunity. The lymphoid clusters may play a role in tumor metastasis as well, since they are
the major site of tumor dissemination in the peritoneal cavity.173

739
considered to be SALCs. The absence of inflammatory findingssuch as adipose tissue necrosis, abscesses, adhesions, increased
granulocytes, and hemorrhages distinguishes SALCs fromperitonitis.
desSALCs, Increased (N) (Figure 121) Other
lymphoid tissues

con
enh

SALCs, Increased

Lymphocytes, Adipose Tissue, Increased

Species
Mouse; rat.

Other Terms
Fat-associated lymphoid clusters (FALCs); milky spots (MSs);
Kampmeier’s foci.

Pathogenesis/Cell of Origin
Precise histogenesis unknown.

Diagnostic Features
Development

In the human omentum, SALCs are present before birth.171
Although considered to be secondary lymphoid organs, they
may develop via different molecular pathways than lymph
nodes and spleen.174 Part of the clusters may develop after birthand only upon an inflammatory stimulus, resembling TLSs.175

Histology







Serosa-associated lymphoid clusters consist of clusters of lymphocytes (including innate lymphoid cells), macrophages,
plasma cells, and mast cells located immediately below, andcovered by, the mesothelium. They can be highly vascularized
and have HEVs and efferent lymphatics. Serosa-associatedlymphoid clusters are not well-organized; distinct T- andB-dominant areas are absent, and follicles with or without
germinal centers are rare or absent.172,176

Differential Diagnoses
Lymphocytic Inflammatory Cell Infiltrates


Sampling and Diagnostic Considerations
Serosa-associated lymphoid clusters are not reported independently, but instead are often a chance finding in the serosa of
the lungs, heart, or abdominal organs. They may increase in
response to intraperitoneal injections. They are generally not
diagnosed unless they are abundant. It is often difficult to distinguish these lymphoid clusters from lymphocyte infiltrates in
H&E-stained sections, let alone to establish whether the clusters are induced or preexistent (hyperplastic). For practicalreasons, in this document, all lymphoid clusters in the adipose
tissue depots of the abdominal and thoracic cavities are

Lymphoid clusters composed of variable populations of
B and T lymphocytes with macrophages and myeloid
cells, depending on the stimulus.
Always in association with the serosa, most often inassociation with adipose tissue depots in the abdominal
and thoracic cavities.
Clusters are covered by mesothelium.
May show some degree of organization.
Macrophages situated at the periphery.
High endothelial venules are often present.
Follicles absent or rare.



Infiltrates lack organization (but so may SALCs).
Necrotic adipocytes surrounded by macrophages andlymphocytes, in case of low-grade inflammatory cell
infiltrates as seen in metabolic disease (called “crownlike structures”).
Absence of a distinct number of innate lymphoid cells(mainly nuocytes/NK cells and B1 B cells), as demonstrated by IHC.

LymphomaPresence of neoplastic lymphocyte accumulations
elsewhere.
Involvement of primary and/or secondary lymphoidorgans.

740

Comment
Immunohistochemical staining for B1 B cells, dendritic cells,
FRCs, HEVs, and other structural elements can help diagnose
SALCs (FALCs/milky spots) and distinguish them from infiltrates and lymphoma, especially when follicle formation isabsent, because it can reveal the lymphoid organ-like organization of the lymphocyte accumulation(s).

HEMATOLYMPHOID NEOPLASMS
The WHO classification system of hematopoietic and lymphoid tumors is an advanced nomenclature for clinical purposes in humans.177-179 In a multidisciplinary approach, thisclassification integrates immunophenotyping, morphology,
degree of differentiation, genetic, and molecular features forrecognition of distinct disease entities and extends to earlylesions identifiable by hematological investigations. The final
goal of the WHO classification is to connect pathology toclinical correlates, treatment, and prognosis.
In an experimental setting with a scope of translational
research, such as transgenic mouse models, the adoption of themost recent human classification could be applied.21,54,108,180 In
these cases, optimal and reproducible fixation conditions can beprovided. The use of formalin-fixed paraffin-embedded tissues
is well established for mice, less so for rats.103,181 An antibody
panel for the investigation of murine hematopoietic tissues has
been described by Kunder181 and immunophenotyping using Tand B-cell markers can be applied. The pan-T-cell marker CD3
can be used after various fixatives such as Bouins, B-5, Zenkers,
paraformaldehyde, and formalin-fixed tissues.
In 18- to 24-month rodent carcinogenicity studies, thefocus is on risk assessment rather than on translational
research for clinical outcome and treatment. To meet therequirements of a classification system for rodent carcinogenicity studies, which are mainly based on H&E morphology, a more practical WHO classification with fewer subtypes
was developed for rat and mouse.182,183 In risk assessment fora carcinogenic effect, fewer categories are advantageous as
the overall incidence of lymphomas is often combined in theinitial assessment of treatment-related effects. If an effect isobserved, a further investigation of subtypes using IHC can beof interest to further characterize the type of tumor. Organs ofdecedents are often stored in formalin for prolonged and variable time periods over the course of the studies, which maylead to inconsistent results of immunohistochemical stains.
However, when fixation conditions are optimal, morphological classification can be further expanded, if desired, byimmunotyping using T/B cell markers.
The progeny of HSCs such as myeloid and lymphoidlineages can give rise to autonomously growing cell clones
forming systemic tumors including myeloproliferative neoplasms, histiocytic sarcoma, and lymphoma. Differentiation
between these tumor entities is often difficult due to morphological similarities arising from relatedness of their cells oforigin. It may be helpful to evaluate bone marrow cytology

Toxicologic Pathology 47(6)or blood smears, but they are not, however, routinely included
in rodent carcinogenicity studies.
Hematopoietic tumors are inducible with various chemicals,
viruses, and radiation, but they are also frequently observed
spontaneously. Particularly, high and variable incidences havebeen observed in aged mice. The etiology of spontaneous hematopoietic and lymphoid tumors in rodents is not well understood,
and historical data from the same strain are helpful for an appraisal of tumor incidences. Notably, such tumors often arise as
sporadic incidental findings earlier than most other tumors inrodents. In mice, T-cell lymphomas may arise spontaneously or
by induction even younger than 3 months of age.
Besides tumors of hematopoietic and lymphoid cells, hematolymphoid organs can develop tumors of their stromal components. In the thymus, the epithelial reticular cells can give rise toneoplastic proliferative lesions. These cells are not merely amechanical scaffold, but support homing, proliferation, andmaturation of mobile cells,184 so proliferative stromal changes
can be associated with increased or altered populations of lymphoid cells. Variable mixtures of epithelial and T cells arereported in thymomas in humans,185 rats,183 and mice,182
according to the WHO classification. Neoplastic stromal cells
may lose their supportive abilities, as in malignant epithelial
thymomas with squamous differentiation in which the lymphocytic component may be virtually absent. Reticulum cell sarcoma was reported in the mouse and rat in the early rodent
literature and was divided into types A and B by Dunn.35 Further
work on these tumors using modern techniques reclassified
Dunn’s type B tumor as B-cell origin lymphoma and Dunn’s
type A tumor as of histiocytic origin.108,186 In recent years, theconcept of reticular cell tumors has been modified and tumors of
the FRC have been described in humans,187-190 but they have notbeen demonstrated in the laboratory mouse or rat to date. Clonesof C57BL/6 non-neoplastic FRCs have been immortalized andcharacterized.190
Finally, vascular proliferative changes such as hemangiomaor hemangiosarcoma often occur in lymphoid organs of aging
rodents. These tumors are beyond the scope of the hematolymphoid system and are presented in the INHAND cardiovascularsystem. Vascular tumors should be considered in the differential diagnosis for tumors with a high amount of connective
tissue and capillary vascular spaces, however.

Hematopoietic Neoplasms
Leukemia, Erythroid (M) (Figures 122-124)
Hematolymphoid neoplasms
Species
Mouse; rat.

Other Terms
Erythroleukemia; erythroblastic leukemia.

Willard-Mack et al.

Pathogenesis/Cell of Origin
Develops from the erythroid cell lineage in the spleen or bonemarrow.

741





Diagnostic Features












Leukemia with excessive proliferation of erythroblasts,
immature erythroid cells, or normoblasts.
Undifferentiated blast cells are more common, but morewell-differentiated tumors may occur exhibiting all
stages of erythroid differentiation.
Immature cells may have signs of atypia.
Often arises in splenic red pulp, particularly in mice;
white pulp is compressed and atrophic.
Preferential spread to liver sinusoids.
Splenomegaly and hepatomegaly may be severe while
lymph nodes are not involved.
Enlarged spleen may have numerous subcapsular
hematomas.
Immature erythrocytic cells can be identified with specific antibodies and/or with benzidine (p-diaminobiphenyl). The expression intensity of antigens varies
depending on the stage of differentiation of the erythroid
cells. Some antigens increase in intensity with maturation (glycoprotein or glycophorin A [CD235a] and TER
119), while the intensity of other antigens decreases as
the erythroid cells mature (GATA1, alpha hemoglobulin
stabilizing protein, CD43, CD71, CD117).
Neoplastic cells may occur in the blood and diagnosis
can be confirmed in blood or bone marrow smears, if
available.

Differential Diagnoses

Usually localized to the spleen, but foci of reactive
hematopoiesis may be present in hepatic sinusoids andoccasionally in other tissues.
Proliferating cells are not invasive and follow the normal distribution of EMH.
When severe, extramedullary erythropoiesis involving
both the spleen and liver may be difficult to distinguishfrom erythroid leukemia.
Refer to EMH in the General Hematolymphoid section
for additional information.

Dyshematopoiesis, Erythroid

Erythroid hypercellularity.
Aberrant erythropoietic cell morphology such as multinuclearity, nuclear fragmentation, megaloblastosis, or
ringed sideroblasts.
Evidence of arrest in a stage of differentiation.44

Leukemia, Megakaryocytic (M) (Figures 125 and126) Hematolymphoid neoplasms
Species
Mouse; rat.

Other Terms
Megakaryoblastic leukemia; megakaryocytic myelosis.

Pathogenesis/Cell of Origin
Develops from the megakaryocytic cell lineage in the spleen
and bone marrow.

LymphomaDiagnostic FeaturesNo evidence of erythroid differentiation.
Differentiation from lymphoma may be difficult iferythroid development is arrested at a very primitive
stage.
Definitive diagnosis of primitive erythroid cells generally requires the use of specific antibodies that recognize
antigens expressed during normal erythropoiesis(CD117, CD43, GATA1, CD71, CD235a, TER119,
alpha hemoglobin stabilizing protein) versus lymphoidantigen antibodies for lymphoid cells (PAX5, CD3,
CD25, CD45, TDT).

Extramedullary Hematopoiesis, Erythroid

A hematopoietic stimulus is present such as anemia or
inflammation.
Mixture of cell lineages is present but erythroid lineage
is predominant.












Increase in megakaryocytes in various stages ofdifferentiation.
Marked increase of large mononuclear megakaryoblasts; smaller immature forms are often present.
Nuclei are often atypical and may or may not be
multinucleated.
Tumor cells may be present in bone marrow, lymph
nodes, spleen, liver, and kidneys.
If lymph nodes are involved, megakaryocytes may bepresent in medullary sinuses.
Large dysplastic platelets are present in circulation.
Thrombocytopenia or pancytopenia may be evident.
Identification of a megakaryocyte lineage often cannot
be made by morphologic features alone and immunophenotyping may be required.191
Positive for platelet glycoprotein IIb/IIIa, CD41,
GATA1, CD61, and von Willebrand factor (Factor VIII)
by IHC.

742


Toxicologic Pathology 47(6)RUNX1 antibody can also be used to identify immaturemegakaryocytic stages of maturation.

Modifier
Neutrophilic; eosinophilic; basophilic; myelomonocytic;
monocytic; NOS (not otherwise specified).

Differential Diagnoses
Extramedullary Hematopoiesis, Megakaryocytic
Megakaryocytic EMH in the splenic red pulp is usuallyassociated with an increase in mature megakaryocyteswith no evidence of blast cells.

Pathogenesis/Cell of Origin
Develops from the granulocytic or monocytic cell lineage inthe spleen (especially in the mouse) or bone marrow.

Dyshematopoiesis, MegakaryocyticIncreased megakaryocytes with abnormal morphology
and thrombocytopenia.
Differentiation arrest.

Leukemia, ErythroidNo multinucleated giant cells resembling normal or atypical megakaryocytes are present.
Differentiating megakaryoblastic leukemia from immature erythroid (erythroblastic) leukemia may requireIHC.
An IHC antibody panel consisting of CD41, CD61, vonWillebrand factor, and RUNX1 for megakaryocytic
cells and glycophorin A (CD 235a) and alpha hemoglobin stabilizing protein for erythroid cells can be helpful
in differentiating these 2 leukemias.

Diagnostic Features











Comment
Megakaryocytic leukemia is rare in conventional mice. It has
been described in GEM and has been reported to be induced
by a recombinant retrovirus (MuLV). Erythroid leukemia is arare spontaneous lesion in the mouse and is also induciblewith MuLVs such as the Friend virus which induces Friend
leukemia. Erythroid leukemia can also be induced by wholebody radiation in C3H/He mice and RF mice. Erythroid leukemia is an extremely rare spontaneous lesion in the rat.192 It
is inducible after radiation and trimethylbenz[a]anthrene
treatment.



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



Leukemia, Myeloid (M) (Figures 127-129)
Hematolymphoid neoplasms

Myeloid cell differentiation varies from immature(poorly differentiated) to mature with variable proportions of blastic to segmented forms.
Nuclei may have immature morphological features
including indented nuclei and doughnut-shaped ringforms with a central opening.
Myeloblast nuclei have fine chromatin and prominent
nucleoli, while promyelocyte nuclei have dense coarse
chromatin and less distinct nucleoli.
Myeloblasts frequently have azurophilic granules containing myeloperoxidase but they may not be visible in
H&E sections.
Blood and/or bone marrow smear preparations may be
helpful for the investigation of cytological details.
Some types of myeloid leukemia can develop very highwhite cell counts (up to 1 million cells/mL) consisting
primarily of myelocytes and mature neutrophils, whileother types (myeloblastic leukemia) consist primarily ofcells resembling myeloblasts and promyelocytes.
Leukemia frequently occurs with diffuse organ enlargement, usually involving the spleen or bone marrow withsecondary involvement of liver and other organs.
The spleen and liver may become very large.
Tissue masses may have a characteristic dull red color;
infiltrated tissues may show a greenish discoloration
macroscopically (hence the name chloroleukemia).
Late promyelocytes and later developmental stages are
positive for lysosome markers, myeloperoxidase, CAE,
lysozyme, and other granulocytic antigens.
Kidneys may have increased eosinophilic granules (hyaline droplets) in the proximal tubules (as also occurs
with histiocytic sarcoma).

Differential Diagnoses
Species
Mouse; rat.

Other Terms
Granulocytic leukemia; acute myeloid leukemia; myeloblastic
leukemia; chloroleukemia; granulocytic sarcoma (for localized
growth).

Extramedullary Hematopoiesis, MyeloidDifferentiating extramedullary hematopoiesis from neutrophilic myeloid leukemia can be difficult.
Usually more mature stages of myeloid cells are present.
Often has an intermixture of relatively high numbers oferythroid precursors and megakaryocytes.
Morphology and maturation sequences are synchronous.

Willard-Mack et al.








Peripheral white blood cell counts usually do not reachthe extremely high levels more commonly seen in myeloid leukemia.
Generally occurs as a reactive response to conditions
such as chronic dermatitis, ulcerated skin tumors,
repeated blood loss, necrotic tumors, abscesses, andincreased red cell destruction.
Lymph nodes are frequently involved, especially mesenteric lymph nodes and regional draining nodes of a lesion.
In rare cases, other organs may be involved such as
adrenal, ovary, pituitary, and perirenal fat.
Distribution tends to follow normal anatomical boundaries and does not appear invasive except for proliferation in the epidural space and in the extramedullary sinus
along bones.55

Dyshematopoiesis, GranulocyticAbnormal or defective differentiation of myeloid
lineage.
Increased numbers of granulocytic precursor cells.
Abnormal nuclear segmentation or asynchrony of chromatin maturation and nuclear segmentation.
Altered granule morphology and/or abnormal cytoplasmic
features (eg, size, shape, number, and/or tinctorial
quality).33

743
including granulocytic, granulocytic/monocytic, erythroid
and megakaryocytic leukemia, mast cell tumors, and histiocytic sarcoma.

Leukemia, NOS (M) Hematolymphoid neoplasms
Species
Mouse; rat.

Synonym(s)Leukemia.

Other Terms
Unclassifiable leukemia; undifferentiated leukemia; anaplastic
leukemia.

Pathogenesis/Cell of Origin
Develops from cells of the hematopoietic tissue.

Diagnostic FeaturesComment
Modifiers may be used to indicate the lineage of the tumor cells
if desired. The modifier NOS (not otherwise specified) can beused when cell lineage is desired but cannot be determined.
Lineage is based on the tinctorial characteristics of the cytoplasmic granules, when present, but definitive subtyping may
require IHC. Smear preparations may be helpful for the investigation of cytological details.
Myeloid leukemias that consist of predominantly blasts andimmature forms, that is, myeloid leukemia without maturation(<10% of mature granulocytes), myeloid leukemia withmaturation (>10% of mature forms), myelomonocytic
leukemia and monocytic leukemia, are extremely difficult todifferentiate from one another without cellular lineage characterization by flow cytometry, IHC, or histochemistry for myeloperoxidase. Therefore, the most appropriate diagnosis forthese types of leukemia is simply myeloid leukemia. Chronic
myeloid leukemias have been described in GEM with a Philadelphia chromosome.193
The term “myeloid” is commonly used to refer to the whiteblood cell component of bone marrow. Histologically, thegranulocytic series is the predominant lineage of the whiteblood cell component. The most common form of myeloid
leukemia arises from the neutrophilic lineage. Thus, myeloid
leukemia, which originates from the myeloblast, is often
called granulocytic leukemia. Myeloid leukemia can also be
used in a broader sense for any leukemic hematopoietic neoplasm derived from a common myeloid progenitor cell,

Leukemia with leukemic infiltration in various organsystems.
Liver, spleen, and/or lymph nodes may be enlarged at
necropsy.

Differential Diagnoses
Leukemoid Reaction (Leukocytosis)

Mature granulocytes in capillary beds.
Most often seen in lungs and liver.
Usually associated with a gross lesion in another tissue,
i.e. abscess, ulcerated skin, etc.

Comment
Leukemia NOS (not otherwise specified) can be used whenclassification is desired but a definitive lineage cannot be determined by IHC or other lab techniques such as FACS and gene
expression studies. Leukemia NOS can also be used when theleukemia is unclassifiable due to the condition of the specimen(autolysis, poor fixation or technique). Leukemia (with nomodification) may be used when classification is not desired.

Lymphoid Neoplasms
Lymphoma (M) (Figures 130-151)
Hematolymphoid Neoplasms
Species
Mouse; rat.

744

Toxicologic Pathology 47(6)Other Terms
Malignant lymphoma; lymphosarcomaModifier
Subtype modifiers: lymphoblastic; pleomorphic; follicular;
immunoblastic; lymphocytic; plasmacytic; epitheliotrophic
cutaneous T-cell; marginal zone; LGL, NOS.

Cell type modifiers: T cell; B cell.

Qualifier (optional): Leukemic (can be used when there is an
obvious leukemia present judging from the peripheral bloodsmears or tissue sections)Pathogenesis/Cell of Origin
Develops from T or B lymphocytes or their precursors in the
spleen, lymph nodes, thymus, MALT or bone marrow.



Diagnostic Features

Lymphoblastic Lymphoma




















Medium-sized to large lymphoblasts.
Cytoplasm is scant to moderate, basophilic, and may bevacuolated.
Nuclei are round, oval, irregular, or convoluted withfinely stippled chromatin.
A central nucleolus ranges from inconspicuous to 1 to 3
distinct small nucleoli.
Nuclear to cytoplasmic ratio is high.
Mitotic figures are variable in number, but are often
numerous.
Cells are noncohesive but form homogeneous sheets.
Starry-sky appearance is often present with tingible
body macrophages and apoptosis.
May become leukemic in later stages.
Aggressive behavior with diffuse infiltration of liver,kidneys, and ovaries and infiltration along the vasculartree in the lung (similar to immunoblastic lymphoma).
May invade the central nervous system when in a leukemic phase.
T-cell lymphoblastic lymphoma.
 Originates in the thymus in young rats and mice.
 Occurs at a high frequency in some strains andgenetic targeted mice.
B-cell lymphoblastic lymphoma.
 Arises in the spleen or lymph nodes in old mice.
 Positive for PAX5 and generally positive for CD45R
but may be negative for CD45R by IHC when cells
are of the pro-B stage.

Pleomorphic Lymphoma

Neoplastic cell population can consist of several cell
types including small lymphocytes, small and large

follicular center cells (centrocytes and centroblasts),
immunoblasts, and small and large cells of nonfollicular origin.
The proportion of each cell type varies in each lymphoma and sometimes even in each anatomical site.
Multinucleated cells may be present.
Blastic cells have prominent nucleoli.
Cells with cleaved nuclei (centrocytes) are seen in variable
proportions to cells with round nuclei (centroblasts).
Cells may be admixed with variable proportions of T
helper CD4þ cells, macrophages, and/or eosinophils.
Single or multicentric origin.
Most common lymphoma subtype in aged mice (>12months old) in most strains.
Early single-site pleomorphic lymphomas common inmesenteric lymph nodes and spleen.
Occur in Peyers patches in aged rodents.
Growth pattern is typically diffuse and generally follicles and germinal centers are not apparent (in contrast tohumans where they are apparent).
When present, follicles may result in a nodular appearance, especially in spleen.
Rarely leukemic in distribution.
Tumor cells are most often of follicular B-cell origin,but may rarely be of T-cell origin.

 IHC or flow cytometry is necessary to determine Bor T-cell origin.

 Pleomorphic lymphoma may be diagnosed when thetumor cell type is mixed or unknown.

 Follicular lymphoma is the preferred terminology if
B-cell origin is determined.

 Pleomorphic lymphomas of B-cell origin may
produce heavy chain Igs and express kappa lightchains far more frequently than lambda lightchains.
Follicular Lymphoma













Follicular lymphoma is of B-cell origin and is the preferred terminology if B-cell origin is determined, especially by IHC and/or with clonal assays.
Neoplastic cell population can consist of several cell
types including small lymphocytes, small and large
follicular center cells (centrocytes and centroblasts).
The proportion of each cell type varies in each lymphoma and sometimes even in each anatomical site.
Multinucleated cells may be present.
Blastic cells have prominent nucleoli.
Cells with cleaved nuclei (centrocytes) are seen invariable proportions to cells with round nuclei(centroblasts).
Cells may be admixed with variable proportions of T
helper CD4þ cells, macrophages, and/or eosinophils.
Single or multicentric origin.
Positive for CD45R/B220, PAX5, CD79a, BCL6, and/or
PNA.

Willard-Mack et al.
Immunoblastic Lymphoma














Cells are large, noncohesive, and monotypic.
Cytoplasm is conspicuously amphophilic.
Nuclei are large and vesicular with one large, sometimes
bar-shaped, central or peripheral nucleolus.
Mitotic figures may be numerous.
May be of B-cell (more commonly) or T-cell origin.
Plasmacytoid cells and plasma cells may be present.
Rare in most nongenetically engineered strains.
Pattern of organ involvement shows diffuse infiltrationof lymph nodes, spleen, liver, kidneys, and ovaries andalong the vascular tree in the lung (similar to lymphoblastic lymphoma).
Not primarily leukemic in distribution.
When of B-cell origin, cells stain positive for heavy
chain Igs or kappa light chains and, rarely, for lambda
light chains.
Has been reported in the BB/E rat in association withtranslocations in the c-Myc oncogene.194

Lymphocytic Lymphoma









Tumor cells are small to medium-sized and well differentiated with a narrow rim of cytoplasm and densely
clumped chromatin.
Cells differ little, if at all, from normal circulating small
lymphocytes.
Cells are uniform and noncohesive.
Mitotic figures are rare.
No tingible body macrophages.
Can be of T- or B-cell origin.
Normal architecture of involved lymphoid organs may
or may not be maintained.

Plasmacytic Lymphoma
Synonyms: Plasma cell tumor; plasmacytoma.












Tumor cells may be mature plasma cells, immature cellswith an obvious plasmacytoid differentiation, or a mixture of mature and immature plasma cells.
Cytoplasm is basophilic and pyroninophilic and asmall perinuclear halo (Golgi apparatus) may bepresent.
Nuclei are round and have a cartwheel appearance.
Mitotic figures are rare in well-differentiated tumors.
Formation of eosinophilic intracytoplasmic crystal-likestructures considered diagnostic for plasmacytic lymphomas, although rarely seen.
May be difficult to differentiate a well-differentiatedtumor from plasma cell hyperplasia based on morphologic features.
 Ki67 can help differentiate because plasma cell
hyperplasia does not have uniform Ki67
immunoreactivity.
May arise in spleen or lymph nodes, rarely in bonemarrow.

745
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



Rare in most mouse strains (more common in NZB miceand inducible in BALB/c mice).
Positive for heavy chain Igs by immunocytochemistry.
More than 95% of tumors are positive for kappa lightchains.
Since the majority of normal mouse cells express
kappa, light chain determination is unsuitable for clonality determination in mice (in contrast to man, where
the kappa to lambda ratio is 2:1). Demonstration of a
restricted IgH isotype (IgG1, IgG2a, IgG2b, or IgA)
would be required for a relatively strong indication of
clonality.195

Epitheliotropic Cutaneous Lymphoma












Presents as a dermal gross lesion with hair loss andencrusted skin.
Tumor cells are small to medium sized with little cytoplasm and indented nuclei.
Mitotic figures are rare.
Infiltration of dermis and squamous epithelium.
Loose lichenoid infiltrates in the epidermis andadnexa.
Pautrier’s microabsesses in the epidermis and hairfollicles.
Inflammatory histiocytes and plasma cells may be present in the dermis.
Resembles “mycosis fungoides.”Later stages may involve multiple extracutaneous sites.
Rare lesion reported in rats.
Tumor cells expressing Pan T-cell markers (CD2,
CD3, and CD8) are consistent with tumors of T-cellorigin.

Marginal Zone LymphomaProgressive lesion.
Arises from marginal zone hyperplasia.
 Widened MZ in the absence of germinal center
hyperplasia.
 Uniform population of lymphocytes with clear cytoplasm (monocytoid cells).
 The width of the MZ in mice varies with thestrain so comparison to concurrent controls is
important.
 Germinal center hyperplasia accompanies MZ
hyperplasia, but not common with MZL.
 May be focal, multifocal, or diffuse.
Marginal zone lymphoma.

 Further widening of the MZ with cellular atypia(medium to large cells with prominent nucleoli andoccasional mitotic figures).
 Very homogenous to pleomorphic population of
medium-sized cells.
 Diffuse infiltration of the red pulp with complete or
incomplete bridging of follicles.

746

Toxicologic Pathology 47(6)Further progression.

 Marginal zones increase in width with coalescenceand bridging of the follicles.

 Larger cells with vesicular nuclei with and withoutprominent nucleoli.

 Invasion of the white pulp, compression of PALS,
obliteration of the white pulp (in rare cases).

 Generally restricted to the spleen but may metasta

size to liver and lymph nodes.
Marginal Zone Lymphomas are of B-cell origin and areoften positive for PAX5, CD45R and/or IgM byimmunohistochemistry.

Large Granular Lymphocyte Leukemia
Synonyms: Large granular lymphocyte lymphoma; LGLNK lymphoma.















Uniform populations of medium-sized lymphocyteswith morphology similar to large granular lymphocytes(NK cells).
Nuclei are round, oval, slightly irregular, or reniformwith varying degrees of differentiation, clumped nuclear
chromatin and small nucleoli.
Cytoplasm is basophilic and has variable numbers/sizeof granules.
 Granules appear reddish in Giemsa-stained peripheral blood smears and tumor imprints.
 Not visible in routine H&E-stained sections.
 Granules differentiate LGL-leukemia from othertypes of lymphoma.
High incidence (10%-50%) in aging Fischer 344 rats.
Reported in other rat strains such as the Wistar or Sprague Dawley.
Not reported as a spontaneous lesion in mice but has
been induced in GEM.
The tumor is almost by definition leukemic but WBC
counts vary.
Liver and spleen most frequently involved, but otherorgans may also be infiltrated.
Early, and possibly primary, lesions are frequently
found in the spleen, especially in the MZ.196
Positive for OX-8 (CD8) by IHC.
Three stages are recognized based upon extent andseverity.197

Stage 1Clinically and morphologically prelymphoma/leukemia.
Spleen is normal in size or only slightly enlarged.
Mild number of LGL cells present in red pulp and MZ.
Few or no neoplastic LGL cells in liver sinusoids.
No identifiable neoplastic LGL cells in any other organ.

Stage 2Moderately enlarged spleen.
Numerous LGL cells in red pulp.



Splenic architecture preserved.
Mild to moderate involvement of liver with aggregatesof LGL cells in sinusoids.
Minimal to no involvement of other organs.

Stage 3








Advanced stage.
Spleen and liver markedly enlarged.
Normal splenic architecture effaced by neoplastic cells.
Liver markedly infiltrated by LGL cells.
Liver frequently has degenerative changes which may
be accompanied by nodular regenerative hyperplastic
lesions.
Neoplastic cell infiltrates in other organs such as lungs,
lymph nodes, kidneys, brain, and adrenals.

Differential Diagnoses
Hyperplasia, LymphoidA reactive response to tumor, ulceration, infection.
Involves the draining lymph node.
Polyclonal proliferation.

Hyperplasia, Marginal Zone


A reactive response to tumor, ulceration, and infection.
Widened MZ with germinal center hyperplasia.
No cellular atypia or increased mitotic figures.
Generally occurs diffusely.

Fibrosarcoma, Pleomorphic








Differential for pleomorphic lymphoma.
Soft tissue tumor (see INHAND soft tissue/skeletal muscle classification).
Also known as pleomorphic/undifferentiated sarcoma.
Tends to have a more marked fibrous structure.
Often has an obvious soft tissue primary site (eg, subcutaneously on the rear leg).
Does not have a primarily lymphoid distribution patternin most cases.
Does not stain with any IHC markers for blood cells.

Histiocytic Sarcoma







Cytoplasm is eosinophilic and abundant.
May sometimes resemble a follicular lymphoma.
May have a more fibrous structure (in rats) and it often
has areas with large multinucleated cells which may
show foreign body–type patterns.
Usually has a different anatomical distribution and distribution pattern compared to lymphomas.
Is sometimes admixed with a lymphoma such that the
animal has 2 systemic tumors simultaneously.

Willard-Mack et al.
Often has a primary site, for example, either retroperitoneally or in the liver, spleen, skin, bone marrow, brain,mesenteric lymph node, uterus, vagina, and so on.
Stains with one or more IHC markers for macrophages/
histiocytes.

Thymoma, Malignant

747

Other Terms
Reticular cell sarcoma type A; malignant histiocytosis; Kupffer
cell sarcoma.

Pathogenesis/Cell of Origin
Cells of the mononuclear phagocyte system.



Has an epithelial component.
Location of lesion is not systemic.

Diagnostic FeaturesComment
The early stages of lymphoma development can be diagnostically challenging. Systemic tumors may start at a single site,
for example, in a solitary lymph node, before spreading to otherorgans. The diagnosis of a single-site tumor can be difficult andis based on abnormal cytological features, disturbance of tissue
architecture, and the absence inflammation or infection. At theother extreme, widely disseminated lymphoma may involve
multiple hematolymphoid organs and extranodal tissues. When
numerous lymph nodes are affected, it is sufficient to evaluate
routine lymph nodes (ie, mesenteric and axillary) and a representative sample of up to 5 other enlarged lymph nodes.
Some types of lymphomas cannot be distinguished morphologically and can only be identified by IHC. For example, pleomorphic lymphoma consists of several subtypes of B- and T-celllineages, the majority of which are the follicular lymphoma type.
Some of the spontaneous B-cell lymphomas in mice resemble
the diffuse large B-cell lymphoma seen commonly in humans.
These include histiocyte-associated B-cell lymphoma and B-cell
lymphoblastic lymphoma, especially in CD-1 mice.180 Lymphoma NOS (not otherwise specified) can be used when classification is desired but a definitive lineage cannot be determined
by IHC or other lab techniques (FACS, gene expression studies).
Lymphoma NOS can also be used when the lymphoma is unclassifiable due to the condition of the specimen (autolysis, poor
fixation or technique). Lymphoma (with no modification) may
be used when classification is not desired.
Differentiating between lymphoma and lymphoid hyperplasia
can be diagnostically challenging. Hyperplasia is usually a reactive change but, on rare occasions, may be a preneoplastic change
if it is severe or prolonged in duration, atypical, distorts normalarchitecture, and exhibits cellular atypia. In cases of concern,
additional diagnostic tests for Ig and TCR clonality should beconsidered to differentiate hyperplasia from lymphoma.

Histiocytic Neoplasm
Histiocytic Sarcoma (m) (figures 152-154)
hematolymphoid neoplasms

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





Differential Diagnoses
Fibrosarcoma, Pleomorphic/Undifferentiated Pleomorphic
SarcomaSpecies
Mouse; rat.

Uniform population of round or oval cells with abundanteosinophilic cytoplasm.
Cells may also be spindle-shaped.
Nuclei are round, irregular, elongated, folded, orindented; ring-shaped nuclei with a central opening may
occasionally be present.
Multinucleate giant cells are often scattered throughout
the tumor.
Phagocytosis may be present.
Mitotic figures may vary from few to numerous.
Atypical cells are sparse and pleomorphism is usuallyabsent.
Areas of necrosis surrounded by palisading tumor cells
are common and characteristic.
Minimal fibrosis may be present.
Perivascular tumor cell infiltrates are commonly foundin the lungs.
Tumor cells may be present in blood smears.
Vascular tumor emboli are common.
Infiltrates can occur in lymphoid organs, in nonlymphoid organs (such as uterus, vagina, liver, skin, brain),retroperitoneally, and on serosal surfaces.
Kidneys often have an increase in intracytoplasmic eosinophilic hyaline granules (hyaline droplets) in the proximal tubules.
 Severity is dependent upon the degree of tumor loadin the animal.
 Hyaline droplets are positive for lysozyme and negative for alpha1-antitrypsin, alpha 2m-globulin, rat
or mouse Ig and albumin.
F4/80, lysozyme and MAC-2 are reliable markers forhistiocytic sarcoma in mice.
ED1 (CD68), ED2 (CD163), ED3 (CD169) and lysozyme
are reliable markers for histiocytic sarcoma in rats.
In a rare histiocytic sarcoma, sporadic neoplastic histiocytes expressed S-100.21



A mixed cell population of histiocyte-like cells, bizarre
tumor giant cells, fibroblasts and undifferentiated cells
in various proportions.
The fibrous component is always prominent.

748

Toxicologic Pathology 47(6)More pleomorphic and more fusiform cell type with lesstendency for multinucleated cells than histiocytic sarcoma.

FibrosarcomaMore uniform fibroblast pattern with long bundles (fascicles) of cells with or without a herring bone pattern.
Fibrous component often prominent.

Lymphoma (pleomorphic, follicular, lymphoblastic)







Lymphoma cells have less cytoplasm.
Multinucleated giant cells are not usually present.
Spleen and lymph nodes are frequently involved.
H&E-stained sections of lymphoma often appear bluishmacroscopically due to the relatively high nuclear:cytoplasmic ratio of the neoplastic lymphocytes.
H&E-stained sections of histiocytic sarcoma appear reddish macroscopically due to the relatively low nuclear:cytoplasmic ratio of the neoplastic histiocytes.

Schwannoma, malignant







Schwannomas have typical growth patterns consistingof solid areas (Antoni A) and areas with large cystic
spaces (Antoni B).
Multinucleate giant cells are not present.
Eosinophilic cytoplasm is not abundant.
Often occurs in the uterus.
S-100 immunoreactivity is diffuse in schwannomas but
is more sporadic in histiocytic sarcoma when it occurs.

Can be differentiated with IHC for melanin (HMB45,
PEP8) and macrophage markers (F4/80).
Mac 2 and CD68 are not helpful since they are expressedin both tumor types.
S100 is also expressed in both tumors, but the pattern ofexpression is sporadic in histiocytic sarcoma and diffuse
in melanoma.

Comment
Histiocytic sarcomas arise most frequently in subcutaneous tissues in the Wistar rat and in the liver and lungs in F344 andSprague Dawley rats. Lymph nodes have been reported as primary sites in some rat strains. The frequency of tumor infiltrates inmice is liver > spleen > lung > bone marrow > uterus > lymph
node > kidney, but primary organ involvement is very much strain
related. In some mouse strains, histiocytic sarcoma and lymphoma may both occur in a single animal, either in the same tissue
or in different tissues. Histiocytic sarcomas most often spread
hematogenously but may also undergo lymphogenous spread.

Mast Cell Neoplasms (Figures 155-157)
Leukemia, Mast Cell (M) Hematolymphoidneoplasms
Species
Mouse; rat.

Tumor, mast cell, malignant









Nuclei are generally bizarre, bilobed, and/or polylobed,
often with prominent nucleoli.
Cytoplasm is granular and basophilic or may be
hypogranular.
Metachromatic cytoplasmic granules are positive for
Giemsa or Toluidine blue.
Solitary type: a single local sarcomatous growth of atypical mast cells.
Systemic type: compact nodular or sheet-like accumulations of round, spindle-shaped, or immature mast cells.
Multiple organs are often involved.
Eosinophils may be associated with the mast cells.
May be difficult to differentiate from other sarcoma
types without the use of IHC.

Adenocarcinoma, spindle cellCan be confused with an immature histiocytic sarcoma
that has spindle cell morphology.
Can be differentiated with IHC for cytokeratin andmacrophage biomarkers.
Growth pattern tends to be more expansive than invasive.

Melanoma, malignant
Nonpigmented melanomas can be confused with amature histiocytic sarcoma with round cells.

Pathogenesis/Cell of Origin
Develops from mast cells and their precursors present in thehematopoietic, mucosal, and/or connective tissues.

Diagnostic Features








Atypical mast cells are present in the liver, spleen, bonemarrow, and/or peripheral blood.
Mast cell accumulations with sheet-like or leukemic
pattern present in one or more hematolymphoid organs.
Histochemical stains and IHC can be used to diagnosis
the atypical cells as mast cells.
Morphological differentiation between mast cells andbasophils is difficult due to the heavy cytoplasm granularity often seen in both cell types.
Unlike mast cells, basophils are generally tryptase negative and are always CD117 negative.
Considered malignant.

Differential Diagnoses
Histiocytic sarcoma

Nuclei are less regular.
Cytoplasm is eosinophilic.
Negative for metachromatic cytoplasmic granules.

Willard-Mack et al.
Melanoma, malignant, amelanoticDifferentiate from mast cells with IHC for expression ofmelanin (HMB45, PEP8).

Comment
Little is known about mast cell proliferations in rodents and theterminology for proliferative conditions is confusing. The classification presented here is proposed as a standardized terminology
for use by the toxicologic pathology community. This classification is considered a work in progress and changes are anticipatedas knowledge and understanding of rodent mast cell proliferations
evolve. Nodular mast cell accumulations are not present in normal
tissue, hence their neoplastic classification. Such lesions are relatively rare, so there is little information on progression. Presently, there are no clear criteria for differentiating benign frommalignant tumors in rodents. In humans, a threshold of 15 or moremast cells is set for aggregates in biopsies indicating systemic
mastocytosis.198 As mast cells are normal tissue constituents, it
may be helpful to define a similar threshold for mast cell lesions inrodents. Because well-differentiated mast cell tumors often show
malignant growth characteristics in other species, a tumor withmultiple nodules in several organs with or without leukemia isconsidered malignant, while a solitary nodular tumor is considered benign in the absence of pleomorphism. Mastocytosis is thediagnosis used in human terminology for a malignant mast celltumor. Mast cell tumors are rare in rats and mice.199 Most reported
cases are generally localized and well differentiated. In rats, 2cases have been reported in the mesentery200,201 and a primarysubcutaneous eyelid nodule generalized to other organs including
lymph nodes, liver, and kidneys.202 “Retikulose mit reicher
Mastzellbeteiligung” (reticulocytosis with prominent mast cells)
was reported by Hunstein after whole-body radiation in a Wistar
rat.203 In mice, reported cases showed distribution to multiple
organs.204
There are at least 3 types of mast cells in the mouse. The mostcommon is the connective tissue type with basophilic metachromatic granules, which is generally seen in the hematolymphoidtissues, interstitial tissues, the serosa, and the intestinal submucosa. There also are 2 mucosal mast cell types, one of which haseosinophilic granules that are not metachromatic and this type isgenerally located within the mucosal epithelium. The othermucosal mast cell type is located in the lamina propria and is
not generally apparent histologically in H&E sections of formalin-fixed paraffin-embedded tissue. The 2 mucosal mast cell
types can be differentiated from one another and the connective
tissue type with immunohistochemical stains.205
Histochemical stains and immunohistochemical antibodies
can be used to differentiate mast cells from histiocytes, lymphocytes, and melanocytes. A panel of histochemical assays that
includes toluidine blue, alcian blue, CAE, and safarin and/or apanel of antibodies for the mast cell markers CD117, CD34, andmast cell proteases (Mcp)-1, 4 and 6 may be used when diagnosing mast cell disorders in rodents. CD117 and CD34 areexpressed in mouse mast cells throughout their development and749
maturation, but they are not specific for mast cells. Anti-tryptase(anti-Mcp-6) has been shown to be the most specific marker for
diagnosing mast cell disorders in tissue or decalcified specimensthat are of the connective tissue mast cell subtype. Both theconnective tissue and lamina propria mucosal mast cells express
Mcp-4 and tryptase, whereas Mcp-1 is the most specific marker
for the intraepithelial mucosal mast cell disorders. Anti-CD117
and anti-tryptase (anti-Mcp-6) will also aid in determining
whether cell infiltrates are mast cells or basophils.21 Note thatsome decalcification solutions may inhibit some mast cell histochemical reagents and IHC antibodies.

Tumor, Mast Cell, Benign (B) Hematolymphoid
Neoplasms
Species
Mouse; rat.

Other Terms
Mastocytoma; systemic mastocytosis.

Pathogenesis/Cell of Origin
Develops from mast cells and their precursors present in thehematopoietic, mucosal, and/or connective tissues.

Diagnostic Features






A single, solitary, compact (dense) aggregate, or
nodule.
Compression of adjacent tissue.
No systemic involvement.
No clear inflammatory stimulus.
Considered benign in the absence of pleomorphism.

Comment
See comment under Mast Cell Leukemia.

Tumor, Mast Cell, Malignant(M) Hematolymphoid Neoplasms
Species
Mouse; rat.

Other Terms
Mastocytoma; mast cell sarcoma; malignant mastocytoma;malignant mastocytosis; systemic mastocytosis.

Pathogenesis/Cell of Origin
Develops from mast cells and their precursors present in thehematopoietic, mucosal, and/or connective tissues.

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Toxicologic Pathology 47(6)Diagnostic Features

Solitary type
A single, local, sarcomatous growth of atypical mast
cells.
Cytoplasm may be hypogranular.
Nuclei are generally bizarre, bilobed, and/or polylobed,
often with prominent nucleoli.
May be difficult to differentiate from other sarcoma
types without the use of IHC.



Systemic type













Compact nodular or sheet-like accumulation(s) ofround, spindle-shaped, or immature mast cells.
Multiple compact/sheet-like accumulations are present
in at least two organs.
Multiple organs are often involved.
In lymph nodes, located in the lymphoid tissue of theparacortex and medullary cords (not exclusively in thesinuses).
Cytoplasm is often hypogranular, but may have typicalbasophilic granules.
May have atypical bilobed or polylobed nuclei.
Destructive growth pattern, may be locally infiltrative.
Eosinophils may be associated with the mast cells.
No bone marrow involvement.
No clear inflammatory stimulus.
Considered malignant.







Comment
See comment under Mast Cell Leukemia.

In rats, tumor differentiation ranges from a predominantly normal thymic structure with medullary differentiation to a mixture of epithelial cells and lymphocytes
without medullary differentiation.
Thymoma with medullary differentiation.
 Tumor subdivided into small lobules.
 Each lobule has a central medullary area of large
pale epithelial cells surrounded by a cortex withlarge numbers of small uniform lymphocytes.
 Thymic corpuscles may be present in medullary areas.
 Medullary areas are associated with fibroustrabeculae.
 Well-circumscribed, noninvasive, may be fully or
partially encapsulated.
 Common in Wistar rats, females more than males.
Thymoma without medullary differentiation.
 Neoplastic epithelial cells are diffusely admixed
with variable numbers of lymphocytes.
 Relative proportions of epithelial cells and lymphocytes vary between tumors and within a given
tumor.
 At least partially encapsulated.
 IHC for Pan-cytokeratin helps visualize the epithelial component in lymphocyte-rich thymomas.
 Relatively rare.
Mice have a solid growth of tubules and epithelial cords
located centrally within the lobules of the thymus.
Slight local invasion beyond the confines of the tumor
capsule or thymus can occur in benign thymomas.
Types of thymoma in rodents.
 Epithelial type: Neoplastic epithelial cells constitute
more than 80% of the tumor.
 Spindeloid type: Neoplastic epithelial cells are fusiform and can mimic mesenchymal cells with a sarcomatous growth pattern.

Thymus Neoplasms
Differential Diagnoses
Thymoma, Benign (B) (Figures 158-162)
ThymusHyperplasia, epithelialSpecies
Mouse; rat.

Modifier
Epithelial; spindeloid.

Pathogenesis/Cell of Origin
Thymic epithelial cells (TEC).

Diagnostic Features

Solitary lesion.
Primarily a tumor of TEC.



Growth is present between and within the lobular structure of the thymus.
Cysts containing eosinophilic colloid are present.

Thymoma, malignantMarked invasion of adjacent tissues.

LymphomaNo neoplastic epithelial component.
Other lymphoid organs often involved.
More common in mice than thymomas.

Hyperplasia, lymphoidNo neoplastic epithelial component.
May have follicle formation.

Willard-Mack et al.


No gross enlargement of the thymus.
Very common in some mouse strains.

751


Mesothelioma, malignantNo neoplastic epithelial component.
Rare tumor with epitheloid or mesenchymal features.

Comment
The epithelial component of a thymoma is an inherent component of the tumor that is present even when the majority ofthe tumor mass is composed of lymphocytes. Special stains
for Pan-cytokeratin can be used to distinguish an early thymoma from lymphoid hyperplasia and lymphoma, especiallywhen the epithelial component is not clearly apparent. The
epithelial component is often more clearly recognizable inrats than in mice. In both species, areas within the lesion thatare densely populated by small lymphocytes can mimic EFAs.
As the proportion of epithelial cells and lymphocytes can varyconsiderably and lymphocytes are often abundant, the use of amodifier “lymphoid” is not encouraged. The incidence ofbenign thymomas varies considerably depending on strain,sex, and breeding source. Benign thymomas with medullary
differentiation are common in certain Wistar strains and rarein Sprague Dawley and Fischer rats and in many mousestrains. In humans, thymomas have been associated withmyasthenia gravis and can include myoid types with striatedmuscle fibers and neuroendocrine types with small epithelial
nests separated by thin bands of connective tissue. Largebenign thymomas may cause dyspnea and other clinical signs
due to compression of thoracic organs but this is not indicative of histologically-based malignancy.

Thymoma, Malignant (M) (Figures 163 and 164)
ThymusSpecies
Mouse; rat.

Modifier








Tumor differentiation ranges from tumors composed ofa mixture of epithelial cells and lymphocytes with orwithout medullary differentiation to tumors composed
exclusively of epithelial cells.
Epithelial cells with squamous differentiation are morecommon in malignant thymomas than in benign
thymomas.
Marked local invasion and/or metastases.
Use of a pan-cytokeratin marker for epithelial cells canbe helpful in determining malignancy in infiltrative orinvasive lymphocyte-rich thymomas.
Additional types of thymoma.
 Epithelial type: Neoplastic epithelial cells constitute
more than 80% of the tumor.
 Spindeloid type: Neoplastic epithelial cells are fusiform and can mimic mesenchymal cells with a sarcomatous growth pattern.

Differential Diagnoses
Hyperplasia, epithelialNo evidence of marked invasion of adjacent tissues.

Thymoma, benign

Differentiation between a malignant thymoma and abenign thymoma is based upon the degree of differentiation and invasive growth pattern.
A malignant thymoma shows a markedly invasive
growth pattern and/or distant metastasis.

Carcinoma, squamous cell (eg, Skin, Carcinoma, squamous cell)

Differentiation between a malignant epithelial thymoma
with squamous differentiation and a squamous cell carcinoma is difficult in the absence of a lymphocytic component and evidence of an extrathymic primarysquamous cell carcinoma.
The diagnosis is made on the anatomical localization of thetumor; thymomas must originate in the thoracic cavity.

Lymphoma Epithelial component is preexisting and not malignant.
Epithelial; spindeloid.

Other lymphoid organs are typically affected.
Lymphoma cells are large with severe atypia, whereas
lymphocytes in thymoma are small with no atypia.206,207

Mesothelioma, malignant

Pathogenesis/Cell of Origin

Mesotheliomas are rare tumors with epitheloid or
mesenchymal features.

No epithelial component.

Thymic epithelial cells.

Diagnostic Features

Thymic epithelial cells are the primary neoplastic cell
type.
Malignant thymomas often have more epithelial cells
than benign thymomas.

Comment
Malignant thymomas are relatively rare tumors in mice and
rats.

752

Toxicologic Pathology 47(6)Figure 1. Rat, mesenteric lymph node, medullary sinus. Reticular meshwork. Figure 2. Rat, lymph node, paracortex. Lymphocyte (arrow) exiting
HEV to the left by squeezing between endothelial cells. Figure 3. Mouse, spleen. Amyloid. Figure 4. Severe combined immune deficient mouse,
thymus. Aplasia/hypoplasia. Figure 5. Severe combined immune deficient mouse, lymph node. Aplasia/hypoplasia. Figure 6. Rat, lymph node,
medullary cord. Apoptosis/necrosis, lymphocyte, increased and tingible body macrophages (arrows), increased.

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753

Figure 7. Mouse, lymph node, medullary cords. Extramedullary hematopoiesis. Figure 8. Rat mesenteric lymph node. Draining neutrophils fromextranodal inflammation (contrast with extramedullary hematopoiesis in Figure 7). Figure 9. Rat, popliteal lymph node. Infiltrate, neutrophil.
Figure 10. Rat, mesenteric lymph node. Inflammation, acute. Figure 11. Rat, mesenteric lymph node. Inflammation, acute (higher magnificationof Figure 10). Figure 12. Mouse, mesenteric lymph node. Abscess.

754

Toxicologic Pathology 47(6)Figure 13. Rat, mesenteric lymph node. Inflammation, chronic. Figure 14. Rat, mesenteric lymph node. Inflammation, chronic (higher magnification of Figure 13). Figure 15. Rat, mesenteric lymph node. Inflammation, granulomatous. Figure 16. Rat, mesenteric lymph node. Inflammation,
granulomatous (higher magnification of Figure 15). Figure 17. Rat, mesenteric lymph node, medullary cord. Granuloma. Figure 18. Mouse,
spleen. Metaplasia, osseous.

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Figure 19. Rat, thymus, cortex. Mineralization. Figure 20. Mouse, mesenteric lymph node. Necrosis, lymphocyte. Figure 21. Rat, mesentericlymph node, medullary cords and sinuses. Pigment, macrophage. Figure 22. Rat, mesenteric lymph node. Vacuolation, macrophage. Figure 23.
Rat, bone marrow. Angiectasis. Abnormally dilated endothelial lined vascular spaces containing red and white blood cells. Figure 24. Rat, bonemarrow. Cellularity, decreased (atrophy), diffuse. Hematopoietic cells decreased in number and density throughout the entire the medullary
cavity.

756

Toxicologic Pathology 47(6)Figure 25. Rat, bone marrow. Cellularity, decreased, bone marrow sternum, focal. Hematopoietic cells absent or decreased within a focal area ofthe medullary cavity. Note prominent stroma and brown pigment. Figure 26. Rat, bone marrow. Fibrosis. Prominent extracellular matrix composedof collagen and fibroblasts focally displace hematopoietic cells within the medullary cavity. Figure 27. Mouse, bone marrow. Granuloma. Lesion
comprised of a central zone of epithelioid macrophages, degenerate neutrophils and necrotic debris surrounded by concentric accumulations of
mixed mononuclear cells. Figure 28. Rat, bone marrow. Necrosis/apoptosis. Dead and dying hematopoietic cells and amorphous eosinophilic
cellular debris. Dying myeloid cells exhibit pyknosis, karyorrhexis, and karolysis. Figure 29. Rat, bone marrow. Serous atrophy of fat. Hematopoietic
cells and adipocytes severely reduced in number with amorphous serous-like eosinophilic extracellular material. Figure 30. Rat, bone marrow.
Cellularity, increased, adipocyte. Increased numbers of contiguous adipocytes displace normally abundant hematopoietic cells within the bonemarrow medullary cavity. Distinguish from increased prominence of adipocytes due to decreased hematopoietic cells.

Willard-Mack et al.

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Figure 31. Rat, bone marrow. Cellularity, increased (hyperplasia). Increased numbers and density of cells of undefined hematopoietic lineages fill
the medullary cavity. Figure 32. Mouse, bone marrow. Cellularity, increased, granulocytic. Increased numbers of densely packed myeloid cells fill
the medullary space, increasing the myeloid to erythroid (M:E) ratio. Megakaryocytes are present (arrows). Abundant band cells identify
granulocytic precursor cells. Response to a peripheral abscess and demand for neutrophils. Figure 33. Rat, bone marrow. Cellularity, increased,megakaryocyte. Increased number of megakaryocytes surrounded by erythroid cells and fewer immature myeloid cells. Note megakaryocyte withmitotic figure (arrow). Figure 34. Mouse, bone marrow. Cellularity, increased, macrophage. Increased numbers of pigmented macrophages
diffusely distributed within the medullary cavity. Figure 35. Rat, bone marrow. Cellularity, increased, mast cells. Increased numbers of mast cells
diffusely distributed among normal appearing hematopoietic cells and adipocytes. Mast cells contain abundant basophilic cytoplasmic granules.
Figure 36. Sprague Dawley rat, thymus, cortex. Lymphocyte apoptosis with tingible body macrophages (arrows).

758

Toxicologic Pathology 47(6)Figure 37. F344 rat, thymus, all compartments. Cellularity decreased (atrophy), lymphocyte. Treatment-related lesion in a subchronic study.
Compare to Figure 49, concurrent control. Figure 38. Rat, thymus cortex and medulla. Corticomedullary ratio, decreased. Figure 39. Rat,
thymus. Corticomedullary ratio, increased. Figure 40. F344/N rat, thymus. Cyst, epithelial. Figure 41. F344/N rat, thymus. Cyst, epithelial.
Higher magnification of Figure 40. Note ciliated epithelial lining (arrows). Figure 42. Mouse, thymus. Ectopic tissue, parathyroid gland.

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Figure 43. F344/N rat, thyroid. Ectopic tissue, thymus. Figure 44. Harlan Sprague Dawley rat, thymus. Ectopic tissue, thyroid. Figure 45.
Severe combined immune deficient mouse, thymus, cortex, and medulla. Hypoplasia. Figure 46. Severe combined immune deficient mouse,
thymus, cortex, and medulla. Hypoplasia. Higher magnification of Figure 45. Figure 47. B6C3F1 mouse, thymus, all compartments. Involution, age
related, moderate severity. Figure 48. B6C3F1 mouse, thymus, all compartments. Involution, age related, marked severity.

760

Toxicologic Pathology 47(6)Figure 49. F344 rat, thymus, all compartments. Normal control. Figure 50. Rat thymus, cortex, and medulla. Loss of corticomedullary
distinction. Figure 51. Sprague Dawley rat, thymus, cortex. Apoptosis, lymphocyte and necrosis, lymphocyte. Mouse was given cyclophosphamide. Because of the overwhelming apoptosis, there was caspase depletion and the apoptotic phenotype eventually became necrosis. Apoptosis
and necrosis may be diagnosed separately or may be diagnosed together as a single entity (apoptosis/necrosis or apoptosis/single cell necrosis).
Figure 52. Sprague Dawley rat, thymus, cortex. Necrosis, lymphocyte. Note presence of cell debris, inflammation and minimal apoptosis (arrows).
Figure 53. Sprague Dawley rat, thymus, cortex. Tingible body macrophages, increased. Figure 54. Sprague Dawley rat, thymus, corticomedullary
junction. Tingible body macrophages. Note tingible body macrophages filled with apoptotic cell debris (arrows).

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Figure 55. Rat, thymus, medulla. Cellularity increased, epithelial cell, focal. Note presence of cysts. Figure 56. Rat, thymus, medulla. Cellularity
increased, epithelial cell, focal. Note presence of tubules. Higher magnification of Figure 55. Figure 57. Rat, thymus, cortex, and medulla.
Cellularity increased, T lymphocyte, cortex, and medulla. Rat was treated with cyclosporine. The original corticomedullary border is still locatedat its original position based on the vasculature. Figure 58. CD1 mouse, thymus, medulla. Cellularity increased, B cell. Medulla expanded bymultiple germinal centers and is often associated with age related involution. Figure 59. CD1 mouse, thymus, medulla. Cellularity increased, B
cell. Note multiple germinal centers with tingible body macrophages. Figure 60. Rat, thymus, medulla. Epithelium-free zones (arrows).

762

Toxicologic Pathology 47(6)Figure 61. Rat, thymus, medulla. Epithelium-free zones. Cytokeratin stain demonstrates absence of epithelium (arrows). Figure 62. Sprague
Dawley rat, thymus, medulla. Thymic (Hassall’s) corpuscle (arrow). Figure 63. B6129 mouse, spleen, white pulp, and red pulp. Cellularity
decreased (atrophy), diffuse with capsule and trabecula contraction. Figure 64. B6129 mouse, spleen, white pulp, marginal zone. Cellularity,
decreased, lymphocyte. Marked decrease in marginal zone lymphocytes exposes the marginal zone macrophages (arrows). Figure 65. B6129
mouse, spleen, white pulp, marginal zone. Cellularity, decreased, lymphocyte. Decrease in marginal zone lymphocytes reveals the marginal zone
macrophage population, the reticulated network of pale pink cells, and prominent marginal metallophilic macrophages that surround the follicle(arrows). Figure 66. B6129 mouse, spleen, white pulp, marginal zone. Cellularity, decreased, lymphocyte. CD45R/B220 stain illustrates marked
decrease in marginal zone lymphocytes and presence of the marginal zone macrophages associated with the marginal zone, CD45R/B220 stain.

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Figure 67. B6129 mouse, spleen, white pulp, marginal zone. Cellularity, decreased, lymphocyte. Ionized calcium-binding adapter molecule 1(IBA1) stain illustrates marked decrease of marginal zone lymphocytes and prominence of the marginal zone macrophages (3,30 -Diaminobenzidene [DAB] positive cells) associated with the marginal zone, IBA1 stain. Figure 68. B6C3F1/N mouse, spleen, red pulp. Angiectasis. Figure 69.
B6C3F1/N mouse, spleen, red pulp. Cellularity, decreased. Note wrinkling of capsule indicative of contraction due to atrophy of red pulp. There isa moderate amount of pigment within the red pulp. Figure 70. F344/N rat, spleen, red pulp. Congestion, sinusoids. Figure 71. Mouse, spleen,capsule, mouse. Contraction. The capsule and trabecula are contracted due to atrophy of the red pulp. Figure 72. B6C3F1/N mouse, mesentery.
Ectopic tissue, spleen.

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Toxicologic Pathology 47(6)Figure 73. Rat, pancreas. Ectopic tissue, spleen. Figure 74. C57BL/6 mouse, spleen, red pulp. Macrophage erythrophagocytosis. Nucleated andmature erythrocytes are engulfed by macrophages in the red pulp (arrows). Figure 75. F344/N rat, spleen, red pulp. Fibrosis, red pulp. Splenicarchitecture is distorted due to contraction of the increased fibrous connective tissue in the red pulp. Figure 76. F344/N rat, spleen, red pulp.
Fibrosis. Splenic white pulp in the upper left corner is atrophied and there is pigment associated with fibrous connective tissue replacing thenormal red pulp elements. Figure 77. CB-17 SCID mouse, spleen, red pulp. Pigment, hemosiderin, macrophages. Figure 78. Rat, spleen.
Macrophage aggregates, increased, red pulp. The animal had an infected catheter.

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Figure 79. Mouse, spleen, periarteriolar lymphoid sheaths (PALS). Cellularity, increased, plasma cells. Figure 80. CD1 mouse, spleen, white
pulp, germinal centers. Cellularity, increased (hyperplasia) and abundant tingible macrophages. Figure 81. Mouse, spleen, white pulp, germinal
centers. Cellularity, increased, lymphocyte. Figure 82. C56BL/6 mouse, spleen, white pulp, marginal zone and germinal center. Hyperplasia.
Mouse was given influenza virus 7 days prior to tissue collection. The marginal zone hyperplasia typifies a T-cell independent innate immune
reaction whereas the germinal center hyperplasia typifies a T-cell dependent adaptive humoral immune reaction. Based on the prominentcentroblasts and high mitotic index the differential diagnosis would be marginal zone (MZ) lymphoma, but marginal zone hyperplasia in conjunction
with germinal center hyperplasia is more consistent with MZ hyperplasia. Figure 83. Rat, spleen, red pulp. Cellularity, increased, adipocytes.
There are multiple foci of vacuolated adipocytes in the red pulp. Figure 84. Mouse, spleen, red pulp. Cellularity, increased, macrophage.

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Toxicologic Pathology 47(6)Figure 85. B6C3F1 mouse, spleen, red pulp. Cellularity, increased, mast cell. Figure 86. Rat, spleen, capsule. Cellularity, increased, mesothelialcell. Figure 87. B6C3F1/N mouse, spleen, red pulp. Cellularity, increased, plasma cell. Figure 88. Rat, spleen, red pulp. Cellularity, increased(hyperplasia), stromal cell. Extravasated red blood cells are interspersed among a proliferation of prominent red pulp stromal cells. Figure 89.
Mouse, spleen, red pulp. Extramedullary hematopoiesis, increased. Figure 90. Rat, spleen, red pulp. Extramedullary hematopoiesis, increased,consists of all 3 lineages with erythropoiesis being dominant.

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Figure 91. Rat, spleen, red pulp. Extramedullary hematopoiesis, increased, consists of all 3 lineages with granulopoiesis being dominant. Figure 92.
NSG (NOD scid gamma) mouse, spleen, red pulp. Extramedullary hematopoiesis, increased, megakaryocyte. Pregnancy-lactation induced.
Figure 93. Rat, mesenteric lymph node. Apoptosis, lymphocyte, increased, severe paracortex. Figure 94. Rat, mesenteric lymph node.
Apoptosis, lymphocyte increased, follicle germinal center. Figure 95. Rat, mesenteric lymph node. Cellularity, decreased, lymphocyte(atrophy), paracortex and interfollicular cortex. Note numerous inactive high endothelial venules (HEVs) crowded together in depleted andcollapsed stroma. Figure 96. Rat, mesenteric lymph node. Cellularity, decreased, paracortex. Note absence of follicles with germinal centers.

768

Toxicologic Pathology 47(6)Figure 97. Rat, mesenteric lymph node. Dilatation, sinus(es). Figure 98. Rat, cervical lymph node. Erythrocytes, intrasinusoidal, medullarysinuses. Figure 99. Mouse, axillary lymph node. Hemorrhage, paracortex, and follicles. Note relative absence of red blood cells in sinuses. Figure100. Rat, mesenteric lymph node. Aggregates, macrophage, increased. Figure 101. Rat, lymph node. Cellularity, increased, interdigitating
dendritic cells, paracortex, lymph node. Note accumulation of pale IDCs above high endothelial venules (arrows). Figure 102. Rat, lymph node.
Cellularity, increased, lymphocyte, cortex, paracortex, medullary cords.

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Figure 103. Rat, submandibular lymph node. Cellularity, increased, plasma cell, medullary cords. Figure 104. Rat, submandibular lymph node,
medullary cord. Cellularity, increased, plasma cell. Note Mott cells containing eosinophilic Russell bodies scattered among plasma cells. Figure105. Rat, mesenteric lymph node. Angiomatous hyperplasia. Figure 106. Rat, mesenteric lymph node. High endothelial venule (HEV) hypertrophy/hyperplasia, cross section. Figure 107. Rat, mesenteric lymph node. High endothelial venule (HEV) hypertrophy/hyperplasia, longitudinal
section. Note the trafficking lymphocytes. Figure 108. Rat, mesenteric lymph node. Cellularity, increased, macrophage, intrasinusoidal, medullarysinuses.

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Toxicologic Pathology 47(6)Figure 109. Rat, mediastinal lymph node. Cellularity, increased, mast cells, subcapsular, and medullary sinuses. Prominent basophilic granules.
Figure 110. Rat, mediastinal lymph node. Cellularity, increased, mast cells, medullary sinuses. Faint basophilic granules. Figure 111. Wistar rat,
Peyer’s patch jejunum, follicle, and interfollicular area. Cellularity decreased, lymphocytes. Figure 112. Wistar rat, Peyer’s patch jejunum, follicle,
and interfollicular area. Cellularity decreased, lymphocytes. Higher magnification of Figure 111. Figure 113. Wistar rat, nasal-associated lymphoidtissue (NALT). Degeneration, follicle-associated epithelium (FAE; arrows). Figure 114. Rat, Peyer’s patch jejunum, subepithelial dome. Hyalinematerial, probably within macrophages (arrows).

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Figure 115. Rat, Peyer’s patch ileum, follicle and subepithelial dome. Hyaline material, capillary wall. Figure 116. Wistar rat, mucosa-associatedlymphoid tissue (MALT) lacrimal duct. Lymphangiectasis. Figure 117. Rat, Peyer’s patch ileum, follicle, interfollicular area, subepithelial dome.
Cellularity increased, lymphocytes and macrophages. Note lymph vessels (lacteals) in villi filled with lymphocytes (arrows). Figure 118. Rat,
gut-associated lymphoid tissue (GALT; also named lymphoglandular complex) colon, all compartments. Cellularity increased, lymphocyte.
Figure 119. Rat, Peyer’s patch ileum, interfollicular area. Hypertrophy/hyperplasia high-endothelial venules (HEV). Figure 120. Mouse, tertiary
lymphoid structure in Harderian gland. High endothelial venules (HEVs) are present (arrows).

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Toxicologic Pathology 47(6)Figure 121. Wistar rat, serosa-associated lymphoid clusters (SALCs, also named fat-associated lymphoid clusters or FALCs) outside the pleura.
Well-vascularized and rich in lymphocytes. Figure 122. Mouse, liver. Erythroid leukemia. Figure 123. Mouse, liver. Erythroid leukemia. Figure124. Mouse, spleen imprint. Erythroid leukemia. Figure 125. Mouse, spleen. Megakaryocytic leukemia. Figure 126. Mouse, spleen. Megakaryocytic leukemia.

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Figure 127. Mouse, liver. Myeloid leukemia. Figure 128. Mouse, spleen. Myeloid leukemia, undifferentiated. Figure 129. Mouse, spleen.
Myeloid leukemia, moderately differentiated. Figure 130. Mouse, spleen. Lymphoblastic lymphoma, with the starry sky effect. Figure 131.
Mouse, liver. Lymphoblastic lymphoma, CD3. Figure 132. Mouse, spleen. Follicular (pleomorphic) lymphoma.

774

Toxicologic Pathology 47(6)Figure 133. Mouse, spleen. Follicular (pleomorphic) lymphoma. Figure 134. Mouse, small intestine. Follicular (pleomorphic) lymphoma.
Figure 135. Mouse, spleen. Follicular (pleomorphic) lymphoma. Figure 136. Mouse, spleen. Follicular (pleomorphic) lymphoma, CD45R(B220). Figure 137. Mouse, spleen. Immunoblastic lymphoma. Figure 138. Mouse, spleen. Lymphocytic lymphoma, diffuse.

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Figure 139. Mouse, spleen. Lymphocytic lymphoma, well differentiated. Figure 140. Mouse, spleen. Plasmacytic lymphoma. Figure 141.
Mouse, skin. Epitheliotrophic cutaneous lymphoma. Figure 142. Mouse, spleen. Marginal zone hyperplasia. Figure 143. Mouse, spleen. Marginalzone lymphoma. Figure 144. Mouse, spleen. Marginal zone lymphoma.

776

Toxicologic Pathology 47(6)Figure 145. Rat, spleen. LGL leukemia, stage 1. Figure 146. Rat, spleen. LGL leukemia, stage 2. Figure 147. Rat, spleen. LGL leukemia, stage 3.
Figure 148. Rat, liver. LGL leukemia. Figure 149. Rat, liver. LGL leukemia, crenated (darker) tumor cells. Figure 150. Rat, blood. LGL
leukemia. LGL indicates large granular lymphocyte.

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Figure 151. Rat, liver. LGL leukemia, OX-8 (CD8a). LGL indicates large granular lymphocyte. Figure 152. Mouse, liver. Histiocytic sarcoma.
Figure 153. Mouse, liver. Histiocytic sarcoma. Figure 154. Mouse, liver. Histiocytic sarcoma with multinucleated tumor cells. Figure 155.
Mouse, spleen. Mast cell tumor, malignant. Figure 156. Mouse, liver. Mast cell tumor, malignant.

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Toxicologic Pathology 47(6)Figure 157. Mouse, spleen. Mast cell tumor, malignant. Giemsa. Figure 158. Rat, thymus. Thymoma, benign. Figure 159. Rat, thymus.
Thymoma, benign with medullary differentiation. Figure 160. Rat, thymus. Thymoma, benign, epithelial. Figure 161. Rat, thymus. Thymoma,
benign, epithelial. Figure 162. Rat, thymus. Thymoma, benign, spindeloid.

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Figure 163. Rat, thymus. Thymoma, malignant, squamous. Figure 164. Rat, thymus. Thymoma, malignant, squamous.

Acknowledgments
The authors wish to express their thanks to the STP membership forcomprehensive reviews, excellent comments, and helpful edits. Photographs used in this document were either provided from coauthors, theNational Toxicology Program Archives, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina,
Drs Armando Irizarry (Eli Lilly and Company), Virginie Piccicuto(Covance, Harrogate, United Kingdom), Christopher Gray (Covance,
Harrogate, United Kingdom), Catherine Ross (Covance, Harrogate,
United Kingdom), and Jonathan Carter (Covance, Harrogate, United
Kingdom).

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect tothe research, authorship, and/or publication of this article.

Funding
The author(s) received the following financial support for the
research, authorship, and/or publication of this article: This research
was supported [in part] by the Intramural Research Program of theNIH, National Institute of Environmental Health Sciences.

ORCID iD
Susan A. Elmore

https://orcid.org/0000-0002-1680-9176

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