Flow cytometry (FCM) is a complex process to examine blood, body fluids,
cerebrospinal fluid (CSF), bone marrow, lymph node, tonsil, spleen and other
solid tissues. The use of peripheral blood and fine needle aspirate material
avoids more invasive procedures for diagnosis.  
  
A flow cytometer evaluates the physical and/or chemical characteristics of
single cells as the cells pass individually in a fluid stream through a
measuring device. Surface receptors, intracellular molecules, and
deoxyribonucleic acid (DNA) bind with fluorescent dyes that allow detection
and evaluation.  
  
When light of 1 wavelength excites electrons of certain chemicals to energy
levels above their ground state and upon return to ground state emits light of
a longer wavelength, fluorescence is produced. A flow cytometer detects cell
characteristics by measuring the fluorescence produced by fluorochromes
conjugated either directly with cell components or conjugated to antibodies
directed against cell components.  
  
 **_Indications_**  
  
 **_Cytopenias and Hypercellular Hematolymphoid Disorders_**  
  
Hematolymphoid neoplasia can present with cytopenias (anemia, leukopenia
and/or thrombocytopenia) or elevated leukocyte counts. If medical review and
preliminary laboratory testing fails to reveal a cause, bone marrow aspiration
and biopsy are indicated to rule out an infiltrative process or a stem cell
disorder. FCM is essential to evaluate hematolymphoid lineages. Although
anemia commonly occurs in nonneoplastic diseases, anemia alone should not
automatically trigger FCM.  
  
FCM may be useful in hypercellular hematolymphoid disorders to differentiate
reactive conditions from neoplastic conditions. In the absence of blasts,
neutrophilic leukocytosis is not generally an indication for FCM. Isolated
polycythemia and basophilia are not sufficient to warrant FCM.  
  
 **_Lymphomas_**  
  
In the current World Health Organization (WHO) classification, all non-Hodgkin
lymphomas (NHLs) are distinct clinicopathologic entities defined by their
clinical features, morpholology, immunophenotype and, where appropriate, their
genetic abnormalities. Immunophenotyping by FCM allows multiparameter
evaluation of single cells and the ability to work on very small samples.  
  
Most new cases of suspected NHL undergo initial immunophenotypic analysis as
part of the routine handling of a specimen. A standard lymphoma panel is
designed to identify abnormal populations of B cells, T cells and/or NK cells.
A standard lymphoma panel might include a combination of markers from the
following categories: T cells (CD2, CD3, CD4, CD5, CD7, CD8); B cells (CD19,
CD20, CD23); Kappa and Lambda surface immunoglobulins light chains; plasma
cells (CD38 and CD138); CALLA (CD10); CD45; CD56: FMC-7, CD103, CD11c, CD13,
CD14, CD15, CD16 and CD34.  
  
The immunophenotypes of lymphomas are widely known and FCM allows appropriate
classification of most cases. However, atypical patterns occur and pose
significant diagnostic difficulties where aberrant antigen expression patterns
must be reconciled with morphology. Additional markers may be required to
characterize the abnormal population of cells including markers of immature
cells (HLA-DR), B cells (CD22) and myeloid cells (CD14, CD15, CD33, CD64,
CD117).  
  
**_Acute Leukemia_**  
  
The diagnosis and management of acute leukemia depends on the detection,
identification and characterization of leukemic cells. The identification of
leukemic cells is straightforward in most occasions. However, each acute
leukemia subgroup has heterogeneous biologic characteristics, many of which
are associated with a different response to therapy.  
  
As part of a routine diagnostic workup, most suspected acute leukemia cases
undergo initial multiparameter immunophenotypic analysis, combined with
morphology, cytochemistry, cytogenetics, and molecular biology.  
  
A standard acute leukemia FCM panel is designed to determine whether leukemic
blasts are of myeloid or lymphoid origin, and then to further classify the
neoplastic cells (myeloid blasts, B lymphoblasts, abnormal promyelocytes,
monoblasts, etc). An acute leukemia panel might include a combination of cell
markers from the following categories: stem cell lineage (CD34), immature cell
lineage (HLA-DR, CD 10); T cell (CD2, CD3, CD4, CD5, CD7 and CD8); B cell
(CD19, CD20); myeloid cell (CD13, CD14, CD15, CD33, CD64 and CD117); CD38,
CD45, and CD56.  
  
When the routine panel is insufficient to characterize the leukemic cells,
additional antibodies including erythroid markers (CD71 and glycophorin A),
megakaryocytic markers (CD41, CD61) or cytoplasmic markers may be indicated.  
  
 **_Chronic Lymphocytic Leukemia (CLL) & Other Chronic Lymphoproliferative
Diseases (CLPD)_**  
  
The history, physical exam (lymphadenopathy, splenomegaly and/or
hepatomegaly), laboratory findings (lymphocytosis, granulocytopenia, anemia,
thrombocytopenia), and lymphocyte morphology are suggestive of CLL. The
diagnosis is established by paradoxical co-expression of CD5 on peripheral
lymphocytes that express B cell markers (CD19, CD20, CD21 and CD23) with Kappa
or Lambda immunoglobulin light chain restriction. Additional markers such as
CD38 and ZAP70 may provide important prognostic information.  
  
FCM can distinguish CLL, the peripheral counterpart of small lymphocytic
lymphoma, often diagnosed in lymph node biopsies, from other indolent
lymphocytic malignancies including prolymphocytic leukemia, Waldenstrom's
macroglobulinemia, leukemic phase of lymphomas, hairy cell leukemia, T-cell
CLL, adult T-cell leukemia, large granulocytic leukemia and cutaneous T-cell
lymphoma and natural killer (NK) disorders including KIR expression.  
  
 **_Plasma Cell Disorders_**  
  
Plasma cell disorders are often identified through a combination of clinical,
laboratory studies (urine or serum gamma globulins), morphologic, and
radiologic findings. FCM immunophenotyping is useful to identify abnormal
plasma cells, and the distinction between lymphoid and plasma cell neoplasms,
and between reactive plasma cells and neoplastic plasma cells.  
  
The initial FCM workup for a plasma cell disorder may include the basic
lymphoma panel markers with additional markers such as CD28 and CD117.  
  
**_Myelodysplastic Syndromes (MDS)_**  
  
The gold standard for an MDS diagnosis is assessment of bone marrow smears for
dysplastic changes. FCM may assist in MDS determination through the
identification of abnormal maturing myeloid cells. An abnormal phenotype by
FCM is a minimal diagnostic MDS criteria to establish a definitive diagnosis.  
  
MDS has a definite risk and rate of progression to acute leukemia. Standard
FCM leukemia panels are indicated to evaluate progression and onset of
leukemia.  
  
**_Chronic Myeloproliferative Disorders (CMPD)_**  
  
Although genetic (Philadelphia chromosome and BCR/ABL) and molecular studies
(JAK 2) are the accepted cornerstone for the identification and classification
of CMPDs, FCM may assist in the distinction from reactive hematopoietic
proliferations and is important in the enumeration of blasts in the
distinction from acute leukemia and an accelerated phase of CMPD.  
  
CMPD also has a definite risk and rate of progression to acute leukemia.
Standard FCM leukemia panels are indicated to evaluate progression and onset
of leukemia.  
  
 **_Mast Cell Neoplasms_**  
  
Mast cell neoplasms are uncommon disorders. Mast cells coexpress multiple
markers including CD9, CD33, CD45, CD68, CD117, but also lack several
myelomonocytic antigens including CD14, CD15, CD16 and most T- and B- cells
antigens. Neoplastic mast cells have a similar antigen profile, but also can
coexpress CD2 and CD25, which help in distinguishing malignant mast cells from
mastocytosis.  
  
**_Paroxysmal Nocturnal Hemoglobinuria (PNH)_**  
  
PNH is a rare clonal hematopoietic disorder of stem cells. This condition is
caused by genetic mutation that results in the absence of over a dozen surface
antigens on red and white blood cells. FCM can diagnose PNH by assessing both
the red and white blood cells for the absence of these antigens.  
  
 **_Minimal Residual Disease (MRD)_**  
  
FCM analysis for MRD must identify phenotypic features characteristic of the
disease of interest. The MRD flow analysis should not rely on an exact match
between the phenotype of the residual disease and the original diagnostic
specimen because phenotypes can change over time and with treatment. The
antibody combinations should be chosen to maximize detection of disease, limit
the impact of phenotypic variation, and permit detection of disease following
antibody directed therapy.  
  
 ** _Human Immunodeficiency Virus (HIV) Infection_**  
  
HIV-1 infection causes significant changes in the number of CD4 and CD8
positive lymphocytes. CD4 count falls roughly 30% while CD8 count increases
within 6 months after seroconversion, causing a decrease in the CD4/CD8 ratio  
  
Following HIV-1 diagnosis, FCM should include enumeration of mature T cells
(CD3), helper T cells (CD4) and suppressor T cells (CD8) to ensure all major T
cell subsets are accounted for (the sum of helper CD4 and suppressor CD8 T
cells is roughly close to the total number of CD3 positive T cells). This
ensures that the absolute CD4 is not artificially decreased due to sample
degradation or other artifact.  
  
A WBC count with differential also needs to be performed to calculate the
absolute CD4 count (absolute lymphocyte count times CD4%).  
  
**_Organ Transplants_**  
  
In order to differentiate early rejection, immunosuppressive therapy toxicity
or infection, FCM may be indicated to monitor postoperative organ transplants.
CD3 is useful to monitor the effectiveness of certain immunosuppressive
therapies. When the transplant patient demonstrates symptoms for the above
conditions, repeated analysis may be required.  
  
**_DNA Analysis_**  
  
 _**Carcinoma, Non-hematolymphoid Tumors**_  
  
DNA analysis of tumor for ploidy and percent S-phase cells may be necessary
for a few selective patients with carcinomas. When the obtained prognostic
information will affect treatment decisions in patients with low stage
(localized) disease, FCM results are useful.  
  
 _**Molar Pregnancy**_  
  
FCM is useful to evaluate molar and partial molar pregnancies. Using a method
to quantify DNA, similar to that used for evaluation of carcinomas, partial
moles (triploid), can be distinguished from normal placenta and complete molar
(diploid) pregnancies.  
  
**_Primary Immunodeficiencies (PIDS)_**  
  
PIDs are rare disorders that reflect inherited abnormalities in the
development and maturation of cells responsible for immune function. More than
120 inherited immunodeficiency disorders are currently recognized. Affected
individuals are prone to repeated infections, allergies, autoimmune disorders,
and malignancies. Diagnosis typically occurs at an early age.  
  
FCM may be indicated for diagnostic purposes and is usually limited to T (CD3,
CD4, CD8), B (CD20) and NK cell (CD56) markers. Additional disease specific
markers may be indicated.  
  
**_Primary Platelet Disorders, Non-neoplastic_**  
  
FCM is used for platelet analysis in quantitative and qualitative disorders
such as Glanzmann Thrombasthenia (GT) and Bernard-Soulier Disease (B-S). GT is
a rare inherited or acquired platelet disorder. Hereditary GT is defined by
platelets with decreased expression or absence of the GPIIa/GPIIIb receptor.
This receptor is responsible for the initial platelet plug at the site of
endothelial injury. Absence of the receptor may result in increased bleeding.  
  
Acquired GT is likely an autoimmune phenomenon with the presence of
GPIIb/GPIIIa blocking antibodies. FCM may be used to determine the functional
effect and identity the molecular targets of these antibodies.  
  
B-S is another rare inherited disorder that prevents the initial binding of
platelets at the site of endothelial injury by absence of or presence of
abnormal surface GPIa/V/IX receptor. Abnormalities of this receptor prevent
attachment of platelets to subendothelial or free von Willebrand's factor with
subsequent tendency to bleed.  
  
FCM may be used to measure antibodies directed at specific loci of the
GPIa/V/IX receptor, which include GPIb (CD42b), GPIX (CD42a), and GPV (CD42d).
FCM is also used to assess the size of platelets in the initial evaluation of
B-S disease. In B-S disease, platelets are generally larger than normal. FCM
can distinguish B-S platelets from fragmented RBCs and debris by antibodies
directed to the GPIb/IX/V receptor.  
  
 **_Red Cell and White Cell Disorders, Non-neoplastic_**  
  
FCM is a valuable tool to establish abnormal or defective red blood cell,
leukocyte and lymphocyte surface receptors, transmembrane molecules, and
intracellular DNA. It may be used in acquired and congenital red cell
conditions such as in quantifying fetomaternal hemorrhage and hereditary
spherocytosis, hereditary elliptocytosis, and hereditary persistence of fetal
hemoglobin in the context of compound hemoglobinopathy syndromes.  
  
FCM is a sensitive and specific method to identify leukocyte receptor
abnormalities for the diagnosis of chronic granulomatous disease and CD11b
deficiency. It is an efficient method to identify lymphocytes HLA B27
associated with uveitis, ankylosing spondylitis, Reiter's syndrome and
sacroiliitis.  
  
 **_Limitations:_**  
  
Since FCM immunophenotypes for most common lymphomas and leukemias are well
characterized, this A/B Medicare Administrative Contractor (MAC) does **NOT**
consider it "reasonable and necessary" to perform more than 24 markers in a
panel. When atypical or unusual FCM results are obtained, the selective
addition of more markers may be indicated.  
  
The flow report must document the specific indication for each marker over the
24 marker limit.  
  
The FCM report must document the specific indication for **_each_** marker
over the 24-marker limit. FCM reports without clear justification for each
marker over 24 will be denied.