# Presentation: 161258

## Paroxysmal Nocturnal Hemoglobinuria (PNH)

**PNH is an acquired chronic hemolytic anemia which arises from a somatic mutation in a hematopoietic stem cell.  Most hematopoitic cell lines may be affected by the intrinsic membrane defect. This defect renders the red cells highly susceptible to complement mediated lysis resulting in the characteristic hemolysis.**

## Paroxysmal Nocturnal Hemoglobinuria (PNH)

- History
- Epidemiology
- Clinical Features
- Relationship to Aplastic Anemia
- other diseases
- Pathogenesis
- Laboratory Diagnosis
- Therapy

**Topics to be considered**

##  History

_**Investigator   Year                            Contribution**_
**Gull    ****	****	****1866     Described nocturnal and paroxysmal nature of ****	****	****	****	****“intermittent haematinuria” in a young ****	****tanner.**
**Strubing         ****	****1882****	****Distinguished PNH from paroxysmal cold ****	****	****	****	****haemoglobinuria and march haemoglobinuria. ****	****	****	****	****Attributed the problem to the red cells. **
**van den Burgh  ****	****1911 ****	****Red cells lysed in acidified serum. Suggested a role ****	****	****	****for complement.**
**Enneking         ****	****1928    ****	****Coined the name “paroxysmal nocturnal ****	****	****	****	****haemoglobinuria”.**
**Marchiafava ****	****1928- ****	****Described perpetual hemosiderinemia in absence of **
**and Micheli ****	****1931****	****hemolysis. Their names became eponymous for PNH ****	****	****	****in Europe.**
**Ham                  ****	****1937-****	****Identified the role of complement in lysis of PNH red ****	****	****1939****	****cells. Developed the acidified serum test, also called ****	****	****	****the Ham test, which is still used to diagnose PNH. ****	****	****	****Demonstrated that only a portion of PNH red cells are ****	****	****	****abnormally sensitive to complement.**
**Davitz****	****	****1986****	****Suggests defect in membrane protein anchoring ****	****	****	****system responsible**
**Hall & Rosse****	****1996****	****Flow cytometry for the diagnosis of PNH**

## Epidemiology

- Rare disease -
- frequency unknown
- thought to be on the same order as aplastic anemia (2-6 per million)
- Median age at diagnosis
- ~ 35 yrs
- PNH reported at extremes of age
- Female:Male ratio = 1.2:1.0
- No increased risk of PNH in patient relatives
- Median Survival after diagnosis ~ 10-15 yrs

## Clinical Features

- Major symptoms (Hemolysis, Cytopenia, and tendency to thrombosis)
- chronic hemolysis with acute exacerbations (hallmark)
- most patient at some stage
- only 1/3 exhibit hemolysis at diagnosis
- Recurrent attacks of intravascular hemolysis are usually associated with;
- hemoglobinuria
- abdominal pain
- dysphagia

## cytopenia (varying severity)
isolated subclinical thrombocytopenia
classical severe aplastic anemia
tendency to thrombosis
venous thrombosis (40%) of patients, main cause of morbidity
Variable expression of above often causes considerable delay in the diagnosis
Major cause of death
venous thrombosis
complications from progressive pancytopenia

- isolated subclinical thrombocytopenia
- classical severe aplastic anemia
- tendency to thrombosis
- venous thrombosis (40%) of patients, main cause of morbidity
- Variable expression of above often causes considerable delay in the diagnosis
- Major cause of death
- venous thrombosis
- complications from progressive pancytopenia

- Clinical Features

## 25% of PNH patients survive >25 years - one half of these go on to spontaneous remission
Remission patients
hematological values revert to normal
no PHN rbcs or granulocytes detected
PNH lymphocytes - still detected but no clinical consequence
Higher incidence of acute leukemia (6%)
“preleukemic condition” most likely bone marrow failure not PNH 

- Remission patients
- hematological values revert to normal
- no PHN rbcs or granulocytes detected
- PNH lymphocytes - still detected but no clinical consequence
- Higher incidence of acute leukemia (6%)
- “preleukemic condition” most likely bone marrow failure not PNH

- Clinical Features - Long term

## AA described as pancytopenia with nonfunctioning bone marrow. Cytopenia in one or all cell lineages also common to PNH
High percentage of patients with AA develop clinical PNH or have lab evidence of PNH abnormality at some point (52%)
Supports the theory that bone marrow failure supports the abnormal PNH cells - more later

- High percentage of patients with AA develop clinical PNH or have lab evidence of PNH abnormality at some point (52%)
- Supports the theory that bone marrow failure supports the abnormal PNH cells - more later

- Clinical Features - Relationship to aplastic anemia (AA)

## Pathogenesis - The Defect

- Defect - Somatic mutation of *PIG-A* gene (phosphatidylinositol glycan complementation group A)  located on the X chromosome in a clone of a hematopoietic stem cell
- >100 mutations in *PIG - A* gene known in PNH
- The mutations (mostly deletions or insertions) generally result in stop codons - yielding truncated proteins which may be non or partially functional - explains heterogeneity seen in PNH

## PIG - A gene codes for 60 kDa protein glycosyltransferase which effects the first step in the synthesis of the glycolipid GPI anchor (glycosylphosphatidylinositol). Results in clones lacking GPI anchor - in turn, attached proteins 

**Pathogenesis - The Defect**

**GPI Anchor**

***PIG - A***** protein**

## PNH blood cells deficient in GPI anchor lack membrane proteins linked via the anchor
Membrane proteins w/o anchor degraded in ER 
Severity & size of deficiency - variable - clinical/diagnostic implications
GPI anchor highly conserved in all eukaryotic cells
Variant surface proteins of Trypanosomes - GPI linked
Shed by cleavage of GPI anchor - immune system avoid
Swapping GPI linked proteins - CD55 complement resistance - Schistosoma mansoni
In Humans
signal transduction, co-receptors
advantage to this type of anchor?

- Membrane proteins w/o anchor degraded in ER
- Severity & size of deficiency - variable - clinical/diagnostic implications
- GPI anchor highly conserved in all eukaryotic cells
- Variant surface proteins of Trypanosomes - GPI linked
- Shed by cleavage of GPI anchor - immune system avoid
- Swapping GPI linked proteins - CD55 complement resistance - *Schistosoma mansoni*
- In Humans
- signal transduction, co-receptors
- advantage to this type of anchor?

**Pathogenesis - The Defect****GPI Anchor deficiency**

## Proteins anchored by GPI Anchorand

**Surface Proteins Missing on PNH Blood Cells**
_**Antigen                                                                 Expression Pattern**_
_**Enzymes**_
**Acetylcholinesterase (AchE)               ****	****	****Red blood cells**
**Ecto-5'-nucleotidase (CD73)       ****	****	****Some B- and T-lymphocytes**
**Neutrophil alkaline phosphatase(NAP) ****	****	****Neutrophils**
**ADP-rybosyl transferase     ****	****	****	****Some T-lymphs, Neutrophils**
_**Adhesion molecules**_
**Blast-I/CD48                              ****	****	****	****Lymphocytes**
**Lymphocyte function- **
**associated antigen-3(LFA-3 or CD58) ****	****	****All blood cells**
**CD66b****	****	****	****	****	****	****Neutrophils**
_**Complement regulating surface proteins**_
**Decay accelerating factor (DAF or CD55)            ****	****All blood cells**
**Homologous restriction factor,****	****	****	**
**Membrance inhibitor of reactive lysis ****	****	****All blood cells**
**(MIRL or CD59)**

## Surface Proteins Missing on PNH Blood Cells

_**Antigen                                                                 Expression Pattern**_
_**Receptors**_
**Fc-******** receptor III (Fc ******** Rlll or CD16)        ****	****Neutrophils, NK-cells, macrophages,**
**	****	****	****	****	****some T-lymphocytes**
**Monocyte differentiation antigen ****	****Monocytes, macrophages**
**(CD14)**
**Urokinase-type Plasminogen ****	****	****Monocytes, granulocytes**
**Activator Receptor (u-PAR, CD87)**
_**Blood group antigens**_
**Comer antigens (DAF)                            ****	****Red blood cells**
**Yt antigens (AchE)                           ****	****Red blood cells**
**Holley Gregory antigen                    ****	****Red blood cells**
**John Milton Hagen antigen (JMH)    ****	****Red blood cells, lymphocytes**
**Dombrock reside                        ****	****	****Red blood cells**
_**Neutrophil antigens**_
**NB1/NB2                                           ****	****Neutrophils**

## Surface Proteins Missing on PNH Blood Cells

_**Antigen                                                                 Expression Pattern**_
_**Other surface proteins **_
_**of unknown functions**_
**CAMPATH-1 antigen (CDw52)                      ****	****Lymphocytes, monocytes**
**CD24 ****	****	****	****	****	****	****B-lymphocytes, Neutrophils, **
**	****	****	****	****	****	****eosinophils**
**p5O-80****	****	****	****	****	****	****Neutrophils**
**GP500                                        ****	****	****	****Platelets**
**GPI75                         ****	****	****	****	****Platelets**

## Pathogenesis - Functional consequences of lack of GPI linked proteins

*In vivo* function of many of these membrane proteins not fully understood
- However, CD55 and CD59 functions are well known
- CD55 (decay accelerating factor) inhibits the formation or destabilizes complement C3 convertase (C4bC2a)
- CD59 (membrane inhibitor of reactive lysis, protectin, homologous restriction factor) Protects the membrane from attack by the C5-C9 complex
- Inherited absences of both proteins in humans have been described
- Most inherited deficiencies of CD55 - no distinct clinical hemolytic syndrome
- Inherited absence of CD59 - produces a clinical disease similar to PNH with hemolysis and recurrent thrombotic events

## Mechanism for hemolysis in PNH via lack of CD59

**Mechanism for hemolysis in PNH via lack of CD59**

**(CD59)**

**(CD59)**

## Pathogenesis - Clonal evolution and cellular selection

- Expansion of abnormal hematopoietic stem cell required for PNH disease expression
- Theories for expansion
- Blood cells lacking GPI-linked proteins have intrinsic ability to grow abnormally fast
*In vitro*  growth studies demonstrate that there are no differences in growth between normal progenitors and PNH phenotype progenitors
*In vivo*  - mice deficient for *PIG -A *gene also demonstrates no growth advantage to repopulation of BM.
- Additional environmental factors exert selective pressure in favor of expansion of GPI anchor deficient blood cells
- PNH hematopoitic cells perferentially engraft SCID mice compared to phenotypically hematopoitic cells
- Close association with AA - PNH hematopoitic cells cells may be more resistant to the IS than normal hematopoitic cells.
- Evidence in AA is that the decrease in hematopoitic cells is due to increased apoptosis via cytotoxic T cells by direct cell contact or cytokines (escape via deficiency in GPI linked protein???)

## Laboratory Evaluation of PNH

- Acidified Serum Test (Ham Test 1939)
- Acidified serum activates alternative complement pathway resulting in lysis of patient’s rbcs
- May be positive in congenitial dyserythropoietic anemia
- Still in use today
- Sucrose Hemolysis Test (1970)
- 10% sucrose provides low ionic strength which promotes complement binding resulting in lysis of patient’s rbcs
- May be positive in megaloblastic anemia, autoimmune hemolytic anemia, others
- Less specific than Ham test

## PNH Diagnosis by Flow Cytometry (1986)
Considered method of choice for diagnosis of PNH (1996) 
Detects actual PNH clones lacking GPI anchored proteins
More sensitive and specific than Ham and sucrose hemolysis test

- Considered method of choice for diagnosis of PNH (1996)
- Detects actual PNH clones lacking GPI anchored proteins
- More sensitive and specific than Ham and sucrose hemolysis test

**Laboratory Evaluation of PNH**

## PNH Diagnosis by Flow Cytometry

_**Antigen**__	__**Cell Lineage**__	__**Function**_
- CD14		monocytes		LPS receptor, MDF
- CD16		neutrophils		FcIII receptor
- CD24		neutrophils		B-cell differentiation marker
- CD55		all lineages		DAF
- CD58		all lineages		possible adhesion
- CD59		all lineages		MIRL, HRF, protectin
- CD66b		neutrophils		CEA-related glycoprotein

- Of the long list of GPI anchored protein, monoclonal antibodies to the following antigens have been used in the diagnosis of PNH
- The most useful Abs are to CD14, 16, 55, 59, and 66.  Are all required?  Probably not - more studies needed

## Antigen expression is generally categorized into three antigen density groups
type I Normal Ag expression
type II Intermediate Ag expression
type III No Ag expression
Patient samples that demonstrate cell populations with diminished or absent GPI-linked proteins (Type II or III cells) with multiple antibodies are considered to be consistent with PNH.
Should examine multiple lineages (ie granulocytes & monocytes)

- type I Normal Ag expression
- type II Intermediate Ag expression
- type III No Ag expression
- Patient samples that demonstrate cell populations with diminished or absent GPI-linked proteins (Type II or III cells) with multiple antibodies are considered to be consistent with PNH.
- Should examine multiple lineages (ie granulocytes & monocytes)

**PNH Diagnosis by Flow Cytometry**

## PNH Diagnosis by Flow Cytometry

- Examples of variable GPI linked CD59 expression on granulocytes on four PNH patients

## PNH Diagnosis by Flow Cytometry

- Example of variable expression of several GPI linked Ags on several lineages

- From Purdue Cytometry CD-ROM vol3 97

## Flow Cytometry is method of choice but only supportive for/against diagnosis
More studies are needed to better define whether the type (I, II, or III), cell lineage, and size of thecirculating clone can provide additional prognosticinformation.
Theoretically - should be very valuable 

- More studies are needed to better define whether the type (I, II, or III), cell lineage, and size of thecirculating clone can provide additional prognosticinformation.
- Theoretically - should be very valuable

**PNH Diagnosis by Flow Cytometry**

## Therapy

- Bone Marrow Transplantation
- Only curative treatment
- chronic condition (possiblity of spontaneous remission) - BMT should be avoided
- Immunosuppressive therapy
- Antilymphocyte globulin &/or cyclosporine A
- Does not alter proportion of PNH hemopoiesis
- Steroids - experimental - controlled studies ??
- Growth Factors
- Some improvement
- no evidence that normal clones respond better than PNH clones