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Paroxysmal Nocturnal Hemoglobinuria

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Published: December 2010

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Dr. Jevremovic discusses utilization of flow cytometry to diagnose and monitor treatment for paroxysmal nocturnal hemoglobinuria, a rare form of acquired hemolytic anemia.

Presenter: Dragan Jevremovic, MD, PhD

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Dragan Jevremovic, Co-Director of the Cell Kinetics Laboratory in the Division of Hematopathology. Dr. Jevremovic will discuss utilization of flow cytometry to diagnosis and monitor treatment for paroxysmal nocturnal hemoglobinuria, a rare form of acquired hemolytic anemia.

Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria is a rare form of acquired hemolytic anemia. The red blood cells get destroyed by complement-mediated lysis which can lead to hemoglobinuria, renal failure and pulmonary hypertension, as well as symptoms such as dysphagia and lethargy. The most serious complication of PNH is venous thrombosis; it often presents in unusual sites such as hepatic, splenic and mesenteric veins. Finally, the disease is characterized by an underlying bone marrow failure presented as severe aplastic anemia in extreme cases. From this variety of symptoms, as well as from the laboratory studies, it is now understood that PNH is a hematopoietic stem cell disorder affecting all blood cell lineages.

Prevalence

PNH is a rare disease; there are about 4 to 6 thousand patients currently in the US, with the annual incidence of about 1.3 per million people. The reports on the overall 5 year mortality from the disease go as high as 35%. It is equally prevalent in men and women and affects all ages, median population being in their early 30s. PNH has a progressive course with episodes of complications and transfusion dependence, severely affecting patient's life.

Pathogenesis

The pathogenesis of PNH starts with an acquired somatic mutation in the hematopoietic stem cell. The mutation is in a gene called PIG-A which stands for X-linked phosphatidylinositol glycan complementation class A. The gene product is an enzyme that catalyzes addition of GPI (glycosyl phosphatidyl inositol) link to proteins, which enables them to be associated with the plasma membrane and expressed on the cell surface. Many proteins utilize GPI link for their expression, including CD55 and CD59 which are involved in protecting normal cells from lysis by complement.

Paroxysmal Nocturnal Hemoglobinuria

As you can see in this diagram, PNH cells lack the expression of GPI-linked proteins on the surface, resulting in susceptibility to complement-mediated lysis, activation of coagulation pathways and other effects.

Progression

Studies have shown that PIG-A mutation is not sufficient to drive survival of the clone, as cells are likely to be lysed by complement. Two proposed mechanisms for clonal progression are immunologic selection and acquisition of additional mutations; however, no definitive cause of clonal progression has been identified.

New Developments

Two recent discoveries have changed PNH practice. First, a therapeutic antibody Eculizumab, which is directed against complement has been developed for clinical use, resulting in less requirements for transfusion and improved life quality. Importantly, for the clinical practice, the use of antibody is associated with less complications, but the percentage of PNH cells often increases due to inhibition of lysis.

Second recent discovery is that small PNH clones can be detected in a significant number of patients with myelodysplastic syndrome and/or aplastic anemia. Although the significance of this finding is not firmly established, it appears that the presence of small PNH clones is associated with responsiveness to immunosuppressive therapy such as ATG and Cyclosporin A.

Role of Laboratory Testing

There are 3 clinical scenarios in which laboratory testing for PNH is appropriate. First, patients with clinical suspicion of having classical PNH should undergo testing for diagnostic purposes. Second, patients with established PNH diagnosis should be monitored regularly, at 1 year intervals if stable, or more frequently, particularly if on therapy. Finally, patients with suspected myelodysplasia and aplastic anemia should undergo testing for detection of small clones; small clones in aplastic anemia should be followed regularly as these clones may progress into classical PNH.

International PNH Group Recommendations: Who to Test?

International PNH group made recommendations on which patients should be tested and these are displayed here. They include patients with signs and symptoms of classical PNH (such as venous thrombosis, hemoglobinuria, Coombs-negative hemolytic anemia and dysphagia with elevated LDH), as well as patients with aplastic anemia and myelodysplasia, as already discussed.

Diagnosis

How to test for PNH? The Ham test for sensitivity of red blood cells to complement has only a historical importance these days, as it is laborious, and lacks sensitivity and specificity. Since early 1990’s flow cytometry immunophenotyping has been the mainstay of PNH testing. Most tests were designed to look for the loss of CD59 and CD55 on RBC, loss of CD59 and fluorescent aerolysin binding to granulocytes, and loss of CD14 on monocytes. Fluorescent aerolysin or FLARE, is a modified Pseudomonas protein, that binds to GPI link.

International PNH Group Recommendation: How to Test?

Besides recommendations who to test, International PNH group came up with recommendations on how to perform flow cytometry testing. The main goal was to try to standardize testing amongst different laboratories. Some of the highlights include use of at least 2 different antibodies against 2 different surface proteins on 2 different cell populations. Also it is recommended to use antibodies for gating, instead of forward and side scatter, as well as minimum sensitivity of 1%.

With these recommendations in mind, we have decided to update our PNH test.

Normal

This slide shows flow cytometry dot-plots of a normal individual without a PNH clone. Without going into details regarding gating strategies, I just want to emphasize that the different cell populations are much more cleanly separated and that we collect many more events, increasing our sensitivity to 0.01% for RBCs and granulocytes and 0.05% for monocytes. Important new element is also that we are able to separate type II RBCs which although have partial loss of GPI-linked proteins on the cell surface, are not susceptible to hemolysis.

New WBC PNH

This slide shows a relatively large PNH clone in granulocytes and monocytes. Again, separation of different cell populations is much better visible and enables easier interpretation.

Sensitivity and Reference Interval

As a part of our validation, we analyzed 108 normal specimens, and established cut-offs for finding rare events in normal individuals versus true PNH population.

Interpretation and Reporting

Based on the data from normal donors, we established reference ranges which are listed here. They are: 0.01% for type III or deficient RBCs and granulocytes, and 0.05 for monocytes. The reason for decreased monocyte sensitivity is in a relatively small number of monocytes routinely available for testing. In addition, there are situations in which we are not able to collect enough granulocytes to reach desired sensitivity, usually because of profound neutropenia of the patient. In these cases, we would indicate in our report that the specimen has low WBC count which may impact the reached sensitivity.

PNH—Summary

In summary, PNH is a rare acquired disorder of hematopoietic cells. It is characterized by hemolysis and many other effects, of which thrombosis is the most serious consequence. Small clones of PNH have been detected in other hematopoietic disorders, such as myelodysplastic syndrome and aplastic anemia. We are able to provide sensitive testing for both early detection and therapeutic monitoring of patients with PNH clones.


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