In February 1999 a 29 year old female attended the Haematology Clinic at the Prince of Wales Hospital. She had a history of anaemia, neutropenia and thrombocytopenia. She was passing dark urine. A full blood count, reticulocyte count and coagulation studies were performed.
The results were as follows:
| Hb 66 g/L | Retic count 16.20% (absolute count 356.40 x 109/L) | |
| MCV 113.6 fL | WBC 3.1 x 109/L | Platelets 55 x 109/L |
| PT 41.5 sec | INR 4.2 | APTT 45.7 sec |
| Fibrinogen 5.6 g/L | D-DIMER LIA <0.19 mg/L | |
A urinary haemosiderin was performed which was strongly positive.
A clinical chemistry profile was also performed:
The LDH was raised at 3436 U/L.
The test results, together with the clinical symptoms of the patient indicated that she had a severe haemolytic anaemia suggestive of Paroxysmal Nocturnal Haemoglobinuria (PNH). A Ham’s test was performed. This is an acidified serum test which detects a population of red cells with sensitivity to complement-mediated lysis. The Ham’s test was positive. A diagnosis of PNH was confirmed on the above patient.
PNH is a rare, acquired, clonal disorder of the bone marrow characterised by a total or partial lack of proteins normally attached to the haemopoietic cell membrane by the glycosylphosphatidylinositol (GPI) anchor. This defect is due to a somatic mutation of the phosphatidylinositol glycan class A (PIG-A) gene on the X chromosome which encodes for a protein needed for the synthesis of GPI anchor. Without GPI anchor, an essential group of membrane proteins are either reduced or absent in all haemopoietic cell lines. These proteins include complement-regulating surface proteins namely decay-accelerating factor (DAF) or CD55, membrane inhibitor of reactive lysis protein (MIRL) or CD59 and the homologous restriction factor (HRF) or CD8 binding protein. All of these proteins interact with complement, particularly C3b and C4b, protecting cells against lysis. In their absence, haemopoietic cells are exposed to complement leading to uncontrolled amplification of the complement cascade and destruction of the red cell membrane leading to intravascular haemolysis.
Figure 1a: Peripheral blood film showing a raised MCV secondary to an increased reticulocyte response ( x 1000)
Figure 1b:
Strongly positive urinary haemosiderin ( x 400)
PNH cells are classified as PNH Type I, Type II and Type III cells. PNH Type I cells have normal levels of CD59; PNH Type II cells have reduced levels while PNH Type III cells completely lack CD59 expression.
Patients with PNH present with symptoms of haemolysis, namely, low haemoglobin, raised reticulocyte count and raised lactate dehydrogenase (LDH). The DAT is negative as there are no antibodies involved. They also present with isolated or multiple cytopenias and/or a history of thrombosis.
Note that in 1999 the Ham’s test was used to make the diagnosis of PNH. Today flow cytometry must be used to make that diagnosis. The presence and size of both red blood cell and granulocyte clones as defined by reduced or absent GPI-linked proteins, must be determined. The Australian Flow Cytometry Group has issued guidelines for the most appropriate flow testing (AFCG Guidelines at www.afcg.org.au). The guidelines recommend that CD59 expression should be determined on red blood cells and that for granulocytes and monocytes the first step is a lineage gate (eg CD33/SS) followed by expression of CD24/FLAER (granulocytes) and CD14/FLAER (monocytes).
Until recently, the treatment for PNH was supportive. Patients were managed with iron and folate supplementation. They were also transfused when appropriate. Transfused red cells do not lack GPI anchor thus are not vulnerable to complement lysis. About 50% of patients responded to corticosteroids. Allogeneic stem cell or bone marrow transplantation was and still is the only cure for PNH.
In March 2007 the use of eculizumab (Soliris) was approved by the FDA. Eculizumab is a monoclonal antibody that targets and prevents cleavage of the terminal complement protein C5. In so doing it inhibits other complement components thus preventing haemolysis. Eculizumab has given patients with PNH a significant improvement in quality of life as well as a marked reduction in the frequency of thrombotic episodes. Many patients are no longer transfusion dependent. Long term treatment with eculizumab is well tolerated however it comes with significant cost consideration.
The above patient was commenced on eculizumab in 2009. It is now September 2010 and she is 39 years old. She has had a remarkable improvement in her quality of life whilst on eculizumab, so much so that she delivered a healthy baby girl on the 23rd of September. At last a Haematology Update with a happy ending!
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Figure 2.
A. CD59 expression on red blood cells of the patient in 2010 showing a Type III PNH clone of 56.5%, a small Type II clone (8.3%) and a clone of Type I red
B. A PNH monocyte clone with reduced CD14 and FLAER expression – about 95% of monocytes.
C. A PNH granulocyte clone with reduced CD24 and FLAER expression – about 95% of granulocytes.
