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 Table of Contents  
REVIEW ARTICLE
Year : 2014  |  Volume : 5  |  Issue : 2  |  Page : 37-44

Paroxysmal nocturnal hemoglobinuria: Diagnosis and management protocol


1 Department of Hematology and Oncology, King Saud University Medical City, Riyadh, Saudi Arabia
2 Departments of Hematology and Pathology, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
3 Departments of Oncology, King Abdulaziz Medical City, Jeddah, Saudi Arabia
4 Department of Hematology, Dubai Hospital, Dubai, Saudi Arabia
5 Department of Oncology, King Abdulaziz Medical City, Riyadh, Saudi Arabia
6 Department of Pathology and Hematology, Mubarak Hospital, Kuwait City, Kuwait
7 Department of Hematology and Pathology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
8 Department of Hematology, Dubai Hospital, Dubai, United Arab Emirates
9 Department of Hematology, College of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
10 Department of Pathology, Rashid Hospital, Dubai, United Arab Emirates
11 Department of Hematology, Qabous University Hospital, Mascut, Oman
12 Department of Hematology, Security Forces Hospital, Riyadh, Saudi Arabia

Date of Web Publication19-Jul-2014

Correspondence Address:
Abdul Kareem Almomen
Center of Excellence in Thrombosis and Hemostasis, King Saud University Medical City, P. O. Box: 66533, Riyadh 11586
Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1658-5127.137081

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  Abstract 

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, rare clonal blood disorder, characterized by chronic intravascular hemolysis, bone marrow failure, renal failure and pulmonary hypertension, and a heightened risk of thrombotic complications. PNH etiology is an Υ-linked gene somatic mutation of the phosphatidylinositol glycan class ΐ (PIG-A ), that results in deficiency of the glycosylphosphatidylinositol anchor structure responsible for fixing a wide spectrum of proteins on blood cell membranes, absence of these proteins, particularly CD55 and CD59, dysregulates the complement on cell membranes and results in significant chronic complement-mediated hemolysis. Early diagnosis of PNH is crucial for effective disease management. However, the heterogeneity of clinical symptoms and rarity of this disease usually results in untimely diagnosis, severe disability of patients, and increased risk of fatal complication. These recommendations are formulated by a panel of experts from the gulf cooperation countries. This information reflects their experience and to assist specialists looking after PNH patients, including hematologists, nephrologists, dialysis specialists, gastroenterologists, cardiologists, and surgeons.

Keywords: Epidemiological studies, paroxysmal, phosphatidylinositol


How to cite this article:
Almomen AK, Al Bakistani AG, Alsaeed A, Al Olama A, Hejazi A, Awarji C, Almhareb F, Alsayegh F, Alzahrani H, Almarashly M, Qari M, Aslam M, Seliem R, Al Kindi S, Abuharbesh S, Owaidah T, Bassuni W. Paroxysmal nocturnal hemoglobinuria: Diagnosis and management protocol. J Appl Hematol 2014;5:37-44

How to cite this URL:
Almomen AK, Al Bakistani AG, Alsaeed A, Al Olama A, Hejazi A, Awarji C, Almhareb F, Alsayegh F, Alzahrani H, Almarashly M, Qari M, Aslam M, Seliem R, Al Kindi S, Abuharbesh S, Owaidah T, Bassuni W. Paroxysmal nocturnal hemoglobinuria: Diagnosis and management protocol. J Appl Hematol [serial online] 2014 [cited 2019 Sep 22];5:37-44. Available from: http://www.jahjournal.org/text.asp?2014/5/2/37/137081


  Definition and diagnostics criteria Top


Definition

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, rare, life-threatening, progressive systemic disorder, characterized by chronic intravascular hemolysis. PNH is associated with a significant increase in mortality, development of arterial and venous thrombosis, visceral organ damage, and rapid deterioration in quality of life. Evidence from major epidemiological studies shows that despite up-to-date management, 35% of patients with classical PNH die within the first 5-year of diagnosis, and the 10-year mortality rate is about 50%. [1],[2] The primary causes of death in PNH patients are thrombotic complications, which account for 40-67% of fatal outcomes. [3],[4],[5],[6]

Other severe complications include renal failure, pulmonary hypertension, peripheral neuropathy and smooth muscle dystonia, which can manifest as chest pain, abdominal pain, dysphagia, and erectile dysfunction. [6],[7]

The main pathogenetic mechanism that mediates organ damage and mortality is the dysregulation of the complement system. The complement system is part of the innate immune system that has three main activation pathways: Classical, alternative, and lectin-mediated. All the pathways lead to the key step that consists of cleavage of complement C3 to C3a and C3b proteins. C3b participates in the formation of C5 convertase, which results in cleavage of the C5 protein to C5a and C5b. The C5a protein is a pro-inflammatory and pro-thrombotic agent, and C5b protein promotes the formation of the membrane attack complex (MAC). The MAC mediates the hemolysis of red blood cell (RBC), the generation of pro-coagulatory membrane microparticles, and initiates the activation of platelets. [8],[9],[10]

C5 convertase activation is normally blocked by a glycosylphosphatidylinositol (GPI)-anchored CD55 (decay-accelerating factor). CD55 is absent or deficient in PNH cells, resulting in an increased susceptibility of the C5 protein on cell surfaces to cleavage. Overproduction of C5a initiates leukocyte and platelet activation and stimulates inflammation and thrombosis. Normally, generation of the MAC on the cell surface is blocked by another GPI-anchored complement inhibitor - CD59. [11],[12] However, in the absence of CD59, the MAC initiates RBCs lysis. Platelets without CD55 and CD59 are affected by complement-dependent aggregation and activation. [12],[13]

Flow cytometry measurement allows identification of the cells with GPI-anchored protein (GPI-AP) 15 deficiency. It is the "gold standard" in PNH diagnosis. [14] RBCs are classified into three types: (i) Type I RBCs with normal GPI-AP expression on cell surface, (ii) Type II RBCs with partial GPI-AP expression and (iii) Type III RBCs with complete GPI-AP deficiency. More informative is the determination of GPI-AP deficiency on granulocytes that allows a definitive diagnosis. [15] It is also reasonable to perform this testing also on monocytes in patients with small populations of GPI-AP deficient cells. [16]

Classification

There are three main forms of PNH:

  • The classical PNH, characterized by clinical laboratory signs of intravascular hemolysis without evidence of other diseases of bone marrow failure (aplastic anemia [AA], myelodysplastic syndrome [MDS], idiopathic myelofibrosis)
  • The PNH, diagnosed in patients with AA (AA/PNH), MDS (MDS/PNH) and very rarely myelofibrosis (idiopathic myelofibrosis/PNH), patients of which have clinical and/or laboratory signs of intravascular hemolysis, and a cell clone with PNH phenotype detected in peripheral blood
  • The sub-clinical form of the disease (AA/sPNH [serum PNH], MDS/sPNH, idiopathic myelofibrosis/sPNH), identified in patients without clinical or laboratory signs of hemolysis, but having a minor cell clone with PNH phenotype (usually < 1%).


The differentiation of the sub-clinical form of PNH does not have clinical significance by itself, but is needed to ensure monitoring of patients' PNH clone size, due to the possibility of progression to a clinical hemolytic form of AA/PNH, which would require appropriate treatment.

Classical paroxysmal nocturnal hemoglobinuria

Patients with classical PNH usually demonstrate significant intravascular hemolysis with increases in the serum lactate dehydrogenase (LDH) level, reticulocytosis and decreases in the haptoglobin level. [12] In this PNH variation, there are no definitive morphologic signs of other bone marrow pathology (AA, MDS, myelofibrosis) and usually no karyotype abnormalities. [16]

Paroxysmal nocturnal hemoglobinuria with underlying bone marrow failure syndrome (aplastic anemia/PNH, myelodysplastic syndrome/PNH)

In patients with AA/PNH and MDS/PNH clinical and laboratory signs of intravascular hemolysis are diagnosed. The symptoms of bone marrow failure or intravascular hemolysis can predominate in different stages of the disease and often in combinations. [5],[13] In spite of usually minimal symptoms and only laboratory signs of intravascular hemolysis in patients with a small PNH clone size, twice-a-year monitoring is necessary due to the possibility of clone expansion with severe hemolysis development and high-risk of thrombotic complications. [17],[18]

Sub-clinical form of paroxysmal nocturnal hemoglobinuria (aplastic anemia/serum PNH, myelodysplastic syndrome/sPNH)

Patients with sub-clinical PNH do not have any clinical or laboratory signs of hemolysis and small populations of GPI-AP deficient cells can only be identified today by flow cytometry. The sub-clinical form of PNH can be diagnosed with underlying diseases characterized by bone marrow dysfunction, primarily AA and MDS. It is very important to perform careful monitoring of these patients (twice yearly) for signs of hemolysis and rapid clone expansion, as 15-17% of patients with AA/sPNH eventually develop the hemolytic form of AA/PNH. [18],[19]

Basic Diagnostic Criteria

Flow cytometry measurement identifies patients with GPI-AP deficient cells in peripheral blood. For more accurate diagnosis, the testing should be performed on both RBCs and leukocytes (granulocytes and monocytes). The testing traditionally uses RBCs, but quantification of GPI-negative (CD59−) cells can be jeopardized by previous transfusions of RBCs or massive hemolysis.

Flow cytometric leukocyte testing in peripheral blood - particularly CD16−, CD66b − and/or CD24 − granulocytes, and CD14 − monocytes - provides the most accurate determination of PNH clone size. [12],[15] The testing is done on mature leukocytes in peripheral blood and not in bone marrow as many GPI-linked antigens are expressed later in maturation. In order to determine the clinical form of PNH in a patient it is necessary to perform a clinical laboratory assessment of hemolysis parameters and histological, cytological, and genetic analysis of bone marrow. The primary diagnostic criteria required to verify diagnoses are shown in [Table 1].
Table 1: Principal techniques and cytometry criteria required for diagnose and classification of PNH

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Early and accurate diagnosis is crucial for effective treatment of PNH patients. [5],[12],[20] PNH diagnosis is further complicated by the diversity of the clinical manifestations and its rare prevalence. As a result, the diagnosis is usually established several years after the disease develops and in some cases with delays of up to 10-year and longer. [3],[12] This panel of experts expressed their concern that many of their patients are detected late due to arising complications, and the disease is underdiagnosed due to similar factors, necessitating an awareness campaign among the concerned practitioners.


  Additional diagnostic techniques to optimize management Top


To choose the optimal PNH management method it is necessary to evaluate a series of factors that influence the prognosis and clinical manifestation of the disease. The list of recommended tests for PNH patients is summarized in [Table 2].
Table 2: Additional testing to optimize patient management

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  Patient selection for paroxysmal nocturnal hemoglobinuria testing Top


While PNH is a rare disease, it often has concurrent symptoms as thrombosis, anemia, and other cytopenias. At the same time, there is no adequate evidence to justify performing PNH screening in all patients with thrombotic complications, anemia or cytopenia, because these manifestations are common in other diseases as well. However, all patients with thrombosis with unknown cytopenias or intravascular hemolysis, especially; Coombs-negative hemolytic anemia, should undergo PNH screening. [18],[14] The presence of other clinical manifestations, as highlighted in the International Clinical Cytometry Society (ICCS) guidelines [14] [Table 3], necessitates the screening for this condition and significantly increases the possibility of PNH clone detection.
Table 3: Clinical signs requiring PNH screening activity

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As hemoglobinuria is seen in 25-27% of PNH patients at the outset of the disease, all patients with this symptom should undergo PNH screening. Significant hemoglobinuria is usually present in all patients with classical PNH, but in patients with AA/PNH or MDS/PNH the signs of hemoglobinuria can be absent due to a relatively small size of the PNH clone. Due to the fact that PNH is characterized by intravascular hemolysis, the LDH levels will almost always rise to variable levels. PNH screening thus far should be initiated in every patient showing signs of intravascular hemolysis with increased LDH levels. However, it should be pointed out that in some patients, the LDH level can marginally increase and even fall into the normal range in spite of a large PNH clone size depending on the type of the red cell clone (Type II vs. Type III).

It is recommended to perform PNH screening in all patients with AA, even in the absence of hemolysis [12] and in all MDS patients. [14] In about 10% patients with classical PNH and 15-25% patients with hemolysis with underlying AA/PNH or MDS/PNH, the disease begins with AA. [16]

Thrombosis is one of the most common complications associated with PNH [14] independent of the PNH form. The disease is diagnosed due to the thrombotic event in <12% of patients. [16] In this respect, it is not recommended to perform routine screening of all patients with thrombosis, except in cases of unusual thrombosis localization ( Budd-Chiari syndrome More Details, cerebral thrombosis, dermal vein thrombosis) or in combination with intravascular hemolysis, concurrent cytopenia after exclusion of other more common causes of thrombophilia. [12],[14] The expert panel opinion is to address question while taking history from the patients with venous thromboembolism to verify if they have noticed a change in the urine color, this hopefully will assist in making decisions as far as the screening for the clone is concerned, yet this requires an elaborate study addressing this issue and providing a higher level of evidence according to the testing guidelines.

An average of 10-20% of PNH patients have abdominal pain and other GI symptoms (dysphagia) at the time the disease is diagnosed. These symptoms are particularly common in patients with classical PNH, in which the frequency of these pathologies reaches 33%. [16] That is why, in spite of a lack of necessity in routine screening in all the patients with GI complaints, the testing should be performed in all patients with abdominal pain and dysphagia in combination with clinical and laboratory signs of intravascular hemolysis.

Data on screening activities in different groups of patients are summarized in [Table 4].
Table 4: Frequency of screening activities

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  Paroxysmal nocturnal hemoglobinuria diagnostics Top


Flow Cytometry Measurement

Flow cytometry has been used in PNH diagnosis for a long time and has shown itself to be the most sensitive and informative technique. [21],[14],[22] The ICCS 2010 guidelines should be used for routine PNH diagnosis and monitoring. Antibodies recommended by ICCS and used for PNH testing.

It is recommended to test for GPI-AP proteins deficiency in both RBCs and leukocytes because PNH RBCs lyse under the action of complement, changing the total clone size. As opposed to RBCs, the lifespan of PNH leukocytes does not differ from the life span of leukocytes in healthy people. [23]

Paroxysmal nocturnal hemoglobinuria diagnosis is confirmed by the presence of ≥ 0.01% leukocytes with complete or partial deficiency of GPI-APs that is, the number of PNH cells or size of PNH clone is at least 0.01%. [14]

To exclude the inherited isolated deficiency of one of the GPI-APs, it is necessary to perform an investigation of two or more GPI-APs. [12] For patients with a stable course of the disease and clone size <0.1%, once yearly study of the peripheral blood for GPI-AP expression is sufficient. Monitoring of patients with clone size >0.1% should be conducted at least every 6 months, because it can increase with time. [18] Moreover, in the presence of clinical deterioration, the appearance of thromboembolic complications or increases in hemolysis, a flow cytometric analysis should be performed immediately. Taking into account the fact that clone size can decrease and even disappear over the course of time, [24],[25] an analysis should be done in the presence of clinical improvement. This recommendation is based on the fact that thrombotic activity depends on the size of a PNH clone. [21],[26],[27] Therefore, decrease of GPI-APs deficient granulocytes may require a modification in preventive anticoagulation therapy.

Identifying different PNH types on RBCs can be complicated by previous RBC transfusions because during the transfusions, the quantity of cells with normal CD55 and CD59 expression increases, and this affects flow cytometry analyses. Taking this into account, it is recommended to perform red cell flow cytometry in a period free of transfusions (>3 months), or immediately before the transfusion to accurately quantify GPI-AP-deficient RBCs.

Flow cytometry allows the identification of cell populations with a lack of GPI-APs, determination of abnormal cells proportions and distinct populations with different severities of GPI-APs deficiency, particularly in RBCs.

High-sensitivity flow cytometry allows a detection of clones with a size of 0.01%. [14] For all patients with AA or refractory cytopenia with unilineage dysplasia (RCUD), high-sensitive flow cytometry is recommended at diagnosis and twice yearly thereafter, even in the absence of obvious clinical and laboratory signs of hemolysis. [12] This recommendation is based on the finding that the clone size is changing with time, and the presence of even a small population of GPI-AP-deficient cells in these patients is of prognostic value and affects the choice of therapy. [12],[25],[28] For example, the presence of PNH cells in patients with AA or RCUD, as demonstrated in some studies [20],[25],[29] is a favorable prognostic factor for patients receiving immunosuppressive therapy.

One of the best reagents for PNH clone detection within leukocytes is FLAER (fluorescent-marked aerolysin; Pinewood Scientific, Vancouver, BC), that directly binds with the GPI-anchor. [30],[12],[14],[31],[32] Compared with other GPI-anchored antibodies tested, the bond formation between FLAER and the GPI-anchor is less dependent on the stage of cell maturity. [14] Moreover, to detect PNH clones, FLAER can be used in multi-color combinations with monoclonal antibodies to GPI-anchored and non-GPI-anchored antigens. [33] However, this method cannot be used for RBC analysis because the reagent poorly binds to the human RBCs in the conditions needed for investigation. The reason for this is unknown as of yet.

Other Diagnostic Techniques

Such techniques as the sucrose hemolysis test and the Ham's test (RBCs lysis on decrease of serum pH) are only of historical interest. These tests are not currently used in PNH testing because they are not sensitive enough compared to flow cytometry. [12]

There is limited data concerning the use of molecular testing in PNH diagnosis.


  Paroxysmal nocturnal hemoglobinuria management Top


Eculizumab

Therapy with eculizumab (Soliris® , Alexion Pharmaceuticals, Cheshire, CT, USA) is indicated in patients with complement-mediated hemolysis. Eculizumab-humanized monoclonal antibody that binds with the C5 component of complement, preventing the formation of C5a and C5b, inhibiting the formation of pro-inflammatory cytokines (through C5a) and MAC (through C5b). [34] The therapy with Eculizumab is indicated in patients with complement-mediated hemolysis. In clinical studies, the use of eculizumab results in significant reduction of hemolysis, thrombosis rate, transfusion dependence, pulmonary hypertension, and severity of symptoms such as dyspnea and weakness. In addition, eculizumab promotes improvement in renal function, and quality of life. [35],[36],[37] Long-term administration of eculizumab was shown to normalize the life expectancy of patients. [38] Though the administration of this medication does not result in complete cure, eculizumab has a very good profile of long-term safety and significantly reduces the rate of complications as well as mortality in PNH patients.

Indications for eculizumab

  • ≥1 thrombotic episode in the past, with LDH level ≥ 1.5 of the upper limit of normal value plus one of the following:
  • Medium and severe anemia (Hb < 90 g/L)
  • Medium and severe pulmonary hypertension (detected on an echocardiogram)
  • Acute renal failure or chronic renal disease
  • Smooth muscle dystonia (abdominal pains, dysphagia, and erectile dysfunction)
  • Pregnancy in classical PNH and AA/PNH.


The standard dosing regimen for eculizumab is sufficient to completely and consistently block complement-mediated hemolysis. Some patients with PNH can experience "breakthrough" hemolysis due to variations in medication metabolism or during infection episodes.

Identification of "breakthrough" hemolysis

  • Signs of hemolysis before next dose of eculizumab is due
  • Hemoglobinuria
  • Recurrence of symptoms
  • Increasing transfusion requirements
  • Increased LDH
  • Decreased haptoglobin
  • Increased reticulocyte number.


Management of "breakthrough" hemolysis

These patients are best managed by reduction in the interval between eculizumab administrations to 12 days, which is more convenient for the patient, or increasing eculizumab dose to 1200 mg (1-2 administrations).

Follow-up of patients treated with eculizumab

Early detection of infections and prompt administration of antibacterial agents are necessary during the eculizumab treatment. When meningococcal infection is diagnosed, the administration of the medication should be stopped.

Recommended laboratory investigations: CBC and reticulocytes count, LDH, creatinine, brain natriuretic peptide B, serum iron, ferritin. PNH clone size control is assessed by flow cytometry performed twice a year.

Supportive management with eculizumab therapy

Cyanocobalamin injection and iron supplements (if deficient) anticoagulant therapy is indicated in thrombotic complications. Primary anticoagulation is not recommended with eculizumab therapy.

Pregnancy in paroxysmal nocturnal hemoglobinuria patients

Pregnancy in PNH patients is characterized by high rate of complications in the form of maternal and newborn mortality. Thus, recommendations for the management of PNH pregnant women include combined care by both obstetricians and hematologists.

Administration of folic acid and iron supplementation are essential due to ongoing intravascular hemolysis, as well as RBC and platelet transfusions if necessary.

Eculizumab is listed as category C product, but has been used in pregnancy (many case reports) and appears to be safe in this setting, and is likely to prevent many of the complications usually observed during pregnancy.

There may be a need to increase the dose to 900 mg/week in the third trimester of pregnancy and in case of "breakthrough" hemolysis. Eculizumab was not found in human milk or in the umbilical vein. Prophylactic anticoagulant therapy with low-molecular weight heparin (LMWH) is recommended.

Bone Marrow Transplant

While eculizumab allows for control of the intravascular hemolysis and PNH complications associated with it, allogeneic bone marrow transplant (BMT) remains the only potentially curative method that allows for the complete recovery of this disease. However, BMT is associated with significant mortality. In a retrospective study of 26 PNH patients in Italy treated with BMTs, the 10-year survival rate was 42. [39] The 2-year survival rate in 48 patients who received BMT from an HLA-matched sibling was 56% according to data from the International BMT Register. [40] Regardless of the PNH manifestations that rationalized the indications of BMT, the rate of complications remains very high. The rate of graft versus host disease in patients with PNH is 42-54%, [41],[39] about one-half of patients develop a veno-occlusive liver disease, [42] and the risk of PNH clone expansion persists. [43] BMT and associated complications negatively affect the patients' quality of life. [44],[45]

In this regard, classical PNH, and PNH with thrombotic complications, is not currently a further indication for BMT. Recently, the results of a retrospective multi-center analysis of BMT in 211 patients by the European Group for Blood and Marrow Transplantation and French Society of Hematology were published. [46] The total 5-year survival of PNH patients following transplants was 68 ± 3% (54 ± 7% in classical PNH group, transplanted due to thrombotic complications, 69 ± 5% in patients with AA/PNH without thrombotic episodes in medical history, and 86 ± 6% in patients with classical PNH with recurrent hemolysis as an indication of BMT). Further, a comparative analysis of the total survival rate in 24 pairs of patients treated and not treated with BMT was performed in a group of PNH patients with thrombosis in their medical histories. The survival rate in the BMT group was consistently lower. These data strongly indicate that thrombotic complications cannot also be an indication for BMT.

Bone marrow transplant is performed as a standard indication in hematopoietic aplasia (AA/PNH and AA/sPNH), rare cases of clonal PNH transformation into MDS/acute leukemia, or when an identical twin sibling is available.

Steroids

Steroid hormones were empirically used in the treatment with no randomized studies to confirm their efficacy. Short courses (2-3 days) of prednisolone 1 mg/kg body weight can be beneficial in ameliorating the severity and duration of an hemolytical crisis according to several experts. Long-term therapy with steroids is not recommended.

Anticoagulant Therapy

Following a thrombotic episode, a long-term (life-long) anticoagulant therapy (warfarin or LMWH) is recommended. Patients with acute and subacute Budd-Chiari syndrome (presenting with 6 weeks) may require hospitalization to a specialized unit to perform local and/or systemic thrombolysis. Anticoagulant therapy for primary thrombosis prevention can be indicated when PNH clone size in granulocytes is ≥50% and in the presence of additional risks of thrombotic complications. [8],[16]

 
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