| Abstract|| |
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, marrow failure, nocturnal hemoglobinuria and thrombophila. This acquired disease caused by a deficiency of glycosylphosphatidylinositol (GPI) anchored proteins on the hematopoietic cells is uncommon in the Indian population. Materials and Methods: Data of patients diagnosed with PNH in the past 1 year were collected. Clinical data (age, gender, various presenting symptoms), treatment information and follow-up data were collected from medical records. Results of relevant diagnostic tests were documented i.e., urine analysis, Ham's test, sucrose lysis test and sephacryl gel card test (GCT) for CD55 and CD59. Results: A total of 5 patients were diagnosed with PNH in the past 1 year. Presenting symptoms were hemolytic anemia (n=4) and bone marrow failure (n=1). A GCT detected CD59 deficiency in all erythrocytes in 4 patients and CD55 deficiency in 2 patients. A weak positive PNH test for CD59 was seen in 1 patient and a weak positive PNH test for CD55 was seen in 3 patients. All patients were negative by sucrose lysis test. Ham's test was positive in two cases. Patients were treated with prednisolone and/or androgen and 1 patient with aplastic anemia was also given antithymocyte globulin. A total of 4 patients responded with a partial recovery of hematopoiesis and 1 patient showed no recovery. None of the patients received a bone marrow transplant. Conclusion: The study highlights the diagnostic methods and treatment protocols undertaken to evaluate the PNH clone in a developing country where advanced methods like flowcytometry immunophenotyping (FCMI) and bone marrow transplants are not routinely available.
Keywords: Paroxysmal nocturnal hemoglobinuria, CD55, CD59
|How to cite this article:|
Gupta P K, Charan V D, Kumar H. PNH revisited: Clinical profile, laboratory diagnosis and follow-up. Indian J Pathol Microbiol 2009;52:38-41
|How to cite this URL:|
Gupta P K, Charan V D, Kumar H. PNH revisited: Clinical profile, laboratory diagnosis and follow-up. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 May 20];52:38-41. Available from: http://www.ijpmonline.org/text.asp?2009/52/1/38/44960
| Introduction|| |
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, nocturnal hemoglobinuria, venous thrombosis and is associated with bone marrow failure. ,,, It is an acquired disease due to non malignant clonal expansion of one or several hemopoietic stem cells that have acquired a somatic mutation of the phosphatidylinositol glycan complementation class A gene (PIG-A). Progeny of the affected stem cells are deficient in glycosyl phosphatidylinositol-anchored protein (GPI-APs).  This results in the deficiency of GPI-AP on hematopoietic cells (CD16, CD24, CD52, CD55, CD59, CD58, CD66b/CD67, CD73, CD87, CD90 and CD108). ,, Deficiency of GPI-AP CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) accounts for the complement-mediated intravascular hemolysis that results in intermittent hemoglobinuria, the main manifestation of the disease. PNH is also frequently (10-50%) associated in patients with bone marrow failure syndromes particularly aplastic anemia., Venous thrombosis is the major cause of morbidity and mortality in PNH. Other complications such as infectious diseases and hematopoietic disorders like myelodysplasia or leukemia may occur.
Various tests are used in the routine diagnosis of PNH. Traditionally, lytic tests like the Ham's test (acidified serum lysis test) and the sugar water test (sucrose hemolysis test) determines the erythrocyte sensitivity to complement-dependent lysis.  Both these tests are used as screening tests to detect the protein defects on erythrocytes. The low specificity and relatively high variability seen due to the different lysing potency of the human sera used, have reduced the interest in these methods. A sephacryl gel card technique using CD55 and CD59 for identification of the PNH defect has gained popularity and is based on antigen-antibody reaction.,, Flowcytometry immunophenotyping (FCMI) is now widely used in the diagnosis of PNH. FCMI can discriminate between cell populations with different expressions or the absence of one or more GPI-APs on erythrocytes, leukocytes and platelets. Apart from measuring the percentage of antigen-deficient cells, flow cytometry allows for the differentiation between PNH subpopulations according to their intensity: PNH I, having normal levels of GPI proteins on their surface; PNH II, intermediate expression; and PNH III, complete deficiency of GPI proteins. This classification of PNH red cells derives from different complement lysis sensitivities with Type III and Type II red cells being 15-25 times and 3-5 times more sensitive to complement than the Type I normal red cells, respectively.  Different PNH clones often coexist in a single patient. The analysis of GPI-APs on the surface of the hematopoietic cells, particularly the red cells, in PNH reveals that approximately 40% of the patients have a combination of Type I, II and III PNH cells. 
This is a retrospective study undertaken to investigate the clinical behavior and outcome of this disease in the past 1 year in a tertiary care hospital. It also highlights the diagnostic methods and treatment protocols undertaken to evaluate the PNH clone in a developing country were advanced methods like FCMI and bone marrow transplants are not routinely available.
| Materials and Methods|| |
Retrospectively, we retrieved the clinical and follow-up data from the medical records of patients diagnosed with PNH in a tertiary care hospital in Western India. Clinical data (age, gender, various presenting symptoms), treatment information and follow-up data were collected from medical records. Results of the relevant diagnostic tests were documented i.e., urine analysis, Ham's test, sucrose lysis test and sephacryl gel card test (GCT) for CD55 and CD59.
A Ham's test and a sucrose hemolysis test (SLT) were performed using previously described methods.  For the GCT, an ID-PNH gel card manufactured by DiaMed-ID (St Morot, Schweiz) was used. A single card contained 6 microtubes (for 2 tests) containing rabbit anti-mouse antibody within the sephacryl gel matrix. Blood samples were drawn into citrate/EDTA or CPD-A anticoagulant. A 0.8% red cell suspension in low ionic strength buffer (ID-diluent 2) was made. A total of 50 ml of red cell suspension was added to 3 appropriate microtubes of the ID-card; 50 ml of anti-DAF (CD55), anti-MIRL (CD59) and PNH-Ctrl (negative control) were added. The microtyping card was incubated at 37 0 C for 15 min. The card was centrifuged in the ID-centrifuge (DiaMed-ID) for 10 min. Normal cell population expressing CD55 and CD59 formed a red line of agglutinated cells on the surface of the gel or agglutinates dispersed in the gel. Pathological cells that lacked CD55 and CD59 and negative control formed a compact button of cells on the bottom of the microtube. A double population of cells indicates that a population of the cells lacks the corresponding antigens DAF and MIRL-this indicates a weak positive PNH test.
| Results|| |
A total of 5 patients with PNH were diagnosed in this hospital in the past 1 year [Table 1]. The delay between the presentation of symptoms and the confirmation of diagnosis ranged from 6 months to 4 years. A total of 4 patients showed signs of hemolytic anemia at presentation and 1 patient had signs of bone marrow failure (aplastic anemia). Hepatosplenomegaly was found in 1 patient.
In all patients, a urine analysis was performed at the time of diagnosis. One patient showed an increase in urobilinogen, 3 patients had hemoglobinuria and 2 patients had both hemoglobinuria and hemosiderinuria. A total of 4 patients had increased indirect hyperbilirubinaemia. A direct Coombs test was negative for all the patients.
The diagnostic evaluations of all 5 patients are shown in [Table 2]. A total of 4 patients were anemic (Hb range: 6.1-10.3 g/dl); all showed elevated HbF (2.9-8.4%) and elevated RDW (50-63.6%). One patient with moderate aplastic anemia with a platelet count of 100 x 10 3 /cmm and who progressed to PNH had normocelluar bone marrow on presentation.
A sephacryl gel card detected CD59 deficiency in all erythrocytes in 4 patients and CD55 deficiency in 2 patients. A weak positive PNH test for CD59 was seen in 1 patient and a weak positive PNH test for CD55 was seen in 3 patients. All patients were negative by sucrose lysis test. A Ham's test was positive in 2 cases. These 2 patients, who showed positivity by Ham's test, showed deficiency in all erythrocytes for CD55 and CD59 by the gel card test. The Ham's test was negative in the presence of weak positivity of either CD55 or CD59 (patients 3, 4 and 5).
Therapy and response data are shown in [Table 1]. Two patients were treated with prednisolone and androgen and 1 patient with aplastic anemia was also given antithymocyte globulin. Others were treated with prednisolone only. Four patients had responded with partial recovery of hematopoiesis and 1 patient (patient 5) showed no recovery. The treatment had been given to the patients for a duration of 1 year and the patients are still in follow-up and are receiving the required treatment. None of the patients received bone marrow transplants. The pre-therapy and post-therapy hematological and bone marrow findings are given in [Table 2] and [Table 3], respectively.
| Discussion|| |
PNH is a rare and serious hematological disorder. Adult patients with PNH suffer from bone marrow failure, serious infection, thrombosis and intravascular hemolysis. PNH seems to occur more frequently in some Asian countries such as China and Thailand than in Western countries.  The natural history of PNH appears different for Americans and Europeans compared with Asians/Pacific Islanders and Hispanics. ,,, The disease in the Orient tends to be more prevalent in the younger age group and males predominate.  In our study, the age at presentation varied from 15 years old to 38 years old and all were males. The reason for the apparent excess of PNH in males versus females in this study is not clear. One possible explanation is the close relationship between PNH and aplastic anemia. The incidence and male:female ratio (1.9:1) of aplastic anemia in some Asian countries are higher than those reported from Europe. 
The manifestations of bone marrow failure are more common in Asians/Pacific Islanders and Hispanics. In contrast, thrombosis and infection appear more common in American and European patients. The basis of these phenotypic differences is unknown, but the relationship of ethnicity and geography to the natural history of PNH should be considered when formulating a management plan. In this study, none of the patients had features of thrombosis and 1 patient (20%) had features of bone marrow failure.
In adults, hemoglobinuria is found as the presenting symptom in the majority of the patients with PNH. , In our study, 3 of the 5 patients presented with features of hemoglobinuria in the diagnostic period. Two of these patients had hemoglobinuria and hemosiderinuria. This is consistent with previous reports., One of our patients, a diagnosed case of aplastic anemia, developed evidence of PNH with minimal hematological recovery after treatment with antithymocyte globulin (ATG). Despite treatment, this patient did not respond to therapy. This may be due to the persistence of the GPI-deficient PNH clone. FCMI studies have brought aplastic anemia closer to PNH. In a study, about 20% of patients with aplastic anemia showed GPI-APs defective granulocytes at the time of first admission and there was little evidence from serial or cohort analysis of evolution to PNH; rather, the two diseases appear to coexist from presentation with pancytopenia. 
The hallmark of PNH is the demonstration of complement sensitive erythrocytes and peripheral blood cells with GPI-membrane protein deficiency. We studied the patterns of CD55 and CD59 expression or PNH phenotypes on erythrocytes using the lytic tests and GCT. Ham's test was positive in 2 patients (40%). A GCT showed positive reactions in all patients; 2 patients (40%) showed a deficiency of both CD55 and CD59 in all erythrocytes and 3 patients (60%) showed weak positivity with either anti-DAF and/or anti-MIRL. A Ham's test and a sugar water test, which are positive only in few cases, have their limitations: firstly, these tests are not specific for PNH; secondly, they can only screen for the protein defect on erythrocytes; and finally, these procedures cannot be applied after a blood transfusion. The GCT also has its limitations but among these three methods, it is the most sensitive and the easiest to perform and interpret. FCMI is the most sensitive method for diagnosis of PNH, as it can quantify and delineate PNH cells with different expressions of GPI-APs. , But, in a developing country where FCMI may not be available in all hospitals, GCT is a useful screening tool. The delay between the presentation of symptoms and diagnosis reported in this study may be due to the lack of sensitivity of the Ham's test and sucrose lysis test, which are still been used in many laboratories in our country.
In conclusion, clinical profile, laboratory diagnosis and follow-up of patients with PNH in the developing world clearly indicates that many patients with a smaller clone of PNH cells may go undiagnosed due to lack of FCMI in many centers. Also, a majority of patients continue to harbor the diseased clone even after diagnosis as many receive only immunosuppressive therapy and not a bone marrow transplant, which is the treatment of choice, due to the lack of infrastructural facilities.
| References|| |
|1.||Flotho C, Strahm B, Kontny U, Duffner U, Peters AM, Dupuis W, et al . Stem cell transplantation for paroxysmal nocturnal hemoglobinuria in childhood. Br J Haematol 2002;118:124-7. [PUBMED] |
|2.||Rizk S, Ibrahim IY, Mansour IM, Kandil D. Screening for paroxysmal nocturnal hemoglobinuria (PNH) clone in Egyptian children with aplastic anemia. J Trop Pediatr 2002;48:132-7. [PUBMED] [FULLTEXT]|
|3.||Meletis J, Terpos E. Recent insights into the pathophysiology of paroxysmal nocturnal hemoglobinuria. Med Sci Monit 2003;9:RA161-72. [PUBMED] [FULLTEXT]|
|4.||Risitano AM, Holada K, Chen G, Simak J, Vostal JG, Young NS, et al . CD34+ cells from paroxysmal nocturnal hemoglobinuria (PNH) patients are deficient in surface expression of cellular prion proein (PrPc). Exp Hematol 2003;31:65-72. [PUBMED] [FULLTEXT]|
|5.||Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, et al . Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 1993;73:703-11. [PUBMED] [FULLTEXT]|
|6.||Hillmen P, Richards SJ. Implications of recent insights into the pathophysiology of paroxysmal nocturnal haemoglobinuria. Br J Haematol 2000;108:470-9. [PUBMED] [FULLTEXT]|
|7.||Karadimitris A, Luzzatto L. The cellular pathogenesis of paroxysmal nocturnal haemoglobinuria. Leukemia 2001;15:1148-52. [PUBMED] |
|8.||Maciejewski JP, Rivera C, Kook H, Dunn D, Young NS. Relationship between bone marrow failure syndromes and the presence of glycophosphatidyl inositol-anchored protein-deficient clones. Br J Hematol 2001;115:1015-22. |
|9.||Azenishi Y, Ueda E, Machii T, Nishimura J, Hirota T, Shibano M, et al . CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia. Br J Haemtol 1999;104:523-9. |
|10.||Socie G, Rosenfeld S, Frickhofen N, Gluckman E, Tichelli A. Late clonal diseases of treated aplastic anemia. Semin Hematol 2000;37:91-101. |
|11.||Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN. Wintrobes Clinical Hematology. Philadelphia: Lea and Febiger; 2003. p. 1203-21. |
|12.||Meletis J, Michali E, Samarkos M, Konstantopoulos K, Meletis C, Terpos E, et al . Detection of ''PNH red cell'' populations in hematological disorders using the sephacryl gel test micro typing system. Leuk Lymph 1997;28:177-82. |
|13.||Zupanska B, Bogdanik B, Pyl H. A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria. Immunohematology 2002;18:9-12. [PUBMED] |
|14.||Nathalang O, Chuansumrit A, Prayoonwiwat W. Rapid screening of PNH red cell populations using the gel test. Southeast Asian J Trop Med Public Health 2003;34:887-90. [PUBMED] |
|15.||Fujioka S, Yamada T. Varying populations of CD59-negative, partly positive and normally positive blood cells in different cell lineages in peripheral blood of paroxysmal nocturnal hemoglobinuria patients. Am J Hematol 1994;45:122-7. [PUBMED] |
|16.||Rosse WF. Phosphatidylinositol-linked proteins and paroxysmal nocturnal hemoglobinuria. Blood 1990;75:1595-601. [PUBMED] [FULLTEXT]|
|17.||Hill A, Richards SJ, Hillmen P. Recent developments in the understanding and management of paroxysmal nocturnal haemoglobinuria. Br J Haematol 2007;137:181-92. [PUBMED] [FULLTEXT]|
|18.||Regan F, Newlands M, Bain BJ. Practical haematology. 9th ed. Churchill Livingstone, Edinburgh; 2001. |
|19.||Pramoonjago P, Pakdeesuwan K, Siripanyaphinyo U, Chinprasertsuk S, Kinoshita T Wanachiwanawin W. Genotypic, immunophenotypic and clinical features of Thai patients with paroxysmal nocturnal haemoglobinuria Br J Haematol 1999;105:497-504. |
|20.||Nishimura J, Kanakura Y, Ware RE, Shichishima T, Nakakuma H, Ninomiya H, et al . Clinical course and flow cytometric analysis of paroxysmal nocturnal hemoglobinuria in the United States and Japan. Medicine (Baltimore) 2004;83:193-207. [PUBMED] [FULLTEXT]|
|21.||Fujioka S, Asai T. Prognostic features of paroxysmal nocturnal hemoglobinuria in Japan. Nippon Ketsueki Gakkai Zasshi 1989;52:1386-94. [PUBMED] |
|22.||Kruatrachue M, Wasi P, Na-Nakorn S. Paroxysmal nocturnal haemoglobinuria in Thailand with special reference to as association with Aplastic anaemia. Br J Haematol 1978;39:267-76. [PUBMED] |
|23.||Araten DJ, Thaler HT, Luzzatto L. High incidence of thrombosis in African-American and Latin- American patients with paroxysmal nocturnal haemoglobinuria. Thromb Haemost 2005;93:88-91. [PUBMED] [FULLTEXT]|
|24.||Le XF, Yang TY, Yang XY, Wang XM. Characteristics of paroxysmal nocturnal hemoglobinuria in China. Clinical analysis of 476 cases. Chin Med J (Engl) 1990;103:885-9. |
|25.||Issaragrisil S, Sriratanasatavorn C, Piankijagum A, Vannasaeng S, Porapakkham Y, Leaverton PE, et al . Incidence of aplastic anemia in Bangkok: The Aplastic Anemia Study Group. Blood 1991;77:2166-8. [PUBMED] [FULLTEXT]|
|26.||Nishimura J, Ware RE, Burnette A, Pendleton AL, Kitano K, Hirota T, et al . The hematopoietic defect in PNH is not due to defective stroma, but is due to defective progenitor cells. Blood Cells Mol Dis 2002;29:159-67. [PUBMED] [FULLTEXT]|
|27.||Zhao M, Shao Z, Li K, Chen G, Liu H, Zhang Y, et al . Clinical analysis of 78 cases of paroxysmal nocturnal hemoglobinuria diagnosed in the past ten years. Chin Med J (Engl) 2002;115:398-401. [PUBMED] [FULLTEXT]|
|28.||Dunn DE, Tanawattanacharoen P, Boccuni P, Nagakura S, Green SW, Kirby MR, et al . Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes. Ann Intern Med 1999;131:401-8. [PUBMED] [FULLTEXT]|
|29.||Gupta R, Pandey P, Choudhry R, Kashyap R, Mehrotra M, Naseem S, et al . A prospective comparison of four techniques for diagnosis of paroxysmal nocturnal hemoglobinuria. Int J Lab Hematol 2007;29:119-26. [PUBMED] [FULLTEXT]|
|30.||Oelschlaegel U, Besson I, Arnoulet C, Sainty D, Nowak R, Naumann R, et al . A standardized flow cytometric method for screening paroxysmal nocturnal haemoglobinuria (PNH) measuring CD55 and CD59 expression on erythrocytes and granulocytes. Clin Lab Haematol 2001;23:81-90. [PUBMED] [FULLTEXT]|
P K Gupta
Department of Transfusion Medicine, Hematology Section, Armed Forces Medical College, Pune 411 040, Maharashtra
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]