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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 55  |  Issue : 2  |  Page : 206-210
Detection of paroxysmal nocturnal hemoglobinuria-phenotype in patients with chronic lymphocytic leukemia and multiple myeloma


Department of Internal Medicine and Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication3-Jul-2012
 

   Abstract 

Background : Paroxysmal nocturnal hemoglobinuria (PNH) results due to decrease or absence of glycosylphosphatidylinositol-anchored (GPI) molecules, such as CD55 and CD59, from the surface of the affected cells. PNH-phenotype has been described in various hematological disorders, mainly aplastic anemia and myelodysplastic syndromes; recently it has been reported in patients with lymphoproliferative syndromes and multiple myeloma (MM). Materials and Methods : We evaluated the presence of CD55 negative and/or CD59 negative red blood cell (RBC) populations in newly diagnosed treatment naive-54 chronic lymphocytic leukemia (CLL) and 29 MM patients by flow cytometry. Results : PNH-phenotype was not reported in any patient; however, RBC populations deficient in CD55 were detected in 16.66% (9/54) CLL and 6.89% (2/29) MM patients. Clinical presentation or the hematological parameters did not show any relationship with the presence of CD55 deficient RBC population. Conclusion : Our study showed absence of PNH-phenotype in patients with CLL and MM; however, isolated CD55 deficient RBC were identified in both CLL and MM. Larger prospective studies by other centers, including simultaneous analysis of granulocytes for the presence of PNH-phenotype, are needed to corroborate these findings and to work out the mechanisms and the significance of the existence of this phenotype in these patients.

Keywords: Chronic lymphocytic leukemia, flow cytometry, multiple myeloma, paroxsymal nocturnal hemoglobinuria

How to cite this article:
Varma S, Varma N, Reddy V V, Naseem S, Bose P, Malhotra P. Detection of paroxysmal nocturnal hemoglobinuria-phenotype in patients with chronic lymphocytic leukemia and multiple myeloma. Indian J Pathol Microbiol 2012;55:206-10

How to cite this URL:
Varma S, Varma N, Reddy V V, Naseem S, Bose P, Malhotra P. Detection of paroxysmal nocturnal hemoglobinuria-phenotype in patients with chronic lymphocytic leukemia and multiple myeloma. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Sep 20];55:206-10. Available from: http://www.ijpmonline.org/text.asp?2012/55/2/206/97871



   Introduction Top


Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder that results in varied clinical manifestations, including chronic intravascular hemolysis, bone marrow failure, and an increased tendency to thrombosis. [1] These manifestations occur as a result of somatic mutation in the X-linked gene, phosphatidylinositolglycan, complement protein A (PIG-A). The PIG-A gene product is responsible for the first step in the biosynthesis of glycosylphosphatidylinositol-anchored proteins (GPI-AP). Hence, the PNH stem cell and all of its progeny have a marked deficiency or absence of all GPI-AP. [2] The absence of certain GPI-anchored complement regulatory proteins, such as CD55 and CD59 accounts for the complement-mediated hemolysis that characterizes PNH; over 20 different antigens (e.g., CD16, CD24, CD52, CD55, CD59, CD58, CD66b/67, CD73, CD87, CD90, CD108, etc) have been described that are missing from PNH cells. [3] Platelets and granulocytes also have reduced GPI proteins; therefore, in addition to hemolysis, these patients are also predisposed to infections and thrombosis.

A classification scheme for PNH has been proposed by the International PNH Interest Group that includes three main categories that cover the spectrum of disease presentation- (i) classical PNH, which includes hemolytic and thrombotic patients; (ii) PNH in the context of other primary disorders, such as aplastic anemia (AA) or myelodysplastic syndrome (MDS); and (iii) subclinical PNH (PNH-sc), in which patients have small PNH clones but no clinical or laboratory evidence of hemolysis or thrombosis. [4]

Classical PNH with clinical syndrome is an uncommon disorder and all individuals having the PNH clones do not have clinical disease. This is because development of clinical manifestation depends on the - (i) somatic mutation of PIG-A gene and hence generation of PHN-phenotype; (ii) size of the PNH clone; (iii) survival advantage of PHN clone, which may result from selection in favor of the PNH clone that allows it to proliferate preferentially compared with the residual hematopoiesis. [5] PNH-phenotype without clinical syndrome has been described in AA and MDS. [6],[7],[8] In addition, studies have also shown the presence of PNH-phenotype in patients with hematological malignancies. [9],[10],[11],[12],[13],[14] Meletis et al. [10],[11] reported a double negative (CD55 and CD59) red blood cell (RBC) population in 9.2% patients with lymphoproliferative syndromes, especially chronic lymphocytic leukemia (CLL) and in 12.9% patients with plasma cell dyscrasias. Seya et al. [12] demonstrated a greater loss of CD55 in non-Hodgkin lymphoma compared with other hematological malignancies; and Terpos et al. [13] showed presence of PNH-phenotype in 56% patients with multiple myeloma (MM).

However, in contrast to patients of AA and MDS, where the identification of PNH-phenotype has been established as clinically relevant, based on the studies which suggest that patients with a small population of PNH cells in combination with either AA or MDS-refractory anemia (MDS-RA) have a high probability of responding to immunosuppressive therapy, the presence of PNH-phenotype, and its clinical impact and biological behavior in CLL and MM patients is not clearly defined. [4],[6] Moreover, the relative frequency of PNH-phenotype in these disorders using flow cytometric detection is not described.

In the present study we analyzed the PNH-phenotype by flow cytometry in red cells of untreated CLL and MM patients and compared the hematological parameters between patients with and without PNH-phenotype.


   Materials and Methods Top


Patients

This study evaluated treatment naive, newly diagnosed 54 CLL and 29 MM patients. The patients were diagnosed as having CLL and MM based on the standard diagnostic criterion. [15],[16] CLL patients were staged according to Binet staging system and Durie Salmon system was used for MM patients. [17],[18] Complete blood counts, renal function tests, serum electrolytes, liver function tests, and bone marrow examination was done in all the patients. Additionally, MM patients also underwent testing for serum and urine electrophoresis, serum and urine immunofixation and skeletal survey.

Test for PNH-Phenotype

PNH-phenotype was tested using flow cytometry technique on fresh ethylenediaminetetra-acetic acid (EDTA) anticoagulated blood samples. Monoclonal antibodies (MoAb) against CD55 and CD59, conjugated with flourescein isothiocyanate (FITC) and phycoerythrin (PE) were used. An isotype-matched IgG1 monoclonal antibody was used as a negative control. The protocol followed was that described by Mitsuhiro et al. [19] Briefly, 1 × 10 6 RBC (washed with phosphate buffered saline-PBS) from control (known normal) and patients were incubated in separate tubes with CD55 and CD59 MoAb for 30 minutes at room temperature in dark. This was followed by washing with PBS and cell button was resuspended in 0.5 ml PBS. The cells were immediately acquired and analyzed on flow cytometer (FACS Calibur using Cell-Quest software; BD Biosciences). The presence of more than 5% RBC deficient in CD55 and CD59 were taken to indicate PNH-phenotype.

Tests to Establish/Exclude Hemolysis

The patients showing deficiency of CD55 and/or CD59 on flow cytometry were subjected to additional conventional tests for documenting/ excluding coexisting hemolysis, including plasma and urine hemoglobin, urine hemosiderin, Hams acidified serum lysis test, and sucrose lysis test. [20],[21]

Statistical Analysis

The results were tabulated and analyzed statistically using Statistical Package for Social Science (SPSS) 13.0 software for Windows (SPSS INC., Chicago, IL, USA). Descriptive statistics were used to analyze the data in both cases and controls. Differences between groups were assessed using the Mann-Whitney test or the chi-square test as indicated. Difference between the different hematological parameters among the positive and negative patients was analyzed by Mann-Whitney test. The correlation between the presence of CD55 deficient and/or CD59 deficient RBC and the stage of disease was evaluated with Spearman's correlation coefficient. A value of P < 0.05 was considered as statistically significant.


   Results Top


In the present study, which was planned to determine the incidence of PNH-phenotype by flow cytometry at initial diagnosis of CLL and MM, total number of 83 patients were studied (CLL = 54, MM = 29). Age of patients ranged from 32-85 years with a median age of 57 years.

Chronic Lymphocytic Leukemia

Fifty four newly diagnosed treatment naive CLL patients were included in the study, having a median age of 64 years with male: female ratio of 2.4:1.

Nine patients were found to have isolated CD55 deficient RBC, CD59 deficiency was not noted in any case. The percentage of CD55 deficient RBC population was less than 10% of the total RBC population. Hams and sucrose lysis tests were negative in all these patients. No statistical significance was found between presence of CD55 deficient RBC and Binet staging [Table 1]. Similarly, the presence of CD55 deficient RBC and requirement of blood transfusions was also not statistically significant [Table 2]. Laboratory parameters when compared between the two groups- CLL patients with and without CD55 deficient RBC, none were found to be statistically significant [Table 3].
Table 1: Statistical Significance of Binet stage distribution in CLL patients with CD55 defi cient RBC

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Table 2: Correlation of presence of CD55 defi cient RBC with blood transfusions in CLL

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Table 3: Laboratory parameters of CLL patients with and without CD55 deficient RBC and their statistical significance

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Multiple Myeloma

Twenty nine newly diagnosed treatment naive MM patients were included in the study, having a median age of 57 years and a male: female ratio of 3.2:1.

Two patients were found to have isolated CD55 deficient RBC, but none had CD59 deficient RBC population. The percentage of CD55 deficient RBC population was less than 10% of the total RBC population. Hams and sucrose lysis tests were negative in 1 MM patient and the other was lost to follow-up. Both MM patients with CD55 deficiency had stage 3b disease. No statistical significance was found between the presence/absence of CD55 deficient RBC and requirement of blood transfusions [Table 4] and between laboratory parameters between two groups of patients [Table 5].
Table 4: Correlation of presence of CD55 defi cient RBC with blood transfusions in MM

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Table 5: Laboratory parameters of MM patients with and without CD55 deficient RBC and their statistical significance

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When presence of CD55 deficient RBC was compared between CLL and MM patients, no statistical significance was observed [Table 6].
Table 6: Statistical significance of presence of CD55 defi cient RBC in MM and CLL

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   Discussion Top


Studies have shown the presence of 'PNH-sc' or the 'PNH-phenotype' of variable degree in a series of patients with lymphoproliferative syndromes and MM. [10],[11],[12],[13],[14] Since the presence of PNH-phenotype has proved to be of clinical importance in AA and MDS-RA patients, its presence correlating with high probability of responding to immunosuppressive therapy; we conducted the present study for detection and determination of relevance of PNH-defect in patients with CLL/MM.

In the current study, we screened RBC for PNH-phenotype in patients with newly diagnosed CLL and MM by flow cytometry to assess the incidence of PNH-phenotype in this group of patients. Isolated CD55 deficiency was observed in 16.66% (9/54) and 6.89% (2/29) CLL and MM patients, respectively. None of the patients had PNH-phenotype, i.e., double negative for CD55 and CD59. In a similar study by Meletis et al. [10],[11] they found isolated CD55 deficiency in 12.2% (6/49) patients with CLL and 46.5% (20/43) patients with MM. However, in their study in addition, PNH-phenotype/double negative RBC population for CD55 and CD59 were reported in 12.2% (6/49) and 9.3% (4/43) patients with CLL and MM, respectively. Isolated CD59 negativity was not found in any patient in the present study and also in study by Meletis et al. [11]

The difference in our study in incidence of PNH-phenotype may be due to a chance association. PNH clone may be incidental in this disease. Another plausible explanation for the low detection of CD55 and CD59 deficient cells may be in vivo lysis of cells showing altered expression of GPI complex and problems with the detection of single cells. RBC showing deficit of GPI-AP are known to be hemolyzed easily. In this study, we found only isolated deficiency of CD55, none had isolated CD59 deficiency. This is in agreement with the findings in literature, where loss of isolated CD55 has more frequently been reported than loss of CD59 alone. [10],[11],[12],[13],[14]

The proportion of the RBC without CD55 did not exceed 10% of the total RBC population in the patients studied by us. This phenomenon supports the hypothesis of a pre-existing clone. On the other hand, expression of CD55 is controlled mainly by transcriptional regulation, and the presence of isolated CD55 deficiency might suggest that a possible mutation in the disease clone may affect the presence of CD55 on the cell membrane. [22]

In our study population, we did not find PNH-phenotype in both CLL and MM patients; however, isolated CD55 deficiency was more frequently observed in CLL than MM patients. However, the difference did not reach statistical significance, but a larger sample size may be needed to look for a true difference or otherwise.

PNH-phenotype has also been reported in CLL and MM patients following therapy. Hertenstein et al. [23] reported presence of GPI-deficient T-lymphocytes in patients with CLL after administration of Campath-1H (anti-CD52 antibody). They hypothesized that an attack against the CD52, another GPI-anchored protein, might lead to the expansion of a GPI-anchor-deficient cell population with the phenotypic characteristics of PNH cells. Kulagin et al. [24] reported a minor PNH clone in a heavily pre-treated (with vincristine, high-dose methylprednisolone, melphalan, bisphosphonates, cyclophosphamide, and bone marrow transplantation) 49-year-old patient with MM who developed severe pancytopenia. The size of PNH clone in their patient was 2.25% in granulocytes and 9.78% in RBC. Hams test and sugar lysis test were negative, and there was no clinical or laboratory manifestations of hemolysis. They also found hematological response to cyclosporin therapy which they associated with the presence of PNH clone. However, they suggested that further investigations are required to clarify the association between MM, chemotherapy-induced marrow failure, and PNH clone expansion.

None of our patients with CD55 deficiency showed evidence of hemolysis; Hams and sucrose lysis test were negative in all these patients; these results are in concordance with the data presented in literature. This is possibly due to the small population of erythrocytes with reduced expression of GPI-AP. On the other hand, it has been shown that CD55 or CD59 deficiency alone is not sufficient to produce homologous hemolysis and this phenomenon may also explain the absence of hemolysis in patients with isolated reduction of these antigens from cell membrane. [25]

Terpos et al. [13] observed a strong correlation between the presence of PNH-phenotype and bone resorption in patients with MM. Because of the small number of patients with CD55 deficiency and no patient with PNH-phenotype in our study, it was not possible to ascertain the clinical significance of the incidence of PNH-phenotype and the stage of disease and disease characteristics like the grade of bone lesions. However, most of our patients had significant bone disease, and hence it may be summarized that PNH-phenotype has no direct relationship with bone disease.

We also did not find any statistically significant differences in the hematological and biochemical parameters; and the transfusion requirements of the patients with and without CD55. Thus, from this study, it appears that the clinical presentations of the disease or the hematological parameters do not seem to have any relationship with the development of the PNH-clone.

This study used the flow cytometry method for identification of PNH-phenotype, using antibodies directed against GPI-AP, which is considered to be the most sensitive and informative assay available for diagnosis of PNH. [2] Additionally, flow cytometric analysis is more than binary (i.e., positive or negative); it identifies a population of GPI-AP-deficient cells and can both determine the percentage of cells that are abnormal and identify discrete population of different degrees of deficiency. Also, flow cytometry is especially useful in detecting PNH-sc as it is sufficiently sensitive to detect accurately 3% GPI-AP-deficient cells. By using 2-color analysis and careful gating, sensitivity can be increased by 3 orders of magnitude. [26] It has been reported that the optimal approach for detection of PNH-sc is the simultaneous evaluation of both RBC and granulocytes for the expression of CD55 and CD59. [27] Detection of cell clone possessing PNH-phenotype may be facilitated by application of MoAb against CD11b and glycophorin in concert with CD55 and CD59, this approach provides for proper gating of the appropriate subpopulations.


   Conclusion Top


The present study has found isolated CD55 deficiency in patients with CLL and MM; however, PNH-phenotype was not reported in any case. Clinical presentation of disease or the hematological parameters do not seem to have any relationship with the development of the PNH-clone. Further studies with large number of patients are needed to validate these findings. In addition, simultaneous evaluation of granulocytes and erythrocytes might increase the sensitivity of the detection of PNH-phenotype.

Since the presence of PNH-phenotype has proved to be of clinical importance in AA and MDS-RA patients, studies for detection of this phenotype and determination of its relevance in patients with other hematological disorders is warranted.

 
   References Top

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Correspondence Address:
S Varma
Professor, Head, Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh-160012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.97871

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

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