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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 699-703
Flow cytometric analysis of erythrocytes in paroxysmal nocturnal hemoglobinuria reveals superiority of CD59 as a diagnostic marker compared to CD55


Hematopathology and Flow Cytometry sections, Super Religare Laboratories, Mumbai, India

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Date of Web Publication27-Oct-2010
 

   Abstract 

Context: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by complement-mediated hemolysis due to reduced expression of glycosyl phosphatidylinositol-anchored complement deactivating proteins such as CD55 and CD59 on RBC. Flow cytometric analysis of CD55 and CD59 expression by RBC is a reliable tool for the diagnosis of PNH. Aims: Detection and quantification of PNH clone and comparison of the relative role of CD55 and CD59 expression by RBC in the diagnosis of PNH. Materials and Methods: Flow cytometric analysis of RBC was performed in blood samples of 239 patients by direct immunofluorescence using monoclonal anti-CD55 and anti-CD59 antibodies. CD55 and CD59 expressions by RBC were compared in 54 cases in which PNH clones were detected. Results: Out of 54 cases, 85% and 72% revealed CD59 and CD55 negative populations, respectively. Various combinations of type II and III erythrocytes could be identified in all cases having CD59 deficient RBC. In contrast, distinct populations of CD55-deficient RBC were seen in only 33% cases. In the remaining (67%) cases, CD55 negative RBC caused sloping of the ascending limb of the histogram resulting in difficulties in interpretation. Fifteen percent cases had false CD55-deficient RBC and in 23% cases anti-CD55 antibody failed to identify PNH clones which were detected by CD59. Conclusion: CD59 is a better marker for the diagnosis of PNH. Although CD55 negativity supported the diagnosis of PNH in cases with CD59-deficient RBC, its role as an independent diagnostic marker for PNH is questionable due to its lower sensitivity and specificity.

Keywords: CD55, CD59, flow cytometry, Paroxysmal nocturnal hemoglobinuria

How to cite this article:
Tembhare P, Ramani M, Syed K, Gupta AD. Flow cytometric analysis of erythrocytes in paroxysmal nocturnal hemoglobinuria reveals superiority of CD59 as a diagnostic marker compared to CD55. Indian J Pathol Microbiol 2010;53:699-703

How to cite this URL:
Tembhare P, Ramani M, Syed K, Gupta AD. Flow cytometric analysis of erythrocytes in paroxysmal nocturnal hemoglobinuria reveals superiority of CD59 as a diagnostic marker compared to CD55. Indian J Pathol Microbiol [serial online] 2010 [cited 2014 Oct 31];53:699-703. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/699/72042



   Introduction Top


Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired stem cell disorder associated with periodic episodes of intravascular hemolysis. [1] This benign clonal disease is caused by abnormalities of the X-linked phosphatidylinositol glycan class A (PIG-A) gene and is associated with cytopenias and thrombosis. This gene which is located on the short arm of the X chromosome (Xp22.1) codes for glycosylphosphatidyl inositol (GPI) that acts as the anchor for many cell surface proteins. [1],[2],[3],[4] Abnormalities of this gene result in total or partial deficiency of blood cell membrane proteins such as decay accelerating factor (DAF, CD55), membrane inhibitor of reactive lysis (MIRL, CD59), and other proteins normally attached to the GPI spine. Three broad types of clinical situations are associated with the presence of PNH clones, (a) hemolytic anemia and hemoglobinuria, (b) venous thrombosis and (c) aplastic anemia and refractory anemia/myelodysplastic syndrome. The presence of very small populations of PNH cells has also been reported in asymptomatic individuals.

The hitherto popular biochemical tests, e.g. Ham's and sucrose lysis tests, are at best presumptive [4] while the newer, flow cytometric (FCM) assays, are more definitive. [1],[2],[3],[4],[5] The latter group includes FCM evaluation of RBC and WBC in the peripheral blood for expression of GPI-linked proteins using monoclonal antibodies against CD55, CD59 (in RBC and/or WBCs), and other markers such as CD14, CD16, CD24, CD64, CD66b (in WBCs). [2],[6],[7] Recently, FCM quantification of specific GPI-anchor binding by fluorochrome-labeled, inactivated bacterial toxin aerolysin (FLAER) has been developed for WBC. [6],[8],[9] The choice of the assay cells and that of the CD markers, alone and/or in combination, in the diagnosis of PNH are still a matter of debate although WBC analysis using FLAER is emerging as the method of choice. The stability of expression of the aforementioned CD markers and viability of cells are areas of concern when dealing with older samples, especially in the context of distant testing. Red cells are more s table in both respects compared to WBC. Hence, the former cells seem more sui table for analysis in samples older than 24 hour. In the present study, we report the results of FCM analysis of RBCs using monoclonal antibodies against CD55 and CD59. We also present our data on the relative role of CD55 and CD59 expression by the red cells in the diagnosis of PNH.


   Materials and Methods Top


Peripheral blood RBC from 239 suspected cases of PNH referred to our laboratory over the past 3 years were examined for CD55 and CD59 expression by FCM using a commercially available reagent kit, "RED QUANT" (procured from M/S Biocytex, France) consisting of a calibrator and monoclonal anti-CD55 and anti-CD59 antibodies. Out of 239 patients, 54 (22.6%) cases (31.5% females and 68.5% males, age range 13-72 years) revealed the presence of one or more clones of PNH cells. The cells were processed as per the manufacturer's instructions. Briefly, one aliquot each (10 μl) of properly mixed anticoagulated (EDTA) blood was diluted 150 times with phosphate buffered saline and was labeled with anti-CD55-fluorescein isothiocyanate (FITC) and anti-CD59-FITC. Samples were analyzed on FC-500 flow cytometer (M/S Beckman Coulter, USA) and a minimum of 10,000 events were acquired. For the purpose of analysis, RBCs were identified by light-scatter properties [Figure 1]a. A cut off for positive and negative cells was set by using the calibrator beads (a and b) included in the reagent kit with a lot-specific multiplier [Figure 1]b and c. Cells with fluorescence intensity below this predetermined cut off were considered deficient in the CD antigen of interest. More than 3% of abnormal cells were required for an abnormal test result for a particular RBC marker. Results were expressed as percent deficient cells. In addition, sucrose hemolysis tests and Ham's acid hemolysis tests were also performed in cases which revealed red cells deficient in one of the two markers tested, i.e. either CD55 or CD59. A comparative analysis of expression of CD55 and CD59 by red cells was performed in all cases.
Figure 1 :(a) Forward scatter (FSC) log vs. side scatter (SSC) log gating strategy for erythrocytes and beads. (b) Setting of a negative limit for anti-CD55-FITC with á beads. (c) Setting of a negative limit for anti-CD55-FITC with â beads.

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Statistical Methods

For statistical evaluation of the data under analysis, range, positive, and negative predictive values were determined using the SPSS and Microsoft Office Excel software programs. The Student t-test was used to perform comparative statistical analysis between the patterns of staining for CD55 and CD59 and P value was calculated. P < 0.01 was considered to be associated with statistical significance.


   Results Top
[Figure 2]a-d

Out of total 54 cases, 85% (46) cases revealed CD59 negative population and 72% (39) cases revealed CD55 negative population. In cases with CD59 negative clones, 5 (10.2%) cases showed all three types (type I, II, and III) of PNH red cells [Figure 2]d, 8 (16.32%) cases showed only type III deficient cells, and 33 (67.34%) cases revealed only type II cells. In contrast, out of the 39 cases with CD55 negative clones, only 13 (33.33%) showed a single distinctly negative population of red cells, possibly representing both type II and III cells [Figure 2]a. In the remaining 26 (66.66%) cases, although there were CD55 negative cells, they did not appear as a separate population. Rather they caused sloping of the ascending limb of the histogram [Figure 2]b [Table 1]. Moreover, the flow cytometric detection rate of PNH red cells using anti-CD59 antibody is found significantly greater than using anti-CD55 antibody (P = 0.0086).
Table 1 :Distribution of PNH red cells percentages and flow cytometric subtypes of anti-CD55 negative and anti-CD59 negative expression


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Figure 2 :RBC analysis in cases of PNH by FCM using REDQUANT Kit. (a) Shows a small anti-CD55-FITC negative PNH clone of RBCs falling the negative cutoff set with α beads. (b) Shows anti-CD55-FITC negative PNH clone of RBCs falling on the left slope of peak of normal RBCs without any distinct population. (c) Shows a small anti-CD59-FITC negative PNH clone of RBCs falling within the "negative" region set with β beads. (d) Shows nicely separated all three types of erythrocytes with anti-CD59-FITC. Type I-anti-CD59-FITC positive RBCs, type II-partially anti-CD59-FITC negative RBCs, and type III-completely anti-CD59-FITC negative RBCs.

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A comparative analysis of CD55 and CD59 deficient RBC revealed 31 (57.4%) cases showing both CD55 and CD59 negative populations. Additionally, in 8 (15%) cases only CD55 negative cells and 15 (22.8%) cases only CD59 negative cells were identified. Hence, in these cases sucrose hemolysis test and Ham's acid hemolysis test were carried out as additional tests for diagnosis [Table 2]. All 8 cases with only CD55 negative cells yielded negative results in these additional tests and out of the 15 cases with only CD59 negative cells, sucrose hemolysis test was positive in 8 and Ham's acid hemolysis test was positive in 12. In the remaining three cases these tests could not be performed due to insufficient sample. Furthermore, in four out of eight cases showing only CD55 negative cells, a reanalysis was performed on fresh samples which did not revealed any PNH clone and in remaining four cases repeat samples were not available. These findings proved that 22.8% cases showed false CD55 negative RBC, whereas in 13% cases CD55 did not contribute to the diagnosis of PNH. Five of these eight cases had a history of recent blood transfusion which may be one of the contributing factors. Predictive values for both CD55 and CD59 were calculated. Positive predictive values for anti-CD55 antibody and anti-CD59 antibody were 79.5% and 100%, respectively, while negative predictive values were 92.8% and 100%, respectively. However, the complete clinical profile, transfusion history, and follow up data of the patients were not available. In 41 cases in which clinical data were available, we correlated types of PNH red cells with their clinical presentation [Table 3]. Most common presentation of PNH patients was anemia (hemolytic anemia) followed by pancytopenia (bone marrow failure) and deep vein thrombosis. The study revealed that patients with signs and symptoms of hemolytic anemia showed the presence of predominantly type III PNH red cells and patients with pancytopenia showed predominantly type II red cells. Unfortunately, complete clinical profile and follow up information was not available in some cases.
Table 2 :Results of sucrose hemolysis test and Ham's acid hemolysis test in cases with either only anti-CD55 negative or only antiCD59 negative red cell population


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Table 3 :Correlation between types PNH red cells and the clinical presentations in the patients. of PNH


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   Discussion Top


PNH is a rare disease whose molecular pathogenesis has been elucidated in great detail over the past decade. [1],[2],[3],[4],[5] Though traditional laboratory methods for diagnosing PNH relied on demonstration of phenotypic vulnerability of the affected red cells to activated complement, it soon became clear that the disease fundamentally and reproducibly affected the expression of GPI-anchored cell surface molecules [2],[3],[4],[6] which can be sensitively identified with FCM. Therefore, FCM-based assays appeared to be the ideal modality for unequivocaldiagnosis of this disease and gained widespread acceptance as the reference method. [2],[4],[5],[6],[8] Unfortunately, as yet there is no consensus on the most appropriate procedure for doing this. [4] Clearly there is need for guidelines for flow cytometric diagnosis of PNH.

PNH clone size can be more accurately enumerated in WBC, because WBC half-life is normal in PNH, whereas RBC half-life, especially for type III RBC, is shortened due to hemolysis. Furthermore, PNH RBCs, as opposed to WBCs, are diluted by transfusion which additionally diminishes the value of RBCs for evaluation of clonal size. However, determination of CD55 and CD59 is technically more convenient in RBC compared to WBC especially in a reference FCM laboratory [10] on account of questionable viability of the WBC more than 24 h after collection of the sample and poor signal to noise ratio for these reagents in WBC. Furthermore, red cell analysis in PNH allows an accurate determination of subpopulations of deficient cells, namely types II and III which in turn can predict the clinical phenotype; patients with >20% type III (completely deficient) cells almost always show clinical evidence of hemolysis. [11] In addition, FCM analysis of RBCs in PNH also helps in monitoring response to treatment with Eculizumab, a humanized antibody that inhibits the activation of terminal complement components thereby prolonging the life of PNH cells. [5]

Several studies involving FCM analysis of RBC as well as WBC have been published in the literature. [2],[5],[7],[8],[9],[11],[12],[13],[14],[15] However, only a few attempts have been made to evaluate the relative contribution of anti-CD55 and anti-CD59 antibodies in the diagnosis of PNH. [3],[16] Hence, there was a need to determine whether there was a synergistic effect between the two antibodies in enhancing the accuracy of diagnosis of suspected cases of PNH when used simultaneously. Cost of reagents would be another area of concern. Some commercial kits contain both the antibodies and ideally they should complement each other in increasing the specificity and the sensitivity of the tests. Otherwise one could use the superior of the two for diagnosis and not both. In this study, we present the largest data on flow cytometric analysis of RBC for the diagnosis of PNH from India with a focus on detecting and quantifying the PNH clones in patients and to compare the relative role of CD55 and CD59 expression by RBC in the diagnosis of PNH.

A number of earlier studies have examined the expression of GPI-linked antigens by red cell in PNH. [3],[15],[16] Although there is no consensus on the gating strategy for red cells in the literature, the use of forward scatter (FSC) and side scatter (SSC) amplification in log mode to establish an acquisition/analysis gate based on physical characteristics of red cells is commonly used. [4],[6] Gating strategy using Glycophorin-A (CD235a) with multicolor FCM has also been tried but the significant technical problem due to red cell agglutination is a major consideration when attempting multicolor staining of red cells. [4] The presence of low levels of protein in cell washing solutions and monoclonal reagents can enhance agglutination of red cells if coated by antibody. Therefore, single-antibody staining of red cells is recommended. [4] In the present study, we too adopted the FSC vs. SSC gating strategy and single-antibody staining of red cells using a commercially available reagent kit, "RED QUANT" (M/S Biocytex, France).

In a flow cytometric analysis of RBC and WBC using anti-CD55 and anti-CD59 antibodies, Hall and Ross reported 39% cases with type II and III PNH clones and the remaining (61%) cases with only type III red cells. [3] Red cells of intermediate abnormality (type II) were identified in flow cytometry by Rosse et al.[17] and Shichishima et al.[15] In the present study using CD59 expression we found 10.2% cases having all three types of erythrocytes (types I, II, and III), 16.3% cases with only type III cells and 67.3% cases revealed only type II cells. Hsi evaluated RED QUANT kit in normal as well as PNH cases and found anti-CD55 less sensitive than anti-CD59 in detecting PNH red cells. [2] Hall and Ross also showed that CD59 was a better and more sensitive diagnostic marker than CD55. [3] In the present study, in the majority (66.6%) of cases CD55 negative cells did not appear as a separate population, rather they caused sloping of the ascending (left) portion of the histogram causing difficulties in interpretation. On one hand anti-CD55 failed to detect the PNH red cells in 22.8% of our cases that were detected by anti-CD59, on the other hand in 15% cases it showed the presence of the CD55-deficient red cells that was not supported by CD59 expression data when a cut off value of >3% was used. In 40% of these cases there was a history of recent blood transfusion which may be one of the contributing factors for false results. Hence our study also revealed that CD55 is a less sensitive marker than CD59 in red cell analysis of PNH (P = 0.0086). In 2001, Oelschlaegel U and co-workers showed that this limitation can be overcome using the combination of the RED QUANT and CELL QUANT kits which enables the differential diagnosis of PNH clones by a standardized, simple and rapid approach. [14] However, in CELLQUANT granulocyte kit there may be a risk of false positivity as it has been shown that CD59 had an elevated false positive rate in WBC analysis. [2] In conclusion, our study revealed that in flow cytometric analysis of erythrocytes for the diagnosis of PNH, CD59 is a superior marker than CD55 and though CD55 negativity supports the diagnosis of PNH cases, in many instances it might yield false positive or false negative results. Hence, the role of CD55 in the red cell analysis of PNH cases seems questionable. In contrast to CD59 which is strongly expressed by the red cells, CD55 is weakly expressed by these cells. Therefore, FITC-conjugated anti-CD55 is an inferior reagent for the diagnosis of PNH compared to anti-CD59. Phycoerythrin-conjugated anti-CD55 antibody could be a better reagent in this respect and would allow dual color analysis of red cells provided the potential problem of excessive agglutination of the red cells on account of the use of two anti-erythroid antibodies is addressed.

 
   References Top

1.Brodsky RA. Paroxysmal nocturnal hemoglobinuria. In: Hoffman R, Benz EJ Jr, Shattil SJ, Furie B, Cohen H, Silberstein LE, et al, editors. Hematology: Basic Principles and Practice. 4th ed. Philadelphia: Elsevier; 2005. p. 419-27..  Back to cited text no. 1
    
2.Hsi ED. Paroxysmal nocturnal hemoglobinuria testing by flow cytometry: Evaluation of the REDQUANT and CELLQUANT kits. Am J Clin Pathol 2000;114:798-806.  Back to cited text no. 2
    
3.SE Hall, WF Rosse. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 1996;87:5332-40.  Back to cited text no. 3
    
4.Richards SJ, Rawstron AC, Hillmen P. Application of Flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry 2000;42:223-33.  Back to cited text no. 4
    
5.Hillmen P, Hall C, Marsh JC, Elebute M, Bombara MP, Petro BE, et al. Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med 2004;350:552-9.  Back to cited text no. 5
    
6.Richards SJ, Hill A, Hillmen P. Recent Advances in the diagnosis, monitoring, and management of patients with paroxysmal nocturnal hemoglobinuria. Cytometry 2007;72B:291-8.  Back to cited text no. 6
    
7.Piedras J, Lo΄pez-Karpovitch X. Flow cytometric analysis of glycosylphosphatidyl-inositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size. Cytometry 2000;42:234-8.  Back to cited text no. 7
    
8.Brodsky RA, Mukhina GL, Li S, Nelson KL, Chiurazzi PL, Buckley JT, et al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000;114:459-66.  Back to cited text no. 8
    
9.Sutherland DR, Kuek N, Davidson J, Barth D, Chang H, Yeo E, et al. Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytometry 2007;72B:167-77.  Back to cited text no. 9
    
10.Kjeldsberg C, Elenitoba-Johnson K, Foucar K, Hussong J, McKenna R, Perkins S, et al. Practical Diagnosis of Hematologic Disorders. 3 rd ed. Chicago: ASCP Press; 2000. p. 171-81.  Back to cited text no. 10
    
11.Hillmen P, Richards S. Flow cytometry in PNH: serial analysis and the predication of outcome. Blood 1999;94:412-5.  Back to cited text no. 11
    
12.Herna΄ndez-Campo PM, Martý΄n-Ayuso M, Almeida J, Lo΄pez A, Orfao A. Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets. Cytometry 2002;50:191-201.  Back to cited text no. 12
    
13.Pakdeesuwan K, Wanachiwanawin W, Siripanyaphinyo U, Pattanpanyasat K, Wilairat P, Issaragrisil S. Immunophenotypic discrepancies between granulocytic and erythroid lineages in peripheral blood of patients with paroxysmal nocturnal hemoglobinuria. Eur J Haematol 2000;65:8-16.  Back to cited text no. 13
    
14.Oelschlaegel U, Besson I, Arnoulet C, Sainty D, Nowak R, Naumann R, et al. A standardized flow cytometric method for screening paroxysmal nocturnal Haemoglobinuria measuring CD55 and CD59 expression on erythrocytes and granulocytes. Clin Lab Haematol 2001;23:81-90.  Back to cited text no. 14
    
15.Shichishima T, Terasawa T, Saitoh Y, Hashimoto C, Ohto H, Maruyama Y. Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypic analysis of erythrocytes using two-colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF. Br J Haematol 1993;85:378-86.   Back to cited text no. 15
    
16.Bessler M, Mason PJ, Hillmen P, Luzzatto L. Mutations in the PIG-A gene causing partial deficiency of GPI-linked surface proteins in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol 1994;87:863-4.  Back to cited text no. 16
    
17.Rosse WF, Hoffman S, Campbell M, Borowitz M, Moore JO, Parker CJ. The erythrocytes in paroxysmal nocturnal haemoglobinuria of intermediate sensitivity to complement lysis. Br J Haemato1 1991;79:97-9.  Back to cited text no. 17
    

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Correspondence Address:
Amar Das Gupta
Executive Director & Mentor-Hematology Services, Super Religare Laboratories, Prime Square 1, Gaiwadi Industrial Estate, S. V. Road, Mumbai 400 063
India
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DOI: 10.4103/0377-4929.72042

PMID: 21045396

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