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Annals of Clinical & Laboratory Science 39:283-288 (2009)
© 2009 Association of Clinical Scientists

Flow Cytometric Detection of Platelet-Associated Immunoglobulin in Patients with Immune Thrombocytopenic Purpura and Nonimmune Thrombocytopenia

Hee Jin Huh1,*, Chan Jeoung Park1, Sang We Kim2, Sang Hee Han1, Seongsoo Jang1 and Hyun Sook Chi1
1 Departments of Laboratory Medicine and 2 Oncology, College of Medicine, University of Ulsan and Asan Medical Center, Seoul, Korea

Address correspondence to Chan Jeoung Park, M.D. Dept. of Laboratory Medicine, Asan Medical Center, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, South Korea; tel 82 2 3010 4508; fax 82 2 478 0884; e-mail cjpark{at}amc.seoul.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We increased the specificity of flow cytometric detection of platelet-associated immunoglobulin (PAIg) by a combination of platelet gating and cutoff for positivity determined by the use of receiver operating characteristic (ROC) curve analysis, and we evaluated the significance of elevated PAIg in non-immune thrombocytopenic purpura (ITP) patients. Blood samples from 118 patients with a platelet count <100 x 109/L were used in this study. Flow cytometric detection of PAIg was performed. To obtain the cutoff of the surface-bound immunoglobulin for the discrimination of ITP and non-ITP, ROC curve analysis was used. The sensitivity of a positive PAIgG and PAIgM test for ITP in thrombocytopenic patients was 74.6%; the specificity was 79.7%; the positive predictive value 78.6%; and the negative predictive value 75.8%. Among 3 patients with myelodysplastic syndrome, 2 showed increased PAIg. Six of 20 patients with benign disease showed positivity for PAIg. Among these patients, 4 with elevated PAIg were diagnosed with liver disease. This study demonstrates that flow cytometric detection of PAIg combined with ROC curve analysis is a convenient, sensitive, and specific test, compared to previous methods, and it is useful for the differential diagnosis of thrombocytopenic patients.

Keywords: platelet associated immunoglobulin, flow cytometry, immune thrombocytopenic purpura


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In 1996, the American Society of Hematology decided that the diagnosis of immune thrombocytopenic purpura (ITP) should exclude other causes of thrombocytopenia, based principally on the patient’s history, normal physical examination, and complete blood count (CBC) that is normal, excepting for the thrombocytopenia [1]. The platelet-associated immunoglobulin (PAIg) study is not defined in the diagnostic algorithm of ITP. Nevertheless, the PAIg test continues to be widely used in patients with thrombocytopenia to distinguish between ITP and non-ITP and to monitor the response of ITP patients during immunosuppressive treatment [2].

Several methods have been employed to detect and characterize the antiplatelet autoantibodies in ITP. An increase of immunoglobulin on platelet surfaces was detected and initially was considered to have the same significance as the direct antiglobulin test in acquired hemolytic anemia. However, it has become evident that elevation of immunoglobulin on platelet surfaces can be a nonspecific phenomenon associated with thrombocytopenic states [3,4]. The phase II assay described by Kelton [5] for the detection of PAIg included radioimmunoassay, enzyme-linked immunoassays, and flow cytometry. Flow cytometry is considered a sensitive method for the measurement of PAIgG [6]. However, PAIgG detection by flow cytometry has low specificity, since elevated levels have been noted in patients with both ITP and non-ITP [7].

Patients with liver cirrhosis or myelodysplasia were included in several studies to evaluate methods for detection of PAIg [7]. It is suggested that these patients have platelet autoantibodies [8,9]. It is reported that 38% of patients with liver cirrhosis had a detectable level of anti-GPIIb IIIa antibodies and that autoantibody mediated platelet destruction may contribute at least in part to cirrhotic thrombocytopenia [8]. It has been proposed that because elevated PAIgG, some of which are antiplatelet autoantibodies, is frequent present in critically ill thrombocytopenic patients, thrombocytopenia may be related to the presence of platelet autoantibodies, as occurs in patients with idiopathic thrombocytopenic purpura [10].

In the present study we increased the specificity of flow cytometric detection of PAIg by a combination of platelet gating and the cutoff value determined by the use of receiver operating characteristic (ROC) curve analysis. Moreover, we delineate the significance of elevated PAIg in non-ITP patients with thrombocytopenia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients.  Blood samples from 118 patients (57 males, 61 females) with a platelet count <<100 x 109/L due to different pathological conditions were used in this study. All patients were diagnosed and enrolled at our institution between May 2004 and December 2004. Their median age was 46 yr (range 1 mo to 88 yr).

For analysis, the patients were assigned to groups with regard to the clinical diagnosis: ITP (n = 59) and non-ITP (n = 59), which included hematologic diseases (n = 19; acute leukemia, 5; lymphoproliferative disease, 3; myelodysplastic syndrome, 3; and other hematologic diseases, 8), malignancy (n = 20; hepatocellular carcinoma, 2; lung cancer, 4; cervical cancer, 2; stomach cancer, 3; and other malignancies, 9), and benign disease (n = 20; infection, 5; liver disease, 4; and other diseases, 11). Diagnoses were not influenced by the results of the current PAIg test. The diagnosis of ITP was made according to American Society of Hematology criteria and the classification of acute and chronic ITP was determined by patient history and follow-up review. Among the patients with ITP, 10 were diagnosed as acute ITP and 49 as chronic ITP.

In order to investigate the influence of plasma globulin fraction, platelet counts, mean platelet volume (MPV) tests, complete blood counts, and relevant chemistry tests were performed.

Flow cytometry of PAIg.  Samples of 2–5 ml of peripheral blood were collected in EDTA. The platelet concentration was determined using an automatic cell counter (XE 2100, Sysmex Corporation, Kobe, Japan). The platelet-rich plasma was made by centrifugation at 100 g for 15 min. The isolated platelets were washed twice with phosphate buffered saline containing 10 mM EDTA and 0.5% bovine albumin (PBS/EDTA) and adjusted to a platelet concentration of 50 x 109/L. Fluorescein isothiocyanate (FITC)-conjugated F(ab)2 fragments of rabbit anti-human IgG, IgA, and IgM (Dako, Glostrup, Denmark) were used to detect PAIg; pycoerythrin (PE)-conjugated CD41 monoclonal antibody (Becton-Dickinson, Franklin Lakes, NJ, USA) was used to identify the platelet population (Fig. 1Go). The nonspecific fluorescence was established by the isotypic control of PE- or FITC-conjugated mouse monoclonal antibody. Platelets were dually stained with FITC-conjugated antibody and PE-conjugated CD41 monoclonal antibody. Three tubes were prepared for 200 µL of platelet suspension mixed with 4 µL of each FITC-conjugated antibody and 4 µL of PE-conjugated CD41 monoclonal antibody. The mixture was incubated at room temperature for 15 min. Flow cytometry was performed using a FACScan cytometer (Becton-Dickinson).


Figure 1
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  		Fig. 1. Flow cytometric detection of platelet-associated immunoglobulins in an ITP patient with positivity of PAIgG, PAIgA, and PAIgM. (A) platelet gating; (B) CD41(+) regating after platelet gating; (C) PAIgG; (D) PAIgA; and (E) PAIgM.

 
The platelet population was gated with forward scatter light and right angle scatter light, and CD41 positive platelets were regated. Blood samples of patients with a normal platelet count range were used as a control and the cutoff line for positivity was positioned with <2% positivity in controls. To obtain the cutoff for positivity of surface-bound immunoglobulin for the discrimination of ITP and non-ITP, ROC curve analysis was used.

Statistics.  Data are presented as median, count, or proportion. Mann-Whitney U test was used to compare the platelet count, MPV, globulin, and positive percentage of PAIg between two groups. The level of significance was set at p <0.05. All statistical analyses were performed using SPSS version 11.5 (SPSS, Chicago, IL, USA). The sensitivity of PAIg was determined by the percentage of ITP samples with PAIg positivity among the total number of ITP samples; the specificity was calculated as the percentage of non-ITP samples with PAIg negativity among the total number of non-ITP samples. Positive predictive value was calculated as the percentage of ITP samples with PAIg positivity among the total number of positive samples; negative predictive value was calculated as the percentage of negative non-ITP samples among the total number of negative samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cutoff value of positivity of PAIg for the discrimination of ITP and non-ITP was 12% by ROC curve analysis and any specimen with >12% was considered to be positive. The areas under the curves of PAIgG, PAIgM, and PAIgA were 0.795, 0.716, and 0.676, respectively.

Forty four (74.6%) of 59 patients with ITP were positive for at least one PAIg: 39 (66.1%) for PAIgG, 15 (25.4%) for PAIgA, 31 (52.5%) for PAIgM, and 44 (74.6%) for PAIgG or PAIgM. The positivities of PAIg in non-ITP patients were as follows; PAIgG, 9 (15.3%); PAIgA, 2 (3.4%); PAIgM, 7 (11.9%); PAIgG or PAIgM, 12 (20.3%); PAIgG, PAIgA, or PAIgM, 13 (22.0%). The PAIgA positive ITP patients also showed positivity for PAIgG or PAIgM (Table 1Go). Four patients with chronic ITP showed PAIgM positivity without PAIgG positivity. The positivity value of PAIgM was higher in those with acute ITP than in chronic ITP (Fig. 2Go).


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  		Table 1. Positivity of platelet-associated immunoglobulin in patients with immune thrombocytopenic purpura and other thrombocytopenic diseases.
 

Figure 2
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  		Fig 2. Distribution of positivity percentages in patients with different diagnoses. (A) Positivity percentages of PAIgG; (B) positivity percentages of PAIgM. Boxplots show the median and interquartile range.

 
The sensitivity of a positive PAIgG/PAIgM test for ITP in thrombocytopenic patients was 74.6%; the specificity was 79.7%, the positive predictive value was 78.6%, and the negative predictive value was 75.8% (Table 2Go).


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  		Table 2. The sensitivity, specificity, and positive and negative predictive values for the diagnosis of ITP.
 
Of 3 patients with myelodysplastic syndrome, 2 showed positivity of PAIg. Six of 20 patients with benign disease showed positivity for PAIg. Among these patients, 2 were diagnosed with an infection or Parkinsonism and another 4 were all diagnosed as liver disease.

Platelet counts and globulin concentrations were not significantly different between the PAIgG or PAIgM positive and negative groups in ITP and non-ITP patients. ITP patients with PAIgG or PAIgM positivity showed lower platelet count than those with PAIgG and PAIgM negativity (p > 0.05). The platelet count was significantly lower in ITP patients with PAIgM positivity than in those without (52.0 x 109/L and 25. x 109/L, respectively; p = 0.01). The MPV was significantly greater in the PAIgG or PAIgM positive groups than in both negative groups of non-ITP patients (Table 3Go).


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  		Table 3. Comparisons of the PAIgG and PAIgM negative group vs the PAIgG or PAIgM positive group in patients with immune thrombocytopenic purpura and non-immune thrombocytopenic purpura.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Flow cytometric detection of PAIg is a convenient, rapid, and relatively cheap method to evaluate patients with thrombocytopenia. However, it has been generally accepted that the specificity of PAIg assays is low [11]. In order to improve the diagnostic efficiency of flow cytometric detection of PAIg, we used the reference value for ITP determined by ROC curve analysis from results of non-ITP patients and ITP patients. In this study, PAIgA was always detected with another immunoglobulin class in ITP patients. Thus, measurement of PAIgG and PAIgM was enough for the evaluation of PAIg. This study confirmed that the measurement of PAIgG/PAIgM detection by flow cytometry is sensitive (74.6%) and specific (79.7%) for the diagnosis of patients with ITP.

Our results are different from those of Romero-Guzman et al [7], who found 39.3% specificity by using a cutoff value determined by the channel fluorescence of mean ± 2 SD in normal controls. Hezard et al [12] reported that flow cytometric PAIg assay showed 58% specificity by using mean fluorescence intensity cutoff based on ROC curve analysis versus MAIPA [12]. PAIg assay has been recommended as a screening test because of its low specificity and high sensitivity in the diagnosis of immune thrombocytopenia [12–14]. It was suggested that because of low specificity, PAIgG may be replaced by tests for platelet-associated GP-specific autoantibodies[15]. However, the determination of a cutoff value for the positivity of PAIg based on ROC analysis provides high specificity. Although the sensitivity of the PAIgG was lower than expected, our results correspond with the results of earlier studies that reported sensitivities of PAIg from 75 to 85% [16,17].

PAIgG is elevated in many conditions that involve increased platelet destruction and thrombocytopenia [18]. Platelet immunoglobulins may represent releasable immunoglobulins stored by endocytosis in the granules, which are related to platelet size, immune complexes bound to platelet Fc receptor, and nonspecific absorption [19,20]. This study demonstrated that large platelets are significantly associated with PAIgG or PAIgM positivity in non-ITP patients, and that platelet counts and globulin concentrations were not significantly different between the PAIgG or PAIgM positive and negative groups in ITP and non-ITP patients. These results are consistent with data that suggest IgG is taken up by megakaryocytes during maturation and gradually decreases during the lifetime of the platelets; thus, younger platelets have more PAIgG than do platelets of average age [19]. Furthermore, it was reported that large platelets bound more IgG than platelets of normal size, which may explain at least in part the reported low specificity of total PAIgG measurement [15].

We found PAIgM without PAIgG in chronic ITP patients (n = 4), but not in acute ITP patients (n = 0). Elevated PAIgM was associated with low platelet count in ITP patients. This finding may represent an interesting role of PAIgM in chronic ITP patients.

The causes and significance of increased PAIg in patients with thrombocytopenia are unknown. In our study, 12 non-ITP patients (20.3%) had elevated PAIgG or PAIgM. Two of 3 patients with myelodysplastic syndrome and all 4 patients with liver disease showed increased PAIgG or PAIgM. Thrombocytopenia in critically ill patients may share similarities with ITP characterized by increased platelet destruction as the result of binding of autoantibodies directed against platelet surface glycoproteins [10,21]. Thrombocytopenia is a common feature of chronic liver disease. Several studies have reported that serum PAIgG levels are elevated in thrombocytopenic patients with cirrhosis with specificity for GPIIb/IIIa or Ib/IX [22,23]. PAIgG has been implicated as a thrombocytopenic factor mediated by an immune mechanism in patients with liver diseases because PAIgG binds to platelets, thus promoting sequestration in the reticuloendothelial system. Moreover, in other studies, elevated PAIgG was described in patients with myelodysplastic syndrome mimicking ITP and myelodysplastic syndrome with auto-immune thrombocytopenia [9,24]. This suggests that peripheral immune destruction of platelets is a major cause of thrombocytopenia in some myelodysplastic syndrome patients, although no precise study of the mechanism of thrombocytopenia was made in the reported cases. Because of the small number of patients in the present study, definitive information concerning the biological implications of the finding of elevated platelet bound immunoglobulin is not provided. Further studies with bigger groups may provide more conclusive evidence of the role of PAIg in thrombocytopenic patients with liver disease or myelodysplastic syndrome.

In conclusion, this study demonstrates that the flow cytometric detection of PAIg combined with ROC curve analysis is a convenient, sensitive, and specific test compared with previous methods and is useful for the differential diagnosis of thrombocytopenic patients.


   Footnotes
 
* Dr Huh’s present address is Dept. of Laboratory Medicine, Dongguk University College of Medicine, Goyang, Korea. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. George JN, Woolf SH, Raskob GE, Wasser JS, Aledort LM, Ballem PJ, Blanchette VS, Bussel JB, Cines DB, Kelton JG, Lichtin AE, McMillan R, Okerbloom JA, Regan DH, Warrier I. Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology. Blood 1996;88:3–40.[Free Full Text]
  2. Berchtold P, Muller D, Beardsley D, Fujisawa K, Kaplan C, Kekomaki R, Lipp E, Morell-Kopp MC, Kiefel V, McMillan R, von dem Borne AE, Imbach P. International study to compare antigen-specific methods used for the measurement of antiplatelet autoantibodies. Br J Haematol 1997;96:477–483.[Medline]
  3. von dem Borne AE, Vos JJ, van der Lelie J, Bossers B, van Dalen CM. Clinical significance of positive platelet immunofluorescence test in thrombocytopenia. Br J Haematol 1986;64:767–776.[Medline]
  4. Hagenstrom H, Schlenke P, Hennig H, Kirchner H, Kluter H. Quantification of platelet-associated IgG for differential diagnosis of patients with thrombocytopenia. Thromb Haemost 2000;84:779–783.[Medline]
  5. Kelton JG. The measurement of platelet-bound immunoglobulins: an overview of the methods and the biological relevance of platelet-associated IgG. Prog Hematol 1983;13:163–199.[Medline]
  6. Rosenfeld CS, Nichols G, Bodensteiner DC. Flow cytometric measurement of antiplatelet antibodies. Am J Clin Pathol 1987;87:518–522.[Medline]
  7. Romero-Guzman LT, Lopez-Karpovitch X, Paredes R, Barrales-Benitez O, Piedras J. Detection of platelet-associated immunoglobulins by flow cytometry for the diagnosis of immune thrombocytopenia. Haematologica 2000;85:627–631.[Abstract/Free Full Text]
  8. Kajihara M, Kato S, Okazaki Y, Kawakami Y, Ishii H, Ikeda Y, Kuwana M. A role of autoantibody-mediated platelet destruction in thrombocytopenia in patients with cirrhosis. Hepatology 2003;37:1267–1276.[Medline]
  9. Kuroda J, Kimura S, Kobayashi Y, Wada K, Uoshima N, Yoshikawa T. Unusual myelodysplastic syndrome with the initial presentation mimicking idiopathic thrombocytopenic purpura. Acta Haematol 2002;108:139–143.[Medline]
  10. Stephan F, Cheffi MA, Kaplan C, Maillet J, Novara A, Fagon J, Bonnet F. Autoantibodies against platelet glycoproteins in critically ill patients with thrombocytopenia. Am J Med 2000;108:554–560.[Medline]
  11. Kelton JG, Powers PJ, Carter CJ. A prospective study of the usefulness of the measurement of platelet-associated IgG for the diagnosis of idiopathic thrombocytopenic purpura. Blood 1982;60:1050–1053.[Abstract/Free Full Text]
  12. Hezard N, Simon G, Mace C, Jallu V, Kaplan C, Nguyen P. Is flow cytometry accurate enough to screen platelet autoantibodies? Transfusion 2008;48:513–518.[Medline]
  13. Janisiw M, Eichelberger B, Koren D, Panzer S. Screening for platelet auto-antibodies by flow cytometry and their evaluation by the MAIPA technique. Wien Klin Wochenschr 1998;110:531–534.[Medline]
  14. Fabris F, Scandellari R, Randi ML, Carraro G, Luzzatto G, Girolami A. Attempt to improve the diagnosis of immune thrombocytopenia by combined use of two different platelet autoantibodies assays (PAIgG and MACE). Haematologica 2002;87:1046–1052.[Abstract/Free Full Text]
  15. Javela K, Kekomaki R. Mean platelet size related to glycoprotein-specific autoantibodies and platelet-associated IgG. Int J Lab Hematol 2007;29:433–441.[Medline]
  16. Christopoulos CG, Kelsey HC, Machin SJ. A flow-cytometric approach to quantitative estimation of platelet surface immunoglobulin G. Vox Sang 1993;64:106–115.[Medline]
  17. Tazzari PL, Ricci F, Vianelli N, Tassi C, Belletti D, Pierri I, Gugliotta L, Gobbi M, Conte R. Detection of platelet-associated antibodies by flow cytometry in hematological autoimmune disorders. Ann Hematol 1995;70:267–272.[Medline]
  18. Beardsley DS, Ertem M. Platelet autoantibodies in immune thrombocytopenic purpura. Transfus Sci 1998; 19:237–244.[Medline]
  19. George JN. Platelet immunoglobulin G: its significance for the evaluation of thrombocytopenia and for understanding the origin of alpha-granule proteins. Blood 1990;76:859–870.[Free Full Text]
  20. Warner M, Kelton JG. Laboratory investigation of immune thrombocytopenia. J Clin Pathol 1997;50:5–12.[Free Full Text]
  21. Brighton TA, Evans S, Castaldi PA, Chesterman CN, Chong BH. Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias. Blood 1996;88:194–201.[Abstract/Free Full Text]
  22. Feistauer SM, Penner E, Mayr WR, Panzer S. Target platelet antigens of autoantibodies in patients with primary biliary cirrhosis. Hepatology 1997;25:1343–1345.[Medline]
  23. Sanjo A, Satoi J, Ohnishi A, Maruno J, Fukata M, Suzuki N. Role of elevated platelet-associated immunoglobulin G and hypersplenism in thrombocytopenia of chronic liver diseases. J Gastroenterol Hepatol 2003;18:638–644.[Medline]
  24. Giannouli S, Voulgarelis M, Zintzaras E, Tzioufas AG, Moutsopoulos HM. Autoimmune phenomena in myelodysplastic syndromes: a 4-year prospective study. Rheumatology (Oxford) 2004;43:626–632.[Medline]




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