From www.bloodjournal.org by guest on January 12, 2015. For personal use only. CORRESPONDENCE chromosomal translocation t(4;14)(p16.3;q32) in multiple myeloma involves the fibroblast growth-factor receptor 3 gene. Blood 10:4062, 1997 9. Tavormina PL, Shiang R, Thompson LM, Zhu YZ, Wilkin DJ, Lachman RS, Wilcox WR, Rimoin DL, Cohn DH, Wasmuth JJ: Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet 9:321, 1995 10. Caligaris-Cappio F, Bergui L, Gregoretti MG, Gaidano G, Gaboli M, Schena M, Zallone AZ, Marchisio PC: Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood 77:2688, 1991 11. Allouche M: Basic fibroblast growth factor and hematopoiesis. Leukemia 9:937, 1995 12. Keegan K, Johnson DE, Williams LT, Hayman MJ: Isolation of 2989 an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci USA 88:1095, 1991 13. Perez-Castro AV, Wilson J, Altherr MR: Genomic organization of the human fibroblast growth factor receptor 3 (FGFR3) gene and comparative sequence analysis with the mouse fgfr3 gene. Genomics 41:10, 1997 14. Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ, Bocian M, Winckur ST, Wasmuth JJ: Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 78:335, 1994 15. Rousseau F, Saugier P, Le Merrer M, Munnich A, Delezoide A-L, Maroteaux P, Bonaventure J, Narcy F, Sanak M: Stop codon FGFR3 mutations in thanatophoric dwarfism type 1. Nat Genet 10:11, 1995 Interleukin-2 Receptor Subunit Expression and Function on Human Peripheral T Cells Is Not Dependent on the Anticoagulant To the Editor: In a recent report, David et al1 addressed the ongoing controversy on expression levels of interleukin-2 receptor (IL-2R) вђЈ, вђ¤, and вђҐ chains on various mononuclear cells from the peripheral blood. Using freshly isolated peripheral blood mononuclear cells (PBMCs) from (sodium) heparinized blood and fluorescein isothiocyanate (FITC)-labeled commercial monoclonal antibodies, they showed that all three IL-2R chains usually are hardly detectable on either CD4 or CD8 T cells from healthy donors and from hemochromatosis patients. These results are in contrast with the much higher levels of IL-2R subunits on T cells observed by several investigators, including ourselves.2-9 David et al1 tentatively explain the discrepancy by invoking effects of anticoagulant and of storage. Indeed, if Ca2П© chelators were used instead of heparin, the levels of all three IL-2R chains on T cells apparently increased, and overnight storage of heparinized blood also seemed to upregulate IL-2R subunit expression. These observations are very important, because they not only seem to settle a long-standing controversy on IL-2R expression, but they also imply that the use of Ca2П© chelators as anticoagulant instead of heparin could dramatically influence the sensitivity of the T cells to IL-2. Because IL-2 and other common вђҐ-chain triggering cytokines are central to almost any T-cell function, EDTA or citrate anticoagulants should be avoided if subsequent functional testing is envisioned. A lot of immunological research is based on buffy coats, which routinely are anticoagulated with citrate. In our own studies of T-cell function during human immunodeficiency virus (HIV) infection, we have systematically used EDTA blood as starting material, because it is readily available and because we did not find a functional difference between lymphocytes derived from blood anticoagulated with heparin, citrate, or EDTA in preliminary experiments. In view of the findings of David et al,1 we felt obliged to carefully control the effect of Ca2П© chelators on IL-2R expression and function and we did not observe any significant influence of the anticoagulant. In three separate experiments, blood from five healthy control subjects (all lab personnel) was drawn at 10 AM in three different tubes from Sarstedt containing either sodium heparin (final concentration, 0.3 mg/mL), potassium-EDTA (final concentration, 1.6 mg/mL), or sodiumcitrate (final concentration, 10.6 mmol/L). The largest part of each tube was immediately processed for mononuclear cell (PBMC) separation, using Histopaque 1077 (Sigma, Bornem, Belgium), whereas the rest was kept at room temperature. At 2 PM, 50 ВµL of whole blood and 50 ВµL of PBMCs (containing 200,000 cells), derived from each of the three anticoagulant tubes, were incubated for 20 minutes at 4В°C with 0.1 Вµg of the nonconjugated reference monoclonals anti-Tac (IL-2RвђЈвЂ“specific; obtained from Dr Thomas Waldman, National Institutes of Health, Bethesda, MD) and with 2R-B (IL-2Rвђ¤вЂ“specific; from Dr Takashi Uchiyama, Institute for Virus Research, Kyoto University, Kyoto, Japan). As an isotypic (IgG1) control, we used purified 56D3 directed against an irrelevant parasitic antigen (provided by Dr J. Brandt, Institute of Tropical Medicine, Antwerpen, Belgium). After washing with phosphate-buffered saline (PBS), containing 0.5% bovine serum albumin, 1 ВµL of FITC-labeled F(abР€) 2 goat antimouse IgG (Tago, Burlingame, CA) was added for another 20 minutes. After washing again, the remaining binding sites on the FITC-conjugate were blocked with 5 ВµL of mouse serum. Next, 5 ВµL of phycoerythrin (PE)-labeled anti-CD4 and 5 ВµL of peridinin-chlorophyll A protein (PercP)-labeled anti-CD3 (both from Becton Dickinson, Erembodegem, Belgium) were added for the last 20 minutes. The tubes with whole blood were then subjected to the Becton Dickinson lysing solution. All preparations were washed once and fixed with 1% paraformaldehyde. The samples were analyzed on a FACScan (Becton Dickinson) using the LYSYS I software. Based on the scatter and the CD3/CD4 expression, the CD4П© and CD4ПЄ T lymphocytes were gated separately and the distribution of the first fluorescence was represented in a histogram for each subset. An example of this analysis is shown in Fig 1. It is evident that, within both the CD4П© and CD4ПЄ T-cell populations, the expression profile of IL-2RвђЈ is rather broad and tends to be bimodal (a negative and a positive subpopulation), whereas the curve of IL-2Rвђ¤ is unimodal and shows a shift to the right, which is most evident in the CD4ПЄ subset. We chose to express the results for both chains as percentage of positive cells, after establishing a narrow threshold at a relative fluorescence intensity of 10, based on the background of the control monoclonal. A summary of the results is shown in Table 1. No significant difference was observed in the level of IL-2R вђЈ and вђ¤ chains on CD4П© or CD4ПЄ T cells, according to the anticoagulant used and regardless of whether the cells were stained in the context of whole blood or PBMCs. Comparing the mean fluorescence intensity of all gated cells (instead of the percentage of positive cells) showed similar results and confirmed that the low level of IL-2Rвђ¤ expression on CD4П© T cells significantly differed from background (data not shown). We next wanted to know whether the anticoagulant influences the sensitivity to IL-2. To this end, we cultured the three preparations of PBMCs at a final concentration of 106/mL in RPMI, supplemented with antibiotics (GIBCO, Paisley, UK) and 10% bovine calf serum (Hyclone, From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 2990 CORRESPONDENCE Fig 1. Representative binding profiles of a control MoAb, the antiвЂ“IL-2Ra MoAb anti-Tac, and the antiвЂ“ IL-2Rb MoAb 2RB on CD4Ш‰ and CD4ШЉ T cells, assessed in three-color flow cytometry. Logan, UT), in the presence or absence of 100 U/mL IL-2 (BoehringerMannheim, Brussels, Belgium) at 37В°C in a 5% CO2 atmosphere. After an overnight incubation, the cells were stained with a combination of either FITC-labeled IgG1 or anti-CD69-FITC and anti-CD4-PE plus anti-CD3-PercP (all from Becton Dickinson). As shown in Table 2, the specific expression of CD69 on CD4П© and CD4ПЄ T cells is similarly upregulated by IL-2, irrespective of the original anticoagulant. The only (nonsignificant) difference was a lower IL-2вЂ“induced CD69 on CD4ПЄ T cells from EDTA blood, as compared with heparinized or citrated blood. The latter observation certainly does not plead for a higher sensitivity of Table 1. Effect of Anticoagulant on T-Cell Expression of IL-2R вђЈ and вђ¤ Chains CD4П© T Cells K-EDTA Whole blood PBMCs Na-heparin Whole blood PBMCs Na-citrate Whole blood PBMCs CD4ПЄ T Cells Control IgG1 AntiвЂ“IL-2R вђЈ AntiвЂ“IL-2R вђ¤ Control IgG1 AntiвЂ“IL-2R вђЈ AntiвЂ“IL-2R вђ¤ 0.3 П® 0.2 0.7 П® 0.3 49.6 П® 8.2 49.2 П® 3.5 2.3 П® 1.3 3.3 П® 0.8 0.7 П® 0.6 1.4 П® 1.2 14.5 П® 5.1 15.5 П® 5.6 28.4 П® 13.4 30.8 П® 10.2 3.4 П® 6.6 1.0 П® 0.6 49.7 П® 6.3 48.4 П® 5.7 2.7 П® 1.0 3.3 П® 0.4 2.8 П® 4.9 1.3 П® 0.6 15.9 П® 5.3 14.5 П® 3.6 32.2 П® 14.5 30.7 П® 11.9 0.3 П® 0.3 0.9 П® 0.5 49.6 П® 7.9 49.7 П® 6.5 2.6 П® 1.0 2.6 П® 0.7 0.9 П® 0.8 1.4 П® 0.7 16.2 П® 6.1 15.3 П® 4.5 31.1 П® 12.6 26.0 П® 8.6 Values are the percentage of cells within the indicated subset showing a relative fluorescence intensity of greater than 10 after incubation with the indicated monoclonal (mean П® SD). From www.bloodjournal.org by guest on January 12, 2015. For personal use only. CORRESPONDENCE 2991 Table 2. Effect of Anticoagulant on T-Cell Sensitivity to IL-2 % CD69 Expression After 1 Day of Culture On CD4П© T Cells Culture Condition Medium Only IL-2 (100 U/mL) On CD4ПЄ T Cells Medium Only IL-2 (100 U/mL) PBMC isolated from K-EDTA blood 3.3 П® 1.5 9.5 П® 5.7 10.7 П® 2.4 28.6 П® 6.4 Na-heparin blood 2.1 П® 0.6 9.8 П® 19.0 9.4 П® 3.2 40.7 П® 18.6 Na-citrate blood 3.3 П® 1.6 10.0 П® 13.9 9.4 П® 1.8 37.6 П® 14.7 Values are the percentage of cells within the indicated subset showing specific binding to anti-CD69-FITC (mean П® SD). T cells from EDTA blood, which is to be expected if the latter anticoagulant was able to upregulate IL-2R expression, as suggested by David et al.1 To make absolutely sure that the storage of 4 hours we used in our standard procedure did not influence the results, we performed two additional experiments in which either whole blood anticoagulated with heparin, EDTA, or citrate or the isolated PBMCs were stained for IL-2RвђЈ or CD69 either immediately or after 4 hours of incubation at room temperature. Again, no differences in CD25 or CD69 expression were observed, regardless of whether completely fresh cells or cells after 4 hours were used, thus excluding spurious activation upon short-term storage of blood or PBMCs. In summary, our well-controlled flow cytometric measurements, using a sensitive indirect labeling technique and reference anti-вђЈ and anti-вђ¤ monoclonal antibodies, confirm our own and other investigatorsвЂ™ previous findings that both IL-2R вђЈ and вђ¤ chains are expressed to a significant extent on both resting CD4П© and CD4ПЄ T cells.2-9 We further confirmed that the вђЈ chain is predominantly present on CD4 T cells, whereas the вђ¤ chain is more prominently expressed on the CD4ПЄ subset, largely corresponding to the CD8П© T cells. Moreover, our present data clearly show that the level of IL-2R chain expression is not influenced by the use of a Ca2П©-chelating anticoagulant and is similar in the context of fresh whole blood and freshly isolated PBMCs. We did not investigate the effect of prolonged storage, because, in our experience, such a treatment results in a variable degree of viability loss, which might induce artifacts. However, short-term storage for 4 hours had no influence on either IL-2RвђЈ or CD69. Our culture experiments with added IL-2 further indicated that the IL-2R function, as measured by upregulation of CD69 on T cells, is also not significantly influenced by the anticoagulant. In our previous studies, we showed that the CD69 upregulation by IL-2 on CD4 T cells could be blocked by a combination of antiвЂ“IL-2R вђЈ and вђ¤ monoclonals, pointing to an implication of the high-affinity receptor. In CD8 T cells, nearly complete blocking could be obtained by anti-вђ¤ monoclonals alone at high IL-2 concentrations, whereas, at lower concentration, a synergistic effect of anti-вђЈ and anti-вђ¤ monoclonals was obvious.2 Thus, the expression of both вђЈ and вђ¤ chains was shown to be of functional relevance in both T-cell subsets. In a separate report, we recently demonstrated IL-2R вђҐ chain expression on at least 60% of both CD4 and CD8 T cells from healthy donors and HIV-infected subjects.10 An incomplete screening of the literature found that at least two other independent groups of investigators who stained PBMCs isolated from heparinized blood with the same reference anti-Tac monoclonal but a different anti-вђ¤ (Mik1-вђ¤) showed expression levels of IL-2R chains on peripheral T cells very similar to those we observed.5,6 However, other investigators, also starting from heparinized blood but using different monoclonals and/or different staining procedures, reported lower or even no measurable expression of the IL-2R subunits, indeed.11-13 The seemingly low expression of IL-2R chains observed by some investigators thus might relate to technical factors, including the affinity of the antibodies used or the sensitivity of the cytofluorometer rather than to the kind of anticoagulant. In conclusion, we feel that the use of sensitive staining procedures is crucial to correctly assess IL-2R expression on resting T cells. Moreover, we are convinced that the anticoagulant does not influence IL-2R expression or function in peripheral blood T cells. ACKNOWLEDGMENT Supported by Grants No. 3.0307.95 and 3.0226.96 of the вЂ�вЂ�Fonds voor Wetenschappelijk Onderzoek VlaanderenвЂ™вЂ™ (Fund for Scientific Research of Flanders). Guido L. Vanham Godelieve Penne Chris Vereecken Johan Vingerhoets Luc Kestens Laboratory of Immunology Department of Microbiology Institute of Tropical Medicine Antwerpen, Belgium REFERENCES 1. David D, Bani L, Moreau J-L, Demaison C, Sun K, Salvucci O, Nakarai T, de Montalembert M, Chouaib S, Joussemet M, Ritz J, The`ze J: Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets. Blood 91:165, 1998 2. Vanham G, Kestens L, Vingerhoets J, Penne G, Colebunders R, Vandebruaene M, Goeman J, Ceuppens JL, Sugamura K, Gigase P: The interleukin-2 receptor expression and function on peripheral blood lymphocytes from HIV-infected and control persons. Clin Immunol Immunopathol 71:60, 1994 3. Zola H, Mantzioris BX, Webster J, Kette FE: Circulating human T and B lymphocytes express the p55 interleukin-2 receptor molecule (TAC, CD25). Immunol Cell Biol 67:233, 1989 4. Zola H, Purling RJ, Koh LY, Tsudo M: Expression of the p70 chain of the IL-2 receptor on human lymphoid cells: Analysis using a monoclonal antibody and high-sensitivity immunofluorescence. Immunol Cell Biol 68:217, 1990 5. Sheldon A, Flego L, Zola H: Coexpression of IL-2 receptor p55 and p75 by circulating blood lymphocytes. J Leukoc Biol 54:161, 1993 6. Taga K, Kasahara Y, Yachie A, Miyawaki T, Taniguchi N: Preferential expression of IL-2 receptor subunits on memory populations within CD4(П©) and CD8(П©) T cells. Immunology 72:15, 1991 7. Kanegana H, Miyawaki T, Kato K, Yokoi T, Uehara T, Yachie A, Taniguchi N: A novel subpopulation of CD45RA(П©) CD4(П©) T cells expressing IL-2 receptor вђЈ-chain and having a functionally transitional nature into memory cells. Int Immunol 12:1349, 1991 8. Zola H, Koh LY, Mantzioris BX, Rhodes D: Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL-2 receptor p22 chain (TAC, CD25). Clin Immunol Immunopathol 59:16, 1991 9. Hofmann B, Nishanian P, Fahey JL, Esmail I, Jackson AL, Detels R, Cumberland W: Serum increases and lymphoid cell surface losses of IL-2 receptor CD25 in HIV infection: Distinctive parameters of HIV-induced change. Clin Immunol Immunopathol 61:212, 1991 10. Vingerhoets J, Bisalinkumi E, Penne G, Colebunders R, Bosmans E, Kestens L, Vanham G: Altered receptor expression and decreased sensitivity of T cells to the stimulatory cytokines IL-2, IL-7 and IL-12 in HIV-infection. Immunol Lett 61:53, 1998 From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 2992 CORRESPONDENCE 11. Ohashi Y, Takeshita T, Nagata K, Mori S, Sugamura K: Differential expression of the IL-2 receptor subunits p55 and p75 on various populations of primary peripheral blood mononuclear cells. J Immunol 143:3548, 1989 12. Yagita H, Nakata M, Azuma A, Nitta T, Takeshita T, Sugamura K, Okumura K: Activation of peripheral blood T cells via the p75 interleukin-2 receptor. J Exp Med 170:1445, 1989 13. Caligiuri MA, Zmuidzinas A, Manley TJ, Levine H, Smith KA, Ritz J: Functional consequences of interleukin-2 receptor expression on resting human lymphocytes. J Exp Med 171:1509, 1990 Response We have recently published in BLOOD the first extensive survey concerning IL-2RвђЈ, IL-2Rвђ¤, and IL-2RвђҐ expression by human PBMC subsets.1 More than 100 donors were examined through a 4-year period and the results are consistent with our published data.1 Throughout the work, care was taken to study healthy donors and resting PBMCs. The reported results were obtained by measuring cell surface and intracellular expression of IL-2RвђЈ, IL-2Rвђ¤, and IL-2RвђҐ by flow cytometry. The data were verified for the three chains by measuring mRNA expression. For intracellular expression of IL-2RвђҐ, Western blot analysis was also performed. Table 1 summarizes the data. Several reasons may explain why Vanham et al were not able to obtain the same results concerning cell surface expression of IL-2RвђЈ by CD4 and CD8 T lymphocytes. (1) Their donors were apparently not controlled. Under some circumstances, we also found IL-2RвђЈ expression by CD4 T cells, for instance during and after winter and spring seasonal infections. In our study, donors went through a rigorous medical check-up before blood collection. (2) Their assay does not identify a clear negative lymphocyte population; therefore, we cannot exclude either that their assay is not reliable or that their cells are activated. Under these conditions, it may not be surprising that they do not find any influence of anticoagulants. In our hands, all the PBMC subsets analyzed from healthy donors were negative for IL-2RвђЈ expression, and we use cells from hemochromatosis patients as a positive control to show that our assay was able to detect IL-2RвђЈ when expressed. These results were misinterpreted by Vanham et al. Furthermore, we show that IL-2RвђЈ could be in vitro induced in IL-2RвђЈвЂ“negative resting CD4 T cells after various stimulations.2 (3) The very poor and inconsistent (high standard deviation) induction of CD69 by CD4 T cells after IL-2 stimulation shown by Vanham et al suggests that their cells do not express IL-2R, and not the contrary. Furthermore, we previously demonstrated that IL-2 reactivity does not always correlate with IL-2R expression.3 In conclusion, it is commonly accepted that CD25 expression correlates with T-cell activation and that resting T cells do not express CD25.4-10 Therefore, we believe that Vanham et al should make a more detailed study before claiming high expression of CD25 by resting CD4 T cells. They should be aware that, in the IL-2R system, there are not yet defined reference reagents and techniques and that their methods may have to be improved and controlled if they intend to modify almost consensual data concerning IL-2RвђЈ expression. Denis David Lynda Bani Jean-Louis Moreau Jacques The`ze UniteВґ dвЂ™ImmunogeВґneВґtique Cellulaire DeВґpartement dвЂ™Immunologie Institut Pasteur Paris, France Table 1. Expression of IL-2R Chains and Specific mRNA by PBMCs of Healthy Adult Donors: Summary of our Data Presented in BLOOD CD4 T Lymphocytes Cell surface expression (flow cytometry)* вђЈ вђ¤ вђҐ Intracellular protein (intracellular flow cytometry) вђЈ вђ¤ вђҐ mRNA (RT-PCR) вђЈ вђ¤ вђҐ B Lymphocytes CD8 T Lymphocytes Monocytes NK Cells ПЅ5% ПЅ5% ПЅ5% F5% 14% П® 5.2%вЂЎ 8.6% П® 7.5%вЂЎ ПЅ5% ПЅ5% 49% П® 6.0%В§ ПЅ5% 78% П® 3.1% ПЅ5% ПЅ5% ПЅ5% Пѕ60% ПЅ5% ПЅ5% Пѕ45% ПЅ5% ПЅ5% Пѕ45% ND ND ND ПЅ5% Пѕ40% Пѕ60% вЂ” вЂ” П© вЂ” вЂ” П© вЂ” вЂ”(П©)аїЈ П© вЂ” вЂ” П© вЂ” П© П© F5%вЂ ПЅ5% ПЅ5% Values in bold and underlined are results in disagreement with the letter of Vanham et al, whereas values in italic and underlined are results in partial agreement with the letter of Vanham et al. Abbreviations: ND, not determined; вЂ”, not detected. *Percentage of positive cells. вЂ Expression of less than 5% is considered nonsignificant. вЂЎTwo kinds of donors can be distinguished. Most of them do not express significantly вђ¤ or вђҐ IL-2R chains, whereas 30% of them highly expressed these chains. This results in an important standard deviation. В§IL-2RвђҐ chain is expressed for all donors but with a variable intensity. аїЈmRNA detected for few donors in agreement with the вЂЎ note. From www.bloodjournal.org by guest on January 12, 2015. For personal use only. CORRESPONDENCE 2993 REFERENCES 1. David D, Bani L, Moreau J-L, Demaison C, Sun K, Salvucci O, Nakarai T, de Montalembert M, Chouaib S, Joussemet M, Ritz J, The`ze J: Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets. Blood 91:165, 1998 2. Bani L, David D, Moreau J-L, Cayota A, Nakarai T, Ritz J, The`ze J: Expression of the IL-2 receptor вђҐ subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component. Int Immunol 9:573, 1997 3. Moreau J-L, Chastagner P, Tanaka T, Miyasaka M, Kondo M, Sugamura K, The`ze J: Control of IL-2 responsiveness of B lymphocytes by IL-2 and IL-4. J Immunol 155:3401, 1995 4. Robb RJ, Munck A, Smith KA: T cell growth factor receptors. J Exp Med 154:1455, 1981 5. Cantrell DA, Smith KA: Transient expression of interleukin-2 receptors. Consequences for T cell growth. J Exp Med 158:1895, 1983 6. Depper JM, Leonard WJ, KroВЁnke M, Noguchi PD, Cunningham RE, Waldmann TA, Greene WC: Regulation of interleukin 2 receptor expression: Effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen. J Immunol 133:3054, 1984 7. Ohashi Y, Takeshita T, Nagata K, Mori S, Sugamura K: Differential expression of the IL-2 receptor subunits, p55 and p75 on various populations of primary blood mononuclear cells. J Immunol 143:3548, 1989 8. Caligiuri MA, Zmuidzinas A, Manley TJ, Levine H, Smith KA, Ritz J: Functional consequences of interleukin 2 receptor expression on resting human lymphocytes. J Exp Med 171:1509, 1990 9. Nakarai T, Robertson MJ, Streuli M, Wu Z, Ciardelli TL, Smith KA, Ritz J: Interleukin 2 receptor вђҐ chain expression on resting and activated lymphoid cells. J Exp Med 180:241, 1994 10. Unutmaz D, Pileri P, Abrignani S: Antigen-independent activation of naive and memory resting T cells by a cytokine combination. J Exp Med 180:1159, 1994 Primary Recurrent Miscarriages: AntiвЂ“вђ¤2-Glycoprotein I IgG Antibodies Induce an Acquired Activated Protein C Resistance That Can Be Detected by the Modified Activated Protein C Resistance Test To the Editor: Using the study of affinity-purified IgG phospholipid-dependent inhibitors on the time-course of factor Va generation and inactivation, Galli et al1 demonstrated convincingly that antiвЂ“вђ¤2-glycoprotein I antibodies induce an acquired resistance to activated protein C (aPC), thus providing a new possible explanation for their thrombogenic potential. We have studied the effect of various antiphospholipid IgG antibodies on the activated protein C-mediated factor Va proteolysis (aPC-FV) using the modified aPC-resistance test that includes predilution of patient plasma in factor V depleted plasma, according to Jorquera et al2 (Coatest APC Resistance V; Chromogenix, MoВЁlndal, Sweden; results given as aPC-FV ratios:time obtained in the presence of aPC divided by the time obtained, using the same plasma, in absence of aPC). All patients were nonthrombotic young women who had experienced primary unexplained recurrent miscarriages. They had been controlled to be negative for the R506Q (factor V Leiden) mutation. Lupus anticoagulant activity (LA) had been tested according to the revised Table 1. Effect of Various Plasmas Positive for Antiphospholipid-Related Markers on the AP C-Mediated Factor Va Proteolysis (aPC-FV) Using the Modified aPC-Resistance Test aPC-FV Ratio Mixing Studies 1:1 Patient Plasmas Control plasmas (n П 200) Whole plasmas NP (pooled control plasmas) Isolated LA (n П 10) Whole plasmas IgG-depleted plasmas Isolated aCLAb (n П 10) Whole plasmas IgG-depleted plasmas Isolated aвђ¤2GP1Ab (n П 10) Whole plasmas IgG-depleted plasmas Isolated aFIIAb (n П 2) Whole plasmas IgG-depleted plasmas Isolated aAnVAb (n П 10) Whole plasmas IgG-depleted plasmas NP 2.54 (2.08-3.36) 2.55 2.55 (2.19-2.96) 2.55 2.64 (2.19-3.24) 2.57 (2.11-3.18) 2.59 (2.23-2.85) 2.55 (2.28-2.80) 2.72 (2.31-3.30) 2.68 (2.35-3.22) 2.63 (2.35-2.96) 2.60 (2.38-3.01) 1.52 (1.11-2.02) [1] 2.42 (2.12-2.65) [2] 1.75 (1.23-2.19) [3] 2.45 (2.23-2.60) [4] (2.76-3.15) (2.80-3.04) (2.65-2.96) (2.71-2.82) 2.61 (2.25-3.00) 2.58 (2.29-3.05) 2.58 (2.36-2.88) 2.55 (2.34-2.79) NP-вђ¤2neg 2.50 (2.10-2.92) 2.51 1.93 (1.54-2.52) [5] 2.47 (2.20-2.71) [6] Results are expressed as aPC-FV ratios (time obtained in the presence of aPC divided by the time obtained in absence of aPC) and are given as median value (range). Statistical analysis (Wilcoxon paired t-test): [1] v [2], [3] v [4], and [5] v [6]: P П .005; [1] v [3], [3] v [5], and [1] v [5]: P П .005. All other comparisons were nonsignificant. Abbreviations: NP, normal plasma; NP-вђ¤2neg, normal plasma after вђ¤2-glycoprotein I depletion; LA, positive lupus anticoagulant; aCLAb, positive anticardiolipin IgG antibodies; aвђ¤2GP1Ab, positive antiвЂ“вђ¤2-glycoprotein I IgG antibodies; aFIIAb, positive antiprothrombin IgG antibodies; aAnVAb, anti-annexin V IgG antibodies. From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 1998 92: 2989-2993 Interleukin-2 Receptor Subunit Expression and Function on Human Peripheral T Cells Is Not Dependent on the Anticoagulant Guido L. Vanham, Godelieve Penne, Chris Vereecken, Johan Vingerhoets and Luc Kestens Updated information and services can be found at: http://www.bloodjournal.org/content/92/8/2989.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.