2306 Letters to the Editor
Factor VIII-specific memory B cells in patients with hemophilia A P . M . W . V A N H E L D E N , * P . H . P . K A I J E N , * K . F I J N V A N D R A A T , H . M . V A N D E N B E R G à and J. VOORBERG* *Department of Plasma Proteins, Sanquin-AMC Landsteiner Laboratory and Van Creveld Laboratory, Amsterdam; Department of Pediatrics, EmmaÕs ChildrenÕs Hospital AMC, Amsterdam; and àVan Creveldkliniek, Department of Internal Medicine, University Medical Center, Utrecht, The Netherlands
To cite this article: van Helden PMW, Kaijen PHP, Fijnvandraat K, van den Berg HM, Voorberg J. Factor VIII-specific memory B cells in patients with hemophilia A. J Thromb Haemost 2007; 5: 2306–8.
Antibody levels in serum, as maintained by plasma cells, are the first line of defense against incoming pathogens, whereas memory B cells are indispensable for long-lasting protection [1]. Upon re-exposure to antigen, memory B cells differentiate into antibody-secreting cells (ASCs), thereby enabling rapid eradication of pathogens that are not cleared by pre-existing antibodies. Additionally, there is evidence that memory B cells contribute to replenishing the pool of long-living plasma cells residing in the bone marrow [2]. Persisting levels of peripheral memory B cells have been identified in the context of vaccination [3] and viral infections [4]. Humoral responses against therapeutic or self-proteins are potentially harmful and in this setting development and persistence of antigen-specific memory B cells is undesirable. Thus far, circulating levels of memory B cells in patients with pathogenic antibodies to therapeutic or self-proteins have not been defined. Here, we assessed the frequency of antigen-specific memory B cells in hemophilia A patients [5] who developed inhibitory antibodies to factor (F) VIII. We used the EL4.B5 system [6] to analyze FVIII-specific memory B cells in the circulation of hemophilia A patients. Previously, it has been shown that CD40 L stimulation, provided by EL4.B5 thymoma cells in combination with a T-cell supernatant, results in activation and clonal expansion of the majority of B cells [6]. Subdivision of the CD19+ fraction revealed that the IgG producing ASCs developed solely from the IgG+ B-cell population (data not shown). Frozen peripheral blood mononuclear cells were carefully thawed and stained for the B-cell marker CD19. CD19+ cells were sorted onto irradiated EL4.B5 cells and cultured for 9–10 days. Culture supernatants were subsequently analyzed for the presence of FVIII-specific IgG using ELISA. Cells were then washed and incubated on ELISpot
Correspondence: Jan Voorberg, Department of Plasma Proteins, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Tel.: +31 20 5123120; fax: +31 20 5123680; e-mail:
[email protected] Received 25 June 2007, accepted 26 July 2007
plates, coated with 5 lg mL)1 of either FVIII or an anti-IgG monoclonal antibody, in order to visualize antibody-secreting cells. Sixteen previously treated adult hemophilia A patients were included from two Dutch Hemophilia Treatment Centers. This study was approved by the institutional review board and informed consent was obtained from all participants. They were divided in three groups: (i) inhibitor patients, (ii) noninhibitor patients that were multi-transfused (>50 exposures) with FVIII without an apparent immune response and (iii) patients successfully treated with immune tolerance induction (ITI) as defined by a negative inhibitor titer. One patient suffering from acquired hemophilia A (A1) and one patient with mild hemophilia A (A2) were included in the group of inhibitor patients. All other patients had severe hemophilia A. In Fig. 1A, results of FVIII ELISpot are depicted for blood samples of five hemophilia A patients with inhibitors. For all five inhibitor patients FVIII-specific ASCs could be detected by ELISpot, although for patient A5 only one well contained spots corresponding to anti-FVIII IgG-producing B cells (Fig. 1A). For all patients the number of IgG-producing ASCs was also determined. These values were used to calculate the percentage of B cells that secreted FVIII-specific IgG. This analysis revealed that for patient A1–A4 0.07 to 0.35% of circulating B cells produces antibodies directed towards FVIII. For patient A5, the percentage of IgG-producing B cells secreting anti-FVIII IgG was 0.01%. To further substantiate our findings, we also determined the frequency of FVIIIspecific memory B cells using an ELISA as a read-out (Fig. 1B). The number of positive wells was used to calculate the frequency of FVIII-specific memory B cells in peripheral blood. A positive well indicates that at least one of the 1000 CD19+ B cells in the well produces anti-FVIII IgG. The number of positive wells divided by the total number of sorted B cells allows for the estimation of the frequency of circulating memory B cells. For patient A1–A4, 5–24 anti-FVIII IgGproducing cells per 105 CD19+ B cells were detected. It has been established that IgG+ memory B cells comprise 10–15% of the peripheral B cell compartment [7]. Assuming a similar frequency in our patients, the percentage of FVIII-specific memory B cells in patients A1–A4 ranges from 0.05 to 0.24% of that of total IgG producing memory B cells. These values are similar to that obtained using ELISpot as a read-out. No 2007 International Society on Thrombosis and Haemostasis
Letters to the Editor 2307 A3
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Fig. 1. Factor (F) VIII-specific memory B cells in patients with hemophilia (A) CD19+ lymphocytes derived from peripheral blood were sorted using a MoFlo cell sorter (Dakocytomation, Glostrup, Denmark) and seeded directly at 1000 per well in 96-well flat-bottom plates on top of preseeded EL4.B5 cells (50 Gy irradiated, 105 per well) in IscoveÕs modified DulbeccoÕs medium containing 10% fetal calf serum and 2.5% T-cell supernatant. Plates were incubated at 37 C in a 5% CO2 humidified atmosphere for 9–10 days. The presence of antibody-secreting cells (ASCs) producing anti-FVIII antibodies was determined by ELISpot. A representative well showing the presence or absence of FVIII-specific ASCs is shown for the different patients. Inhibitor titers (Bethesda units) during sampling are indicated under the wells for the inhibitor patients. For detection of IgG-producing ASCs only 1/5 of the cells from one well were incubated in the ELISpot plate. (B) Frequencies of FVIII-specific memory B cells in hemophilia A patients using ELISA as a read-out. Values are expressed in number of wells containing FVIII-specific IgG per 105 CD19+ cells. Closed circles (d) represent samples containing at least one positive well as detected by ELISA. Open circles (s) represent samples for which no positive wells were observed. Differences in frequency of FVIIIspecific memory B cells between hemophilia A patients with inhibitors and hemophilia A patients with pre-existing inhibitors that were successfully treated by immune tolerance induction (ITI) were statistically significant (Mann–Whitney U-test, P < 0.05). Differences between hemophilia A patients with and without inhibitors did not reach statistical significance (Mann–Whitney U-test, P = 0.06).
2007 International Society on Thrombosis and Haemostasis
positive wells were observed upon analysis of patient A5 (Fig. 1B; open circle). Therefore, the number of FVIII-specific IgG-producing B cells is below 1 per 105 sorted B cells for this patient. We next addressed the presence of FVIII-specific memory B cells in multi-transfused hemophilia A patients without inhibitors. In one out of five patients (B1) without inhibitors, low levels of circulating FVIII-specific memory B cells were detected by ELISpot. Only a single well from patient B1 contained anti-FVIII ASCs (Fig. 1A). For two hemophilia A patients without inhibitors low levels of FVIII-specific memory B cells were detected by ELISA (Fig. 1B; closed circles). These results show that FVIII-specific memory B cells can occur at low frequency in hemophilia A patients without inhibitors. In comparison a low frequency of FVIII-specific memory B cells was also observed in one out of five healthy individuals tested (data not shown).Tolerance to FVIII can be induced in hemophilia A patients with inhibitors so-called immune tolerance induction which consists of frequent administration of high doses of FVIII [8,9]. Six patients are included in our study that had undergone successful ITI as defined by a negative inhibitor titer. Also in this group low numbers of FVIII-specific memory B cells just above the limit of detection were present. One out of 84 sorted wells (with each well containing 1000 CD19+ B cells) was positive for both patient C5 and C6 (Fig. 1A). In a single patient who was successfully treated by ITI low numbers of circulating memory B cells were observed using ELISA as a read-out (Fig. 1B). These data show that FVIII-specific memory B cells are absent or only present at very low levels in hemophilia A patients who have been successfully treated by ITI. Previous studies in a murine model for hemophilia A showed that high concentrations of FVIII prevent in vitro and in vivo differentiation of FVIIIspecific memory B cells into plasma cells [10]. Our results are consistent with the rapid elimination of FVIII-specific memory B cells after the onset of ITI. The dynamics of the pool of circulating FVIII-specific memory B cells during ITI will be addressed in future studies. These studies are likely to generate additional insight into the molecular mechanism underlying ITI and may suggest novel approaches to more efficiently induce tolerance in hemophilia A patients with inhibitors [9]. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. References 1 Tarlinton D. B-cell memory: are subsets necessary? Nat Rev Immunol 2006; 6: 785–90. 2 Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 2002; 298: 2199–202. 3 Crotty S, Felgner P, Davies H, Glidewell J, Villarreal L, Ahmed R. Cutting edge: long-term B cell memory in humans after smallpox vaccination. J Immunol 2003; 171: 4969–73.
2308 Letters to the Editor 4 Tuaillon E, Tabaa YA, Petitjean G, Huguet MF, Pajeaux G, Fondere JM, Ponseille B, Ducos J, Blanc P, Vendrell JP. Detection of memory B lymphocytes specific to hepatitis B virus (HBV) surface antigen (HBsAg) from HBsAg-vaccinated or HBV-immunized subjects by ELISPOT assay. J Immunol Methods 2006; 315: 144–52. 5 Mannucci PM, Tuddenham EG. The hemophilias – from royal genes to gene therapy. N Engl J Med 2001; 344: 1773–9. 6 Wen L, Hanvanich M, Werner-Favre C, Brouwers N, Perrin LH, Zubler RH. Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total and antimalaria responder B cell frequencies. Eur J Immunol 1987; 17: 887–92. 7 Klein U, Rajewsky K, Kuppers R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface
antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J Exp Med 1998; 188: 1679–89. 8 Brackmann HH, Gormsen J. Massive factor-VIII infusion in haemophiliac with factor-VIII inhibitor, high responder. Lancet 1977; 2: 933. 9 DiMichele D. Immune tolerance therapy for factor VIII inhibitors: moving from empiricism to an evidence-based approach. J Thromb Haemost 2007; 5: 143–50. 10 Hausl C, Ahmad RU, Sasgary M, Doering CB, Lollar P, Richter G, Schwarz HP, Turecek PL, Reipert BM. High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors. Blood 2005; 106: 3415–22.
Performance characteristics of a rapid assay for anti-PF4/ heparin antibodies: the particle immunofiltration assay T . E . W A R K E N T I N , * J . I . S H E P P A R D , * R . R A S C H K E and A . G R E I N A C H E R *Department of Pathology and Molecular Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada; and Department of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt University, Greifswald, Germany
To cite this article: Warkentin TE, Sheppard JI, Raschke R, Greinacher A. Performance characteristics of a rapid assay for anti-PF4/heparin antibodies: the particle immunofiltration assay. J Thromb Haemost 2007; 5: 2308–10.
In May 2004, the United States Food and Drug Administration approved for marketing the HealthTEST Heparin/ Platelet Factor 4 Antibody Assay (Akers Biosciences, Inc., Thorofare, NJ, USA), based upon this novel assay being Ôsubstantially equivalent to the GTI PF4 Elisa Assay, Genetic Testing Institute (GTI), Waukesha, WI, USAÕ (http:// www.fda.gov/cdrh/pdf4/k040293.pdf; accessed 14 August 2007). This rapid immunoassay (results usually available within 10 min after collecting serum) utilizes a system known as the Ôparticle immunofiltration assayÕ (PIFA), wherein patient serum is added to a reaction well containing dyed particles coated with platelet factor 4 (PF4; not PF4/heparin) [1]. Whereas non-agglutinated blue-colored particles will migrate through the membrane filter, agglutinated beads will not. Thus, agglutination of the beads is believed to indicate the presence of anti-PF4/heparin antibodies within patient serum, with a negative test shown by a blue color in the result well, whereas no color indicates a positive test. However, the operating characteristics (sensitivity–specificity trade-offs) of
Correspondence: Theodore E. Warkentin, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences (General Site), 237 Barton St. E., Hamilton, ON L8L2X2, Canada. Tel.:+1 905 527 0271 ext. 46139; fax: +1 905 577 1421; e-mail:
[email protected] Received 25 June 2007, accepted 19 July 2007
this qualitative assay for the diagnosis of heparin-induced thrombocytopenia (HIT) remain largely undefined. We used two approaches to evaluate the operating characteristics of this assay. Firstly, we evaluated 93 frozen sera available from a prospective study performed in Hamilton, Canada (from January 2002 until May 2003). This study evaluated a clinical scoring system (the 4 TÕs) in which systematic testing had been performed using three established assays for HIT antibodies [2]: the platelet serotonin-release assay (SRA) [3], a commercial enzyme-immunoassay (EIA) that detects antibodies against PF4/polyvinyl sulfonate available from the GTI [4], and an in-house EIA utilizing PF4/ heparin complexes, which detects IgG class antibodies (PF4/ heparin-IgG EIA) [5]. For this analysis, we used as our definition of a positive test for HIT antibodies those sera that caused >50% serotonin release in the SRA. For each thawed serum tested in the PIFA, three different volumes of serum were added to the reaction well: 20 lL (standard volume recommended by the manufacturer), 10 and 5 lL (mimicking 2- and 4-fold sample dilutions). The second strategy we used was to test 199 consecutive fresh serum samples (received unfrozen), which were referred for diagnostic testing for HIT antibodies in Greifswald, Germany, and 137 fresh unfrozen samples obtained in ongoing prospective studies of PF4/heparin antibody formation during heparin therapy. All of the samples were also tested using the heparin-induced platelet activation (HIPA) test [6] and an in-house anti-PF4/heparin–IgG EIA [7]. This in-house EIA correlates well with the EIA from the GTI [8]. 2007 International Society on Thrombosis and Haemostasis
