Yves Gruel

Antibodies to Platelet Factor 4–Heparin After Cardiopulmonary Bypass in Patients Anticoagulated With Unfractionated Heparin or a Low-Molecular-Weight Heparin Clinical Implications for Heparin-Induced Thrombocytopenia

BACKGROUND Cardiopulmonary bypass (CPB) induces platelet activation with release of platelet factor 4 (PF4), and patients are exposed to high doses of heparin (H). We investigated whether this contributes to the development of antibodies to H-PF4 and heparin-induced thrombocytopenia (HIT). METHODS AND RESULTS CPB was performed with unfractionated heparin (UFH) in 328 patients. After surgery, patients received UFH (calcium heparin, 200 IU. kg-1. d-1) (group 1, n=157) or low-molecular-weight heparin (LMWH, Dalteparin, 5000 IU once daily) (group 2, n=171). Eight days after surgery, antibodies to H-PF4 were present in 83 patients (25.3%), 46 in group 1 and 37 in group 2 (P=0.12). Most patients (61%) had IgG1 to H-PF4, but only 8 samples with antibodies induced platelet activation with positive results on serotonin release assay. HIT occurred in 6 patients in group 1, but no thrombocytopenia was observed in subjects receiving LMWH, although 2 had high levels of antibodies with positive serotonin release assay results. When antibodies to H-PF4 were present, mean platelet counts were lower only in patients with FcgammaRIIA R/R131 platelets. CONCLUSIONS These results provide evidence that the development of antibodies to H-PF4 after CPB performed with UFH is not influenced by the postoperative heparin treatment. The antibodies associated with high risk of HIT are mainly IgG1, which is present at high titers in the plasma of patients continuously treated with UFH.

Prospective evaluation of the ‘4Ts’ score and particle gel immunoassay specific to heparin/PF4 for the diagnosis of heparin-induced thrombocytopenia

Summary.  Background: Heparin‐induced thrombocytopenia (HIT) is a severe disease that is often difficult to diagnose. A clinical scoring system, the ‘4Ts’ score, has been proposed to estimate its probability before laboratory testing, and a particle gel immunoassay (H/PF4 PaGIA®) has also been developed for rapid detection of HIT antibodies. Aim: To evaluate the performance of both methods when HIT is suspected clinically. Methods: Two hundred thirteen consecutive patients were included in four centers. The probability of HIT was evaluated using the 4Ts score blind to antibody test results. HIT was confirmed only when the serotonin release assay (SRA) was positive. Results: The risk of HIT was evaluated by the 4Ts score as low (LowR), intermediate (IR) or high (HR) in 34.7%, 60.6% and 4.7% of patients, respectively. The negative predictive value (NPV) of the 4Ts score was 100%, as the SRA was negative in all LowR patients. PaGIA® was negative in 176 patients without HIT (99.4%, NPV) and the negative likelihood ratio (LR–) was 0.05. PaGIA® was positive in 37 patients, including 21 with HIT (positive predictive value = 56.8%), with a positive LR of 11.4. A negative PaGIA® result decreased the probability of HIT in IR patients from 10.9% before assay to 0.6%, whereas a positive result did not substantially increase the likelihood for HIT. Conclusion: The use of the 4Ts score with PaGIA® appears to be a reliable strategy to rule out HIT.

Changes in platelet count after cardiac surgery can effectively predict the development of pathogenic heparin-dependent antibodies

Cardiopulmonary bypass (CPB) induces the release of platelet factor 4 (PF4) and patients are at risk of heparin‐induced thrombocytopenia (HIT). This study was aimed to determine whether an abnormal evolution in platelet count (PC) after CPB is predictive of the development of HIT antibodies. Two abnormal PC patterns were defined: pattern P1, characterized by a decrease in PC following previous correction of thrombocytopenia occurring during CPB, and pattern P2, defined as a persistent low PC in the days following CPB. PC was evaluated for 10 d in 305 consecutive patients before and after CPB. Serotonin release assay (SRA) was carried out between days 8 and 10 to detect pathogenic heparin‐dependent antibodies. Moreover, antibodies to heparin–PF4 (H–PF4) complexes were assayed by enzyme‐linked immunosorbent assay. PC evolution after CPB was normal in 300 patients although antibodies to H–PF4 were frequently present (53·4%). Changes in PC were abnormal in five patients with pattern P1 (n = 4) or P2 (n = 1). As SRA was positive in four of the five cases, the positive predictive value of abnormal PC pattern for pathogenic HIT antibodies was 80%. Careful follow‐up of PC after CPB makes it possible to predict with high specificity (99%) for those patients who develop pathogenic HIT antibodies.

Laboratory testing for heparin-induced thrombocytopenia: a conceptual framework and implications for diagnosis

*Department of Pathology and Molecular Medicine and Department of Medicine, McMaster University, Hamilton, Canada; Department ofImmunology and Transfusion Medicine, Ernst-Moritz-Arndt University-Greifswald, Greifswald, Germany; Department of Hematology, HoˆpitalTrousseau, Tours, France; §Blood Research Institute, Blood Center of Wisconsin, and Department of Medicine, Medical College of Wisconsin,Milwaukee, WI, USA; and –Department of Medicine, St George Clinical School, University of New South Wales, and St George Hospital, Sydney,Australia.To cite this article: Warkentin TE, Greinacher A, Gruel Y, Aster RH, Chong BH, on behalf of The Scientific and Standardization Committee of theInternational Society on Thrombosis and Haemostasis. Laboratory testing for heparin-induced thrombocytopenia: a conceptual framework andimplications for diagnosis. J Thromb Haemost 2011; 9: 2498–500.

Biological and clinical features of low-molecular-weight heparin-induced thrombocytopenia

Summary. Heparin‐induced thrombocytopenia (HIT) is a common adverse effect of unfractionated heparin (UFH) therapy. In contrast, only a few patients have been reported with HIT following low‐molecular‐weight heparin (LMWH) therapy (LMW‐HIT). To define the clinical and biological characteristics of LMW‐HIT, 180 patients treated for suspected HIT at 15 French centres were investigated. Clinical history was recorded and HIT was confirmed in 59 patients with positive serotonin release assay results: 57 of them had high levels of antibodies (Abs) to heparin–platelet factor 4 complexes (H/PF4) and two had Abs to interleukin 8. Eleven patients were treated exclusively with LMWH (LMW‐HIT) and 48 with UFH either alone (UF‐HIT, n = 34) or combined with LMWH (UF/LMW‐HIT, n = 14). The LMW‐HIT and UF‐HIT groups were similar with respect to sex, age, platelet count before heparin therapy, frequency of bleeding and occurrence of disseminated intravascular coagulation. The interval to onset of HIT was longer in LMW‐HIT patients compared with UF‐HIT patients (P = 0·03). Severe thrombocytopenia (platelets < 15 × 109/l) was more frequent in the LMW‐HIT group (P = 0·04). Thrombosis occurred in three of 11 LMW‐HIT patients, i.e. as frequently as in UF‐HIT patients. LMW‐HIT is potentially severe and may be observed after longer heparin treatment compared with UF‐HIT. It is highly recommended, therefore, that platelet counts be monitored carefully whenever LMWH is administered.

Changes in antithrombin and fibrinogen levels during induction chemotherapy with L-asparaginase in adult patients with acute lymphoblastic leukemia or lymphoblastic lymphoma. Use of supportive coagulation therapy and clinical outcome: the CAPELAL study

The effects of L-asparaginase on hemostasis during induction chemotherapy of acute lymphoblastic leukemia of lymphoblastic lymphoma are less defined in adults than in children. This retrospective study suggests that antithrombin concentrates may have a beneficial effect on the outcome of adults treated for acute lymphoblastic leukemia with L-asparaginase. Background The effects of L-asparaginase on hemostasis during induction chemotherapy are less defined in adults than in children. We, therefore, studied the effects of L-asparaginase in adult patients. Design and Methods This was a retrospective analysis of 214 patients treated with L-asparaginase (7500 IU/m2 x 6) for acute lymphoblastic leukemia or lymphoblastic lymphoma. Between day 1 of the induction course and discharge, clinical events, and biological and therapeutic modifications were reviewed. Results Antithrombin and fibrinogen levels were lower than 60% and 1 g/L in 71% and 73% of patients, respectively. Twenty thromboses occurred in 9.3% of the patients; these patients had a median antithrombin level of 53% (range, 21–111) at the time of the event. Forty-two episodes of bleeding occurred in 31 patients with a median fibrinogen level of 1.3 g/L. Infusions of L-asparaginase were reduced or delayed in 64% of patients due to low fibrinogen and/or antithrombin levels. Fresh-frozen plasma, antithrombin and fibrinogen were infused in 31%, 41% and 52% of patients, respectively. The mean antithrombin and fibrinogen levels increased from 61% to 88% and from 1 to 1.4 g/L after infusion of antithrombin or fibrinogen respectively, while both levels remained unchanged after the infusion of fresh-frozen plasma. In patients who received antithrombin concentrates L-asparaginase injections were less frequently omitted or delayed (53% vs. 72%, p=0.005), the rate of thrombosis was lower (4.8% vs. 12.2%, p=0.04) and the disease-free survival was also reduced (p=0.05). Conclusions This retrospective study suggests that antithrombin concentrates may have a beneficial effect on the outcome of adults treated for acute lymphoblastic leukemia with L-asparaginase; prospective studies are essential to confirm this hypothesis.

Human NK cell-mediated direct and IgG-dependent cytotoxicity against xenogeneic porcine endothelial cells

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking Fc gamma RIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16+ natural killer (NK) cells. These results argue that CD16+ NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.

Affinity purification of heparin‐dependent antibodies to platelet factor 4 developed in heparin‐induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H‐PF4) complexes were purified from the plasma of three patients with heparin‐induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 m NaCl at pH 7·5 (peak 1) and the remainder was obtained using 0·1 m glycine/0·5 m NaCl at pH 2·5 (peak 2). In an enzyme‐linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H‐PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H‐PF4 complexes. Finally, IgG in patient 3 only bound to H‐PF4 and was unreactive with PF4 alone. Using [14C]‐serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H‐PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.

Differences in specificity of heparin-dependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium

Heparin‐induced thrombocytopenia (HIT) is frequently associated with antibodies (Abs) to heparin–PF4 complexes (H‐PF4). In order to investigate whether there are variations in specificity of Abs, we studied 63 samples from patients with suspected HIT. Two groups of samples were separated after comparing their reactivity against H‐PF4 or recombinant PF4 (r‐PF4) using ELISA. In group Ab1 (n = 46), Abs only or mainly bound to H‐PF4 complexes and thus most of the epitopes recognized probably involved both heparin and PF4. In group Ab2 (n = 17), Abs exhibited similar reactivity to r‐PF4 and H‐PF4, and the antigens recognized were possibly neoepitopes mainly expressed by modified PF4 and by H‐PF4 complexes. Platelet activation tests were positive with 56 samples containing high titres of Abs to H‐PF4. Most samples (n = 59) contained IgG antibodies, often associated with IgA antibodies which were more frequently found in group Ab2, and/or IgM. With unfractionated heparin treatment, HIT was associated with Ab1 or Ab2 antibodies, whereas only Ab1 antibodies were detected after low‐molecular‐weight heparin (LMWH). Furthermore, cross‐reactivity with danaparoid sodium was present only in group Ab1 and mainly involved LMWH‐treated patients.

JAK2 V617F is specifically associated with idiopathic splanchnic vein thrombosis.

Splanchnic vein thrombosis, that is hepatic, portal, mesenteric or splenic vein thrombosis, is a rare event associated with highly variable clinical expression. Indeed, some patients are asymptomatic, but others develop serious and sometimes lifethreatening complications. Putative causes, including overt myeloproliferative disorders (MPDs) and congenital or acquired risk factors for thrombosis, are identified in 75% of patients [1]. Polycythemia vera (PE) and essential thrombocythaemia (ET) are the most frequently detected MPDs after splanchnic vein thrombosis, and their diagnosis is based on well-defined criteria [2]. However, no obvious cause can be identified in at least 25% of cases, and latentMPD is systematically suspected even if the peripheral blood count is normal. Although the mechanisms involved have not been fully defined, latent MPD has been detected in 25–65% of patients with splanchnic vein thrombosis, the diagnosis being supported in most cases by bone marrow histology and erythroid progenitor cell cultures [3–5]. In addition, overt MPD has also been reported in some patients several years after thrombosis. A single somatic mutation localized in exon 12 of the gene coding for tyrosine kinase JAK-2 (Janus Kinase 2) has recently been reported by four independent teams [6–9]. JAK2 is a cytoplasmic signaling tyrosine kinase involved in hematopoietic cell growth, and the guanine-to-thymine mutation that leads to a valine-to-phenylalanine substitution at position 617 (pseudokinase JH2 domain) is associated with a growth factorindependent phenotype promoting differentiation and proliferation of hematopoietic precursors. The V617F mutation has been associated with MPD without the Philadelphia chromosome in 65% to 97% of patients with PV, 23–75% in ET and 30–50% in idiopathic myelofibrosis [10]. We therefore investigated in the present study whether the V617F mutation of JAK2 could be detected in splanchnic vein thrombosis and thus provide a marker of latent MPD in this particular clinical situation. We studied blood samples collected from 44 patients who had developed portal (n 1⁄4 42) or hepatic (n 1⁄4 2) thrombosis between January 2001 and December 2005, and for whom the most frequent causes of portal vein thrombosis (i.e. cirrhosis, infection and tumors) were ruled out. To evaluate whether the V617F JAK2 mutation could be specific to splanchnic vein thrombosis, we also examined 44 control subjects who had presented spontaneous DVT of the lower limbs (with pulmonary embolism in eight cases), matched with our patients for age and gender. All subjects studied were French Caucasian. Laboratory investigations were performed for hereditary and acquired risk factors for thrombosis in all patients and controls, including genotyping for factor (F)V Leiden and the 20210A prothrombin gene allele, measurement of antithrombin, protein C and protein S levels, screening for antiphospholipid antibodies and assay for total homocystein level. Screening for paroxysmal nocturnal hemoglobinuria was also performed by flow cytometry for five patients. In addition, bone marrow investigations (i.e. histological examination and erythroid progenitor cell culture) were also performed in 18 subjects for whom splanchnic vein thrombosis had remained unexplained. Genotyping for detection of the JAK2 mutation was performed on genomic DNA (gDNA) extracted from leukocytes (Flexigene DNA Kit; Qiagen, Courtaboeuf, France). Yield and purity were determined by spectrophotometry (with A260/A280 ratio > 1.6) and reactions were performed with 500 ng of gDNA. Allele-specific polymerase chain reaction (PCR) and restriction fragment length polymorphism PCR using the restriction endonuclease BsaXI were performed as recently described by Baxter et al. [6]. These methods were combined with nano-electrophoresis (Bioanalyzer 2100; Agilent, Massy, France) to increase assay sensitivity and this allowed detection of one homozygous mutation among 200 non-mutated cells. For each PCR, HEL92.1.7 and HL60 cell lines were used as positive and negative controls, respectively. Statistical analysis was assessed using the Mann–Whitney and Correspondence: Yves Gruel, Department of HaematologyHaemostasis, CHU Trousseau, route de Loches, 37044 Tours, France. Tel.: +33 02 47 47 46 72; fax: +33 02 47 47 59 04; e-mail: gruel@med.univ-tours.fr