scholarly journals Indeterminate Serotonin Release Assays Are Associated with a High Mortality Rate

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Shawn Jindal ◽  
Christopher Leyton ◽  
Fred Cohen ◽  
Morayma Reyes Gil ◽  
Henny H. Billett
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Clinical Course ◽  
Conflicts Of Interest ◽  
Serotonin Release ◽  
Categorical Variables ◽  
High Dose ◽  
Heparin Binding ◽  
Antibody Testing ◽  
High Concentrations

INTRODUCTION: The serotonin release assay (SRA) is considered the gold standard for diagnosis of heparin-induced thrombocytopenia (HIT). While the SRA holds high sensitivity and specificity when results are definitive, up to 10% of samples from patients with suspected HIT yield "indeterminate" results. In classic HIT, the SRA typically reveals high levels of serotonin release when serum is mixed with low concentrations of heparin and low levels of serotonin release when serum is mixed with high concentrations of heparin. Results are considered "indeterminate" when high levels of serotonin release are seen at both low and high concentrations of heparin, indicating a failure of high-dose heparin to saturate the heparin binding sites of PF4 molecules and inhibit platelet activation. Explanations for indeterminate assays include the presence of heparin-binding proteins that interfere with the assay, high titers of HLA class I alloantibodies, or immune complexes. Since the diagnosis of HIT carries such significance and has so many ramifications, an indeterminate SRA may leave therapeutic indecision. The etiologies, platelet trends, clinical course and outcomes of patients that receive indeterminate SRA results are not well-understood. We conducted a retrospective review of 2,056 patients that underwent SRA testing as part of their evaluation for HIT. METHODS: Using the electronic medical record data extraction software Clinical Looking Glass, we identified patients that underwent SRA testing between 1/1/2014 and 12/31/2018. SRA results were considered "indeterminate" when serotonin release exceeded 19% at all heparin dilutions (0.1 U/mL, 0.5 U/mL and 10 U/mL). We conducted a retrospective chart review to study the clinical course among patients who had "indeterminate" SRA results, the trends in platelet count, the timing of platelet drops and the physician response to this result. Statistical analysis was performed using chi-square testing for categorical variables. RESULTS: We identified 2,056 patients that underwent SRA testing between 2014 and 2019. Of these, 90 patients (4.4%) had "positive" SRA results and 1,814 patients (88.2%) had "negative" SRA results. Among the 152 patients (7.4%) with "indeterminate" SRA results, the mean 4T score was just 2.9, corresponding to a HIT probability of <5%. One-hundred and twenty of these 152 patients (78.9%) had heparin-PF4 antibody testing with optical densities below 0.60 OD, while only 4 of 152 patients (2.6%) had optical densities above 2.00 OD. Seventy eight of 152 patients (51.3%) either continued or were re-exposed to heparin after the indeterminate SRA result, and in 71 of 78 cases (91.0%), the platelet count stabilized or improved despite heparin exposure. In the remaining 7 cases, no acute VTE were found and no diagnosis of HIT was made. Four of the 71 patients (5.6%) that continued or were re-exposed to heparin were noted to have an acute VTE. A significantly higher portion of patients with an indeterminate SRA died during admission compared to those with a positive or negative SRA (49.3% vs. 21.1% and 27.2%, p <2.4 x10-10). The prevalence of thrombocytopenia <50,000 was substantially higher in patients with an indeterminate or positive SRA, compared to those with a negative SRA (39.5% and 40.0% vs. 27.5%, p <4.0 x 10-4). Re-exposure or continuation of heparin did not affect mortality; patients that were given heparin after an indeterminate SRA had a 47.4% mortality, compared to 51.4% in those given no more heparin (p = 0.63). Table 1 summarizes these findings. CONCLUSIONS: This is the largest study to date looking at patients with indeterminate SRA testing results, in which platelets exhibit high levels of serotonin release at both low and high heparin concentrations. Our data suggest 1) the majority of patients with indeterminate SRAs likely did not have HIT, 2) as evidenced by the low 4T scores and heparin-PF4 antibody levels, an indeterminate SRA suggests thrombocytopenia related to in-vivo platelet activation, and 3) an indeterminate SRA is associated with increased mortality. SRA results warrant a case-by-case assessment of the clinical picture in order to avoid unnecessary cessation of heparin products. Further studies exploring the mechanism leading to high serotonin release at both low and high heparin concentrations, causing an "indeterminate" SRA result, are warranted. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Case Report of Refractory Immune Thrombocytopenia (ITP) Following Reduced Intensity Conditioning (RIC) Hematopoietic Cell Transplantation (HCT) for Myelodysplastic Syndrome (MDS) Successfully Treated with Off-Label Use of Daratumumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4976-4976 ◽  
Author(s):  
Yazan Migdady ◽  
Ridhi Gupta ◽  
Asiri Ediriwickrema ◽  
Francisco Socola ◽  
Sally Arai ◽  
...  
Keyword(s):  
Platelet Count ◽  
Case Report ◽  
Peripheral Blood ◽  
Hematopoietic Cell Transplantation ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
High Dose ◽  
Severe Thrombocytopenia ◽  
Antibody Testing ◽  
Bone Marrow Biopsies

Abstract Background: A source of treatment refractoriness in immune cytopenias appears to be residual CD138/38 positive lymphocyte populations (Audia S et al, Blood 118:4394-400,2011; Mahevas M et al, J Clin Invest 123:432-442, 2013). Persistence of recipient's plasma cells can lead to prolonged refractory thrombocytopenia following RIC-HCT. (Fasano RM et al, Br J Haematol 166(3):425-34, 2014). Daratumumab was effective in the treatment of a child with refractory autoimmune hemolytic anemia after HCT (Tolbert et al, Blood 128:4819, 2016). Case Report: The patient is a 60-year-old man with intermediate-high risk MDS who underwent RIC-HCT with total lymphoid irradiation and antithymocyte globulin with peripheral blood graft from a fully matched unrelated male donor. The patient had mild thrombocytopenia prior to HCT consistent with MDS and had not received platelet transfusions. He had not received any prior therapy for MDS. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mycophenolate mofetil. Cytomegalovirus (CMV) serologic testing for exposure was negative for the recipient and positive for the donor. Both the patient and the donor had evidence for prior exposure to Epstein-Barr virus (EBV). He achieved engraftment on day +12. His peripheral blood chimerism on day + 30 showed full donor origin (WB 98%,CD3 96%,CD15 95%, CD19 98%, CD56 95%) and has been maintained to date. Acute skin GVHD responded to corticosteroids. While on corticosteroid therapy, he developed an abrupt decline in platelet count from 156,000/mcl on day +152 to 9, 000/mcl on Day + 166 without evidence for recurrent or active GVHD. While this was initially attributed to simultaneous EBV and CMV reactivations, severe thrombocytopenia persisted after viral clearance. An extensive work up for other etiologies of thrombocytopenia was negative. Repeated bone marrow biopsies were normal, including adequate megakaryocytosis and no MDS recurrence. Platelet associated antibody testing and platelet antigen genotyping were not conclusive for autoimmune versus alloimmune etiology. Testing for platelet HLA antibodies showed calculated Panel Reactive Antibody of 31% and unsatisfactory corrected count increment after transfusion of HLA compatible platelets units. The patient experienced prolonged severe thrombocytopenia for over 26 weeks with platelet count less than 5000/mcl for 22 weeks and only above 10,000 /mcl on 6 occasions. Potentially responsible medications were discontinued serially, but testing for drug inducted ITP was not conducted. Therapy included high dose corticosteroids, high dose immune globulin, rituximab, plasma exchange, splenectomy, romiplostim 10 mcg/kg/week, eltrombopag 100 mg to 150 mg daily for over 24 weeks, and low dose danazol. Fostamatinib was not available. Prednisone dose was tapered over many weeks to 5 mg daily. The patient experienced recurrent life-threatening and vision-threatening bleeding. Cumulative transfusions following Day + 166 were 133 single donor platelet units and 42 red cell units. All products were from CMV negative donors. Eltrombopag and danazol were deemed ineffective and tapered to discontinuation. CD38 positive cells were present in spleen and marrow by immunohistochemistry. Daratumumab 1300 mg was infused weekly x 4. Four weeks after the last dose of daratumumab, his platelet count increased to 91,000/mcl. Platelet count normalized to 150,000/mcl in week 5 or HCT Day + 383. Hypogammaglobulinemia has been the only detectable toxicity. Testing to determine autoimmune versus alloimmune origin is ongoing. Conclusion: Clinical trials of daratumumab for the treatment of severe refractory ITP are indicated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Significance of the Serotonin Release Assay and Platelet Count Monitoring After Cardiac Surgery

2017 ◽  
Vol 24 (6) ◽  
pp. 944-949 ◽  
Author(s):  
Shinya Motohashi ◽  
Takefumi Matsuo ◽  
Hidenori Inoue ◽  
Makoto Kaneko ◽  
Shunya Shindo
Keyword(s):  
Cardiac Surgery ◽  
Platelet Count ◽  
Clinical Course ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heparin Induced Thrombocytopenia ◽  
Release Assay ◽  
Immunological Assays ◽  
Platelet Count Monitoring ◽  
The Relationship

Heparin-induced thrombocytopenia (HIT) is one of the serious complications in patients who undergo cardiac surgery. However, there remains a major problem in diagnosing HIT because the current immunological assays for detection of HIT antibody have limitations. Furthermore, the clinical course of thrombocytopenia in this surgery makes it increasingly difficult to diagnose HIT. We investigated the relationship between platelet count and HIT antibody in 59 patients who underwent cardiac surgery using cardiopulmonary bypass (CPB). The number of postoperative HIT antibody-positive patients evaluated using enzyme-linked immunosorbent assay kit (polyanion IgG/IgA/IgM complex antibodies/antiplatelet factor 4 enhanced) was 37 (62.7%). In contrast, platelet activation by HIT antibody was evaluated using the serotonin release assay (SRA). More than 20% and 50% release of serotonin was obtained from 12 patients (20.3%) and 8 patients (13.6%), respectively. The levels of d-dimer were significantly different on postoperative day 14 between SRA-positive and SRA-negative groups; however, postoperative thrombus complication was not detected using sonography in the patients with positive serotonin release at all. After being decreased by the operation, their platelet count recovered within 2 weeks in both groups equally. In our study, although the patients were positive in the platelet activating HIT antibody assay, they remained free from thrombosis and their platelet count recovered after early postoperative platelet decrease. Therefore, in addition to the SRA, monitoring of platelet count might be still considered an indispensable factor to facilitate the prediction of HIT thrombosis prior to manifestation in the patients undergoing cardiac surgery using CPB.

Assessment of Thalidomide Therapy Effect on Platelet Activation and Selected Blood Coagulation Parameters in Multiple Myeloma Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5181-5181
Author(s):  
Marta Robak ◽  
Jacek Trelinski ◽  
Krzysztof Chojnowski
Keyword(s):  
Multiple Myeloma ◽  
Platelet Count ◽  
Factor Viii ◽  
Platelet Activation ◽  
Factor Vii ◽  
Control Group ◽  
High Dose ◽  
Closure Time ◽  
Therapy Effect ◽  
D Dimer

Abstract Background: Patients with multiple myeloma are at relatively high risk of developing thromboembolic events (TEE). These life-threatening complications may arise from hypercoagulability associated with malignancy and/or may be connected with anticancer therapy. The risk of developing TEE appears to be particularly high during treatment with thalidomide alone or combined with chemotherapy and/or high-dose dexamethasone. The pathogenesis of thalidomide-related thrombosis in myeloma patients remains unexplained. Some authors suggest that platelet activation can contribute to development of this complication in multiple myeloma patients on thalidomide therapy but until now it has not been a subject of investigation. Patients and methods: The study was performed in 20 patients with multiple myeloma. The tests were done at diagnosis and after one month of thalidomide therapy at a dose of 100–200 mg/24h. All patients had normal renal function and did not take drugs affecting platelet function. The control group consisted of 15 healthy subjects of similar age. In each patient closure time with ADP/Collagen and Epinephrine/Collagen cartridges by PFA-100 method was assessed. Platelet expression of membrane activation marker P-selectin (CD62p) on resting platelets and after stimulation with ADP/Collagen and Epinephrine/Collagen was analyzed by flow cytometry. Additionally, activity of factor VII, factor VIII and von Willebrand factor (vWF), concentration of fibrinogen and D-dimer, and platelet count were evaluated. Results: The mean PFA-100 closure time was significantly shortened with ADP/Collagen (87.2 ±17.1 s vs 100.4 ± 19.3 s, p=0.008) and Epinephrine/Collagen cartridges (118.5 ± 20.3 s vs 132.2 ± 27.9 s, p=0.04) after one month of therapy in comparison to baseline. The median CD62p percentage increased markedly after treatment-on resting platelets 5.1 (0.76–22.2) vs 3.6 (0.1–21.5) p=0.03. and after stimulation with Epinephrine/Collagen 16.6 (2.3–57.3) vs 11.1 (1.4–19.5) p=0.03. The observed increased P-selectin expression after ADP/Collagen stimulation 26.3 (8.5–42.8) vs 19.7 (1.0–35.4) was not statistically significant. The median values of P-selectin expression at diagnosis and after thalidomide therapy were also higher than in the control group. The results of factor VIII, vWF activity, fibrinogen and D-dimer concentration did not differ markedly before and after therapy. Significantly lower mean activity of factor VII (p=0.004) and higher mean platelet count (p=0.03) after therapy were observed. Conclusions: These results demonstrate that platelet activation is one of the pathogenetic factor of thalidomide-related thrombotic complications and can explain some observations that acetylsalicylic acid may protect against TEE during myeloma treatment with thalidomide.

N-Acetylcysteine Treatment in Two Patients with Relapsed Thrombotic Thrombocytopenic Purpura Increased ADAMTS13 Activity, Free Thiol Concentration in Plasma, and Inhibited Platelet Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 239-239
Author(s):  
Junmei Chen ◽  
Tahsin Özpolat ◽  
Colette Norby ◽  
Jennie Le ◽  
Minhua Ling ◽  
...  
Keyword(s):  
Platelet Count ◽  
Plasma Exchange ◽  
Platelet Activation ◽  
Research Funding ◽  
Specific Activity ◽  
High Dose ◽  
Adamts13 Activity ◽  
Thrombocytopenic Purpura ◽  
Platelet Counts ◽  
Free Thiol

Abstract Introduction: Thrombotic thrombocytopenic purpura (TTP) is a catastrophic and potentially fatal disorder caused by systemic microvascular thrombosis due to von Willebrand factor (VWF)-platelet thrombi. TTP is caused by congenital or acquired deficiency of the plasma metalloprotease ADAMTS13. Based on an earlier study (Chen J et al., J Clin Invest 2011, 121:593-603), we proposed N-acetylcysteine (NAC) as an adjunct treatment for TTP. This study showed that NAC reduced the size and activity of VWF in vitro in human plasma and in vivo in a TTP mouse model. In 2013 and 2014, two case reports described treatment of refractory TTP patients with NAC, one receiving a low dose of NAC [300 mg/kg (total 15 g) for the 1st 24 hrs, followed by 2.5 g/day for two weeks concurrently with plasma exchange] (Shortt J et al., N Engl J Med 2013, 368: 90-92; Shortt J et al., Transfusion 2014, 54:2362-2363) and the other receiving high-dose NAC [300 mg/kg/day (11 g/day) for 10 days between plasma exchanges] (Li GW et al. Transfusion 2014, 54: 1221-1224). The patient treated with high-dose NAC improved rapidly (the patient woke up from coma 18 hr after NAC treatment was initiated), but the patient treated with lower dose NAC did not appear to respond. Thus, it is as yet unclear whether NAC is an effective treatment for TTP. Therefore, more clinical studies and detailed analyses are required to examine the effects of NAC in TTP patients. Here we report the results of clinical and biochemical studies on two patients with relapsed TTP treated with NAC. Before, during, and after NAC treatment, we determined the concentrations of NAC, cysteine, and glutathione in plasma; VWF concentration, multimer structure, and functions; ADAMTS13 concentration and activity; and platelet counts and activation status (P-selectin expression and phosphatidylserine exposure). Methods: Two females with a history of prior episodes of TTP presented with acute TTP [ADAMTS13 < 10%, positive for ADAMTS13 inhibitors, platelet count ≤ 10,000/uL, lactate dehydrogenase (LDH) > 600 IU/L] and both were treated with NAC per IRB-approved protocol [150 mg/kg bolus over 1 hr and 150 mg/kg as continuous infusion until the next therapeutic plasma exchange (TPE)]. They received daily TPE until their platelet counts normalized, and intravenous NAC during days 2-5. Blood was collected daily for 8 days for research assays. ADAMTS13 concentrations in patient plasma were measured by ELISA. ADAMTS13 activity was measured using HRP-conjugated A2 peptide substrate (Wu J-J et al. J Thromb Haemost 2006, 4:129-136). Concentrations of NAC, total cysteine, and total free thiols (free thiol cysteine and free thiol NAC) in plasma were determined by mass spectrometry. Plasma VWF multimer patterns were analyzed by 1.5% agarose gel electrophoresis followed by western blotting with an HRP-conjugated polyclonal VWF antibody. Platelets in whole blood were labeled for platelet markers (CD41a or CD42b) together with one of the activation markers, P-selectin or phosphatidylserine (lactadherin). The labeled platelets were analyzed by flow cytometry. Results: Platelet counts in both patients started to increase 1 day after NAC infusion and continued to increase after discontinuation of NAC and TPE. After NAC infusion, the free thiol concentration (NAC and cysteine) in plasma increased 4 and 59 fold in patients 1 and 2, respectively. This was accompanied by increasing ADAMTS13 specific activity (ADAMTS13 activity/ADAMTS13 antigen). In patient 1, the specific activity increased from 127% (prior to NAC infusion but after TPE) to 270% during NAC infusion; in patient 2, the specific activity increased from 56% to 86%. In patient 1, the VWF multimer size decreased during NAC treatment and the VWF multimers migrated slightly faster. NAC also appeared to inhibit platelet activation. Before NAC infusion, the platelets in both patients were positive for phosphatidylserine (PS, > 30%) and P-selectin (> 15%), compared to 2% and 5%, respectively, in a normal control. The percentages of PS- and P-selectin-positive platelets decreased to less than 18% and 10% respectively, during NAC treatment. Summary: NAC treatment of two patients with TTP in conjunction with TPE was well tolerated and associated with recovery of platelet count and LDH, increased ADAMTS13 specific activity and total free thiol concentration in plasma, reduced platelet activation, and decreased VWF multimer size in one patient. Disclosures Konkle: CSL Behring: Consultancy; Pfizer: Consultancy; Baxalta: Consultancy, Research Funding; Biogen: Consultancy, Research Funding; Octapharma: Research Funding; Novo Nordisk: Consultancy.

scholarly journals Increased FVIII and Anti-Phospholipid Antibodies Do Not Modify the Clinical Course of Chronic Immune Thrombocytopenia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5013-5013
Author(s):  
Meet Kumar ◽  
Maitryee Bhattyacharyya ◽  
Shyamali Datta
Keyword(s):  
Platelet Count ◽  
Lupus Anticoagulant ◽  
Immune Thrombocytopenia ◽  
Clinical Course ◽  
Conflicts Of Interest ◽  
Age Groups ◽  
Platelet Counts ◽  
Bleeding Score ◽  
Chronic Immune Thrombocytopenia

Abstract INTRODUCTION: Immune thrombocytopenia is a heterogenous disease with majority patients having a mild bleeding phenotype and one-fourth being asymptomatic. Bleeding episodes are usually seen in patients with platelet counts typically <30,000/cumm. There is no study till date to identify patients with platelet count <30,000/cumm and at high risk for bleeding. Although patients who harbor anti phospholipid antibodies have a higher risk of arterial and venous thrombosis, it is not known whether presence of acquired thrombophilia modifies the clinical course of bleeding in low platelet count ITP patients. We evaluated the role of FVIII and lupus anticoagulant in modifying the clinical course of such patients. MATERIALS AND METHODS: Patients of all age groups with persistent and chronic immune thrombocytopenia were eligible for study enrolment. Patients with acute ITP and secondary ITP were excluded. Eligible patients were evaluated with baseline parameters, ITP bleeding score (ITP-BAT, version 1.0 by IWG on ITP) at baseline and then at every visit and FVIII and lupus anticoagulant at baseline and repeat at six months.Patients were called for monthly scheduled visits if platelet counts were >30,000/cummand more frequently at lower platelet counts. Patients with any evidence of underlying infection or raised c-reactive protein and procalcitonin were deferred evaluation.All patients were treated as per institutional protocol to avoid treatment bias. Patients were followed up for one year. We finally calculated the average bleeding scores of all patients at different platelet counts (for eg. average of skin, mucosal and organ bleeding scores of all patients that had platelet counts <10,000/cumm, 10-30,000/cumm etc) and analysed it with FVIII and anti-phospholipid antibody levels. RESULTS: A total of 45 patients were enrolled with M:F=1:3.2. Median age of patients is 28years (3-72 years). Two patients were excluded (both progressed to SLE) and another two were lost to follow-up. Median duration from ITP diagnosis to study enrolment was 62.4months (8-590 months). Median follow-up of all patients was 14.2 months (13.2-16.4 months). Nine patients had persistent and 32 patients had chronic ITP. ITP-BAT could differentiate intensity of bleeding at different platelet counts by means of bleeding scores (Table 1). Correlation of bleeding scores with prothrombotic markers could not establish a disease course modifying relationship between the two (Table 2). CONCLUSION: Presence of high FVIII and anti phosphoilipid antibodies donot modify the bleeding risks in patients with ITP and low platelet counts. Table 1Platelet count (x109/cumm)Total episodesAverage bleeding scoreP value<1014S2.4 M1.4 O0 0.00210-3015S1.4 M0.9 O030-6018S0.3 M0.1 O0>600S0 M0 O0 Table 2 Platelet count <10,000/cumm Platelet count 10-30,000/cumm N Av BS P= N Av BS P= FVIII>150 5 S2.5M1.1O0 0.02 6 S1.1M0.8O0 0.1 FVIII<150 9 S2M0.7O0 11 S1.4M0.6O0 LA1:LA2>2 1 S2.2M1.5O0 0.03 S1.1M0.8O0 0.2 LA1:LA2<1 12 S2.6M1.2O0 S1.2M0.6O0 Table 3 Platelet count 30-60,000/cumm Av BS P= FVIII>150 4 S0.9M0.1O0 0.2 FVIII<150 11 S0.3M0.1O0 LA1:LA2>2 Nil LA1:LA2<1 9 S1.1M0.4O0 Disclosures No relevant conflicts of interest to declare.

Abstract 451: Platelet Activation and Thrombosis by Antibodies against VEGF and CD40 Ligand: Extending the mechanism of HIT

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ali Amirkhosravi ◽  
Todd Meyer ◽  
Florian Langer ◽  
Theresa Robson ◽  
Liza Robles ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Cd40 Ligand ◽  
Serotonin Release ◽  
Heparin Binding ◽  
Wild Type ◽  
Platelet Factor ◽  
Cardiovascular Comorbidity ◽  
Platelet Aggregometry

Introduction: The vascular endothelial growth factor (VEGF) monoclonal antibody, bevaci-zumab (Avastin), has been associated with arterial thromboembolic events in some cancer patients. Another therapeutic antibody, hu5c8, which targets CD40L, also produced unexpected thrombosis in lupus clinical trials. Because platelets play a crucial role in arterial thrombosis, we hypothesized that antibodies against VEGF and CD40L may activate platelets via a mechanism similar to that responsible for thrombosis in Heparin-Induced Thrombocytopenia (HIT). Methods: Immune complexes (ICs) were prepared by combining these monoclonal IgG antibodies with their antigens: M90 (or hu5c8) with CD40L (“M90+CD40L”), or bevacizumab with VEGF and heparin (“BVH”). VEGF binds heparin (as does platelet factor 4, the HIT antigen). We measured platelet activation by serotonin release assay, platelet aggregometry, and flow cytometry. We also evaluated IC-induced thrombosis in hFc mice, transgenic for the human IgG receptor, CD32. Results: Similar to HIT antibodies, these ICs potently induced platelet activation dependent on CD32, IC concentration (>10nM) and optimal stoichiometry. Intravenous injection of M90+CD40L or BVH into hFc, but not wild-type mice rapidly produced signs of thrombotic shock, thrombocytopenia and pulmonary thrombosis. However, wild-type control mice (lacking platelet CD32) were unaffected by IC injection. Similarly as with HIT antibodies, bevacizumab IC activity was reduced in the absence or excess of heparin both in vitro and in vivo, whilst M90+CD40L-induced platelet activation was abolished in vitro by blockade of the platelet CD40L receptor, CD40, demonstrating a requirement for Fab-dependent anchoring. Furthermore, VEGF 121 (which lacks the heparin-binding domain of VEGF 165 ) and bevacizumab with or without heparin failed to activate platelets or cause thrombosis in hFc mice. Conclusions: Together, these findings demonstrate that Fab-dependent anchoring of anti-CD40L and anti-VEGF ICs is required for potent platelet activation and thrombosis, as is the case in HIT, suggesting common mechanistic elements. Clinical implications may apply in patients with cardiovascular comorbidity receiving immunotherapy.

Cold Hemagglutinin Hemolysis and Hypercoagulable State Due to Mycoplasma Pneumoniae.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5094-5094
Author(s):  
Gaurav Gulati ◽  
Anthony A Donato ◽  
Shuchi Gulati ◽  
Daniel A Forman
Keyword(s):  
Platelet Count ◽  
Hemolytic Anemia ◽  
Mycoplasma Pneumoniae ◽  
Skin Necrosis ◽  
Conflicts Of Interest ◽  
Anticardiolipin Antibody ◽  
The United States ◽  
Cold Agglutinin ◽  
High Dose ◽  
Superficial Skin

Abstract Abstract 5094 Introduction Mycoplasma pneumoniae is responsible for 7 to 20% of community-acquired pneumonia in the United States. Up to 25% of patients develop extra-pulmonary complications. Hemolytic anemia associated with multiple vascular thromboses is a rare but severe complication of Mycoplasma pneumoniae. We present a case of cold agglutinin-associated hemolysis and skin necrosis. Case Presentation An 81 year-old male on Coumadin for atrial fibrillation who recently returned from a cruise, developed non productive cough without fever and dyspnea. He was treated with a course of azithromycin. One week later he developed bruising and skin de-pigmentation of the tips of the pinnae and tip of the nose. On presentation to the emergency department, his physical exam was unremarkable except for these finding and mild rhonchi over his right posterior chest. Laboratory workup revealed a Hemoglobin of 13.5 g/ dL, leukocytosis of 23,700 c/mm3 and platelet count of 57,000 c/mm3. Moderate renal insufficiency was also present with a creatinine of 2.02. Evaluation of peripheral smear revealed mild Schistiocytosis with anisopoikilocytosis. Within 1 week, hemoglobin dropped to 7.4, platelet count reached a nadir of 16,000. Hemolytic parameters included LDH of 647 IU/L with haptoglobin of 26 mg/dL, and reticulocyte count of 1.3%. Total complement levels were severely depressed at 4 U/ml (normal: 30 to 75 U/ml), while there was a slight decrease in C4 complement levels and the C3 levels were normal. Immunoglobulin levels were within limits and cryofibrogen was negative. ANA, anticardiolipin antibody, myeloperoxidase and p ANCA levels showed normal titers. A bone marrow biopsy showed hypercellular marrow with erythroid hyperplasia. Urine Strep. pneumoniae antigen was negative. Cold agglutinin titers were done, which were negative. Due to our strong suspicion for mycoplasma-related cold agglutinin hemolysis, we repeated cold agglutinin titers which turned positive very slowly on prolonged standing. The patient was treated with high dose prednisone and received multiple sessions of plasmapheresis which improved his condition and platelet counts returned to baseline with LDH and haptoglobin levels trending back to normal. Discussion Our patient had a wide differential ranging from Coumadin induced skin necrosis, auricular perichondritis, frost bite, local infections, TTP/ HUS syndrome, and Wegner's granulomatosis. But the clinical presentation of superficial skin necrosis of peripheral skin along with the presence of low complement, recent chest infection, hemolytic anemia and thrombocytopenia with acute renal failure all contributed to our final diagnosis confirmed by the presence of slow reactive Coomb's direct anticoagulation test and supported by improvement with plasmapheresis and steroid therapy. The astute clinician should always remember mycoplasma infection in setting of acute onset hemolytic anemia and suspect it in recently treated chest and sinus infections. Disclosures No relevant conflicts of interest to declare.

Outcomes and Prognostic Factors Following Double-Induction Chemotherapy in AML – A Large, Single Institutional Experience,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3605-3605
Author(s):  
Jeffrey E. Lancet ◽  
Alan F. List ◽  
Celeste M. Bello ◽  
Najla H Al Ali ◽  
Rami S. Komrokji
Keyword(s):  
Bone Marrow ◽  
Logistic Regression ◽  
Induction Chemotherapy ◽  
Odds Ratio ◽  
Conflicts Of Interest ◽  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Categorical Variables ◽  
High Dose ◽  
Cox Regression Analysis

Abstract Abstract 3605 Background: Although a majority of patients with AML achieve complete response (CR) following 1 or 2 cycles of induction chemotherapy, rates of relapse-free and overall survival remain poor. In the US, typically the decision to administer re-induction chemotherapy depends upon the degree of leukemic cell clearance from the bone marrow at 10–14 days after initial induction. In this single-institution study, we assessed patients who underwent double induction chemotherapy for AML in an attempt to delineate specific clinical variables that might influence outcomes and decisions to utilize re-induction chemotherapy. Methods: Between 2004 and 2010, patients who received 2 courses of induction chemotherapy for AML at the H. Lee Moffitt Cancer Center were analyzed. Individual charts were reviewed. Chi square test was used to compare categorical variables in univariate analysis. Kaplan Meier estimates were used to calculate OS. Log rank test was used for comparison between the 2 groups and Cox regression analysis was used for multivariable analysis of survival. Binomial logistic regression was used for multivariate assessment of response rates. All analyses were conducted using SPSS version 19.0 software. Results: We identified 164 patients with previously untreated AML who underwent double-induction chemotherapy at our center, 127 of whom had residual blasts ≥ 10% following initial induction. Baseline characteristics (%): male:female (68%:32%), age less than:greater than 60 (57%:43%), adverse:non adverse karyotype (39%:58%), de-novo:secondary AML (66%:32%). The majority (97%) initially received anthracycline + cytarabine (“7+3”) based induction chemotherapy. Second induction utilized a high-dose cytarabine based regimen in 65% of patients. Overall response rate (CR + CRi) was 62%. Median survival for the entire cohort was 13.3 months (95% CI 11.4–15.3). Univariate analysis of prognostic variables associated with response and survival are shown in Table 1. On multivariate analysis, only adverse karyotype (p=0.02, HR 1.67) and non-hypocellular (≥ 20%) bone marrow at day 14 after 1st induction (p=0.002, HR 1.934) were statistically significant predictors of inferior survival (figure 1), but there was a trend toward inferior OS for re-induction beginning after day 21. The only statistically significant predictors for response (CR+CRi) in the logistic regression model were age < 60 (p=0.034, odds ratio 2.850) and hypocellular day 14 bone marrow after 1st induction (p <0.005, odds ratio 7.87). Conclusions: In patients who received double induction therapy for AML, response was achieved in the majority, and the bone marrow cellularity at day 14 after induction cycle 1 was the strongest predictor of response and survival, strongly suggesting its consideration as a prognostic/stratification factor in future outcomes studies as well as studies testing new agents in refractory disease. Future analyses will include direct comparisons of outcomes and analysis of risk factors between patients receiving 1 versus 2 cycles of induction. Disclosures: No relevant conflicts of interest to declare.

Heparin-Associated Thrombocytopenia: The Effects of Various Intravenous lgG Preparations on Antibody Mediated Platelet Activation - A Possible New Indication for High Dose i.v. IgG

1994 ◽  
Vol 71 (05) ◽  
pp. 641-645 ◽  
Author(s):  
A Greinacher ◽  
U Liebenhoff ◽  
V Kiefel ◽  
P Presek ◽  
C Mueller-Eckhardt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Activation ◽  
Manufacturing Process ◽  
Inhibitory Effect ◽  
Serotonin Release ◽  
Low Ph ◽  
High Dose ◽  
Human Igg ◽  
Platelet Protein

SummaryThe immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-re- ceptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP Ilb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann’s thrombasthenia (GT) lacking GP Ilb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab’)2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-seroto- nin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP Ilb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT. This is, however, possible by high dose i.v. IgG, whereby inhibition of FcRII on platelets strongly depends upon the process by which the i.v. IgG preparation was manufactured.

scholarly journals Platelet Activating Immune Complexes Identified in COVID-19 Associated Coagulopathy

2020 ◽  
Author(s):  
Ishac Nazy ◽  
Stefan D Jevtic ◽  
Jane C Moore ◽  
Angela Huynh ◽  
James W Smith ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Endothelial Activation ◽  
Serotonin Release ◽  
High Dose ◽  
List Type ◽  
Key Points ◽  
Release Assay ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThrombosis is a prominent feature of coronavirus disease 2019 (COVID-19) and often occurs in patients who are critically ill; however, the mechanism is unclear. This COVID-19 associated coagulopathy (CAC) shares features with heparin-induced thrombocytopenia (HIT), including mild thrombocytopenia and thrombosis. We thus tested 10 CAC patients for anti-PF4/heparin antibodies and functional platelet activation. HIT was excluded in all samples based on anti-PF4/heparin antibody and serotonin release assay results. Of note, 6 CAC patients demonstrated platelet activation by the serotonin release assay that was inhibited by FcγRIIA receptor blockade, confirming an IgG-specific immune complex (IC)-mediated reaction. Platelet activation was independent of heparin, but inhibitable by both therapeutic and high dose heparin. All 6 samples were positive for IgG-specific antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples were additionally characterized by significant endothelial activation, shown by increased von Willebrand factor antigen and activity. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, ruling out thrombotic thrombocytopenic purpura. Our study thus identifies platelet-activating ICs as a mechanism that contributes to CAC thrombosis.Scientific CategoryThrombosis and HemostasisKey PointsPatients with COVID-19 thrombosis have immune complexes that activate platelets through FcγRIIA signallingPatients with COVID-19 thrombosis demonstrate increased VWF antigen and activity that is not related to severe ADAMTS13 reduction

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