Karina Althaus

MYH9-Related Platelet Disorders

Myosin heavy chain 9 (MYH9)-related platelet disorders belong to the group of inherited thrombocytopenias. The MYH9 gene encodes the nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cytoskeletal contractile protein. Several mutations in the MYH9 gene lead to premature release of platelets from the bone marrow, macrothrombocytopenia, and cytoplasmic inclusion bodies within leukocytes. Four overlapping syndromes, known as May-Hegglin anomaly, Epstein syndrome, Fechtner syndrome, and Sebastian platelet syndrome, describe different clinical manifestations of MYH9 gene mutations. Macrothrombocytopenia is present in all affected individuals, whereas only some develop additional clinical manifestations such as renal failure, hearing loss, and presenile cataracts. The bleeding tendency is usually moderate, with menorrhagia and easy bruising being most frequent. The biggest risk for the individual is inappropriate treatment due to misdiagnosis of chronic autoimmune thrombocytopenia. To date, 31 mutations of the MYH9 gene leading to macrothrombocytopenia have been identified, of which the upstream mutations up to amino acid approximately 1400 are more likely associated with syndromic manifestations than the downstream mutations. This review provides a short history of MYH9-related disorders, summarizes the clinical and laboratory characteristics, describes a diagnostic algorithm, presents recent results of animal models, and discusses aspects of therapeutic management.

Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests

Summary.  Background: Laboratory confirmation of heparin‐induced thrombocytopenia (HIT) is based on detection of heparin‐dependent platelet‐activating antibodies. Platelet factor 4 (PF4)/heparin enzyme‐immunoassays (EIA) are a widely available surrogate for platelet‐activating antibodies. Objective: Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet‐activating antibodies. Patients/methods: Using quantile regression we determined the 97.5th percentile of PF4/heparin‐immunoglobulin G (IgG) EIA reactivities in non‐heparin‐treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA‐IgG reactivities (Greifswald laboratory; n = 2821) and the heparin‐induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA‐IgG (McMaster laboratory; n = 1956) with the serotonin‐release assay (SRA). Results: PF4/heparin‐IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P < 0.001). The percentage of sera testing positive in the functional assays strongly correlated with PF4/heparin‐IgG EIA OD reactivities in both laboratories with very similar results (correlation coefficient > 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. Conclusions: Results of PF4/heparin‐IgG EIA should not be reported as only positive or negative as there is no single acceptable cut‐off value. Instead, reporting PF4/heparin‐IgG EIA OD results in ranges allows for risk‐stratified prediction for presence of platelet‐activating antibodies. Use of normalized OD ranges permits a standardized approach for inter‐laboratory comparisons.

Evaluation of automated immunoassays in the diagnosis of heparin induced thrombocytopenia

BACKGROUND Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep) complexes. The in vitro demonstration of PF4/hep antibodies using functional and immunological methods is essential for optimal management of patients suspected to have HIT. Since functional assays are technically challenging and limited to specialized laboratories, antigen-binding assays are commonly used in routine laboratories. STUDY DESIGN Blood samples from 448 consecutive patients in whom HIT was suspected were investigated using a latex agglutination test HemosIL® HIT-Ab(PF4-H) (HemosIL-Ab), two chemiluminescence tests HemosIL AcuStar HIT-Ab(PF4-H) (HemosIL AcuStar-Ab) and AcuStar HIT-IgG(PF4-H) (HemosIL AcuStar-IgG), an in-house PF4/hep IgG enzyme immunoassay (EIA) and the heparin induced platelet aggregation (HIPA) test. RESULTS Antibodies against PF4/hep were detectable in 44 out of 119 samples using HemosIL-Ab among which 20 samples were also reactive in the HIPA; and in 122, 64 and 108 out of 448 sera using HemosIL AcuStar-Ab, HemosIL AcuStar-IgG and in-house PF4/hep IgG-EIA, respectively, among which 52 sera were also reactive in the HIPA. All assays had high sensitivities of >95% for platelet activating antibodies; however, they differed in their specificities. The highest specificity and positive predictive value was observed by HemosIL AcuStar-IgG (96% and 78%, respectively). CONCLUSION Automated immunoassays are useful in the laboratory investigations of HIT and present a potential improvement toward standardization of laboratory investigations of HIT. The high positive predictive capability may justify treating the patient with alternative anticoagulants without waiting for the results of a functional assay.

Platelet transfusion for reversal of dual antiplatelet therapy in patients requiring urgent surgery: a pilot study.

1 Food and Drug Administration. Pradaxa (dabigatran etexilate mesylate): drug safety communication-safety review of post-market reports of serious bleeding events. http://www.fda.gov/Safety/MedWatch/ SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm 282820.htm. Accessed 10 March 2012. 2 Therapeutic Goods Administration, Australia. Dabigatran (Pradaxa): risk of bleeding relating to use. http://www.tga.gov.au/safety/alertsmedicine-dabigatran-111005.htm. Accessed 10 March 2012. 3 EuropeanMedicines Agency. EuropeanMedicines Agency updates on safety of Pradaxa [press release]. http://www.ema.europa.eu/docs/ en_GB/document_library/Press_release/2011/11/WC500117818.pdf. Accessed 10 March 2012. 4 The ACTIVE Investigators, Connolly SJ, Pogue J, Hart RG, Hohnloser SH, Pfeffer M, Chrolavicius S, Yusuf S. Effect of clopidogrel added to aspirin in patients with atrial fibrillation. N Engl J Med 2009;360:2066–78. 5 Connolly SJ, Eikelboom J, Joyner C, Diener HC, Hart R, Golitsyn S, FlakerG, AvezumA,Hohnloser SH,DiazR, TalajicM, Zhu J, Pais P, Budaj A, Parkhomenko A, Jansky P, Commerford P, Tan RS, Sim KH, Lewis BS, et al. Apixaban in patients with atrial fibrillation. N Engl J Med 2011; 364: 806–17. 6 ACTIVE Writing Group of the ACTIVE Investigators, Connolly S, Pogue J, Hart R, Pfeffer M, Hohnloser S, Chrolavicius S, Pfeffer M, Hohnloser S, Yusuf S. Clopidogrel plus aspirin versus oral anticoagulation for atrial fibrillation in the Atrial fibrillation Clopidogrel Trial with Irbesartan for prevention of Vascular Events (ACTIVE W): a randomised controlled trial. Lancet 2006; 367: 1903–125. 7 Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A, Pogue J, Reilly PA, Themeles E, Varrone J, Wang S, Alings M, Xavier D, Zhu J, Diaz R, Lewis BS, Darius H, Diener HC, Joyner CD, Wallentin L, et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 2009; 361: 1139–51. 8 Granger CB, Alexander JH, McMurray JJ, Lopes RD, Hylek EM, Hanna M, Al-Khalidi HR, Ansell J, Atar D, Avezum A, Bahit MC, Diaz R, Easton JD, Ezekowitz JA, Flaker G, Garcia D, Geraldes M, Gersh BJ, Golitsyn S, Goto S, et al. Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med 2011; 365: 981–926. 9 Eikelboom JW, Wallentin L, Connolly SJ, Ezekowitz M, Healey JS, Oldgren J, Yang S, Alings M, Kaatz S, Hohnloser SH, Diener HC, FranzosiMG,Huber K, Reilly P, Varrone J, Yusuf S. Risk of bleeding with 2 doses of dabigatran compared with warfarin in older and younger patients with atrial fibrillation: an analysis of the Randomized Evaluation of Long-term anticoagulant Therapy (RE-LY) Trial. Circulation 2011; 123: 2363–72. 10 Hart RG, Pearce LA, Aguilar MI. Meta-analysis: antithrombotic therapy to prevent stroke in patients who have nonvalvular atrial fibrillation. Ann Intern Med 2007; 146: 857–67. 11 Wallentin L, Yusuf S, Ezekowitz MD, Alings M, Flather M, Franzosi MG, Pais P, Dans A, Eikelboom J, Oldgren J, Pogue J, Reilly PA, Yang S, Connolly SJ. Efficacy and safety of dabigatran compared with warfarin at different levels of international normalised ratio control for stroke prevention in atrial fibrillation: an analysis of the RE-LY trial. Lancet 2010; 376: 975–83. 12 Wang TJ,Massaro JM, Levy D, Vasan RS,Wolf PA, D Agostino RB, Larson MG, Kannel WB, Benjamin EJ. A risk score for predicting stroke or death in individuals with new-onset atrial fibrillation in the community: the FraminghamHeart Study. JAMA 2003; 290: 1049–56.

MYH-9 Related Platelet Disorders: Strategies for Management and Diagnosis

MYH-9 related platelet disorders belong to the group of inherited giant platelet disorders. The MYH-9 gene encodes the non-muscular myosin heavy chain IIA (NMMHCIIA), a cytoskeletal contractile protein. Several mutations in the MYH-9 gene lead to macrothrombocytopenia, and cytoplasmic inclusion bodies within leukocytes, while the number of megakaryocytes in the bone marrow is normal. Four overlapping syndromes, known as May-Hegglin anomaly, Epstein syndrome, Fechtner syndrome and Sebastian platelet syndrome, describe different clinical manifestations of MYH9 gene mutations. Macrothrombocytopenia is present in all affected individuals, whereas only some develop additional clinical manifestations such as renal failure, hearing loss and presenile cataracts. The bleeding tendency is usually moderate, with menorrhagia and easy bruising being most frequent. The biggest risk for the individual is inappropriate treatment due to misdiagnosis of chronic autoimmune thrombocytopenia. More than 30 mutations within the 40 exons of the MYH-9 gene leading to macrothrombocytopenia have been identified, of which the upstream mutations up to amino acid ∼1400 are more likely associated with syndromic manifestations than the downstream mutations. Diagnosis is based on identification of the granulocyte inclusion bodies using blood smears and immunofluorescence and is finally confirmed by identifying the mutation. Treatment is supportive and should be aimed to prevent iron deficiency anemia. Beside renal failure, the biggest risk for patients affected by a MYH-9 disorder are the adverse effects resulting form treatment based on the misdiagnosis of immune thrombocytopenia.

Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay

BACKGROUND IgG-specific anti-PF4/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain. OBJECTIVE To determine for an IgG-specific EIA how the OD values of a positive reaction and its inhibition by high heparin can be optimally combined. METHODS We screened 1,000 consecutive patients with suspected HIT using an IgG-specific PF4/heparin in-house EIA with and without high heparin (100 IU/mL); and by the heparin-induced platelet activation test. RESULTS Platelet-activating antibodies were rarely detected (<0.2%) when the IgG-specific EIA was negative at the conventional cut-off (OD, 0.5). However, an OD cut-off of 1.0 resulted in an unacceptable loss of sensitivity (14/83=17%) for detecting platelet-activating antibodies. The high heparin step increased specificity for platelet-activating antibodies from 72% to 89% without loss of sensitivity when applied to weak-positive sera (OD≤1.0). However, decreased sensitivity was observed with strong-positive sera (OD>1.0): 11/69 such sera (16%) that did not show >40% inhibition by high heparin nevertheless contained platelet-activating antibodies. CONCLUSION Specificity of an IgG-specific EIA for detecting platelet-activating antibodies can be optimized by applying the high heparin inhibition step to weak-positive reactions (0.5-≤1.0 OD). However, applying the high heparin inhibition step to strong-positive reactions (>1.0 OD) in our in-house assay risks falsely classifying a serum as negative for platelet-activating antibodies.

Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders

Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25–30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world.

Alternative diagnosis to heparin-induced thrombocytopenia in two critically ill patients despite a positive PF4/heparin-antibody test

Abstract Thrombocytopenia can cause diagnostic challenges in patients who have received heparin. Heparin-induced thrombocytopenia (HIT) is often considered in the differential diagnosis, and a positive screening can be mistaken as confirmation of the disorder. We present two patients who both received low-molecular-weight heparin for several days. In the first patient, clinical judgment rejected the suspicion of HIT despite a positive screening assay, and treatment for the alternative diagnosis of post-transfusion purpura was correctly initiated. In the second patient, the inaccurate diagnosis HIT was pursued due to a positive screening assay, while the alternative diagnosis of drug-dependent thrombocytopenia caused by piperacillin/tazobactam was rejected. This resulted in re-exposure to piperacillin/tazobactam which caused a second episode of severe thrombocytopenia. A positive screening assay for platelet factor 4/heparin-antibody should be verified by a functional assay, especially in patients with low pretest probability for HIT.

Recessive grey platelet-like syndrome with unaffected erythropoiesis in the absence of the splice isoform GFI1B-p37

Gene expression during hematopoietic lineage commitment is tightly regulated by a complex network of transcriptional regulators. Growth Factor Independent 1 (GFI1) and GFI1B are recruiters of histone deacetylases, histone methyltransferases, and key transcriptional repressors during hematopoiesis.[1

Flucloxacillin-induced immune thrombocytopenia.

Drug‐induced immune thrombocytopenia (DITP) is an adverse drug reaction associated with platelet (PLT) destruction by drug‐dependent antibodies. Demonstration of drug‐dependent PLT antibodies is often difficult and can only be rendered by extensive laboratory testing. In this report, we present the first serologically confirmed case of DITP caused by the antibiotic flucloxacillin.