Assessment of Time-Dependent Platelet Activation Using Extracellular Vesicles, CD62P Exposure, and Soluble Glycoprotein V Content of Platelet Concentrates with Two Different Platelet Additive Solutions.

Novel analytical measures are needed to accurately monitor the properties of platelet concentrates (PCs). Since activated platelets produce platelet-derived extracellular vesicles (EVs), analyzing EVs of PCs may provide additional information about the condition of platelets. The prospect of using EVs as an auxiliary measure of platelet activation state was investigated by examining the effect of platelet additive solutions (PASs) on EV formation and platelet activation during PC storage. The time-dependent activation of platelets in PCs with PAS-B or with the further developed PAS-E was compared by measuring the exposure of CD62P by flow cytometry and the content of soluble glycoprotein V (sGPV) of PCs by an immunoassay. Changes in the concentration and size distribution of EVs were determined using nanoparticle tracking analysis. A time-dependent increase in platelet activation in PCs was demonstrated by increased CD62P ex-posure, sGPV content, and EV concentration. Using these strongly correlating parameters, PAS-B platelets were shown to be more activated compared to PAS-E platelets. Since the EV concentration correlated well with the established platelet activation markers CD62P and sGPV, it could potentially be used as a complementary parameter for platelet activation for PCs. More detailed characterization of the resulting EVs could help to understand how the PC components contribute the functional effects of transfused PCs.

Autoimmune heparin-induced thrombocytopenia of delayed onset: a clinical challenge.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) usually appears at 5 to 10 days after initiation of heparin. Autoimmune HIT can arise after discontinuation of heparin treatment (delayed-onset HIT) or without any preceding heparin exposure (spontaneous HIT syndrome). CASE REPORT: This case presents a course of autoimmune HIT with delayed onset. The patient was hospitalized due to influenza pneumonia and received low-molecular-weight heparin thromboprophylaxis for 9 days. Seven days after discharge, she was readmitted because of a cerebral sinus vein thrombosis and severe thrombocytopenia. Intracranial bleeding and brain infarction caused her death. DISCUSSION: Autoimmune HIT was confirmed by functional heparin-induced platelet (PLT) activation test. Intracranial bleeding prevented continuous and effective anticoagulation. PLT transfusions were given, although they are generally advised against in HIT patients due to potential risk of thromboembolic events. CONCLUSION: This case presents that testing PLT-activating antibodies both in the presence and in the absence of current heparin treatment helps to diagnose patients with autoimmune HIT. There is conflicting evidence to refuse PLT transfusion when HIT is complicated with life-threatening bleeding.

Maternal HLA genotyping is not useful for predicting severity of fetal and neonatal alloimmune thrombocytopenia.

Lack of reliable laboratory parameters is the main challenge in the management of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Despite the long-known association between the HLA-DRB3*01:01 allele and human platelet antigen 1a (HPA-1a) alloimmunisation, maternal human leucocyte antigen (HLA) typing has been of little clinical value. Recently, other DRB3 allele variants have been suggested to predict the severity of FNAIT. In this nationwide population-based retrospective cohort study, we performed extensive HLA typing of 96 women, accounting for 87% of our cohort of 110 families with confirmed or possible HPA-1a-immunisation. The HLA type was compared with anti-HPA-1a levels, severity of neonatal disease and responsiveness to maternally administrated intravenous gammaglobulin (IVIG). HLA haplotypes were constructed to investigate further HLA associations. Despite significantly lower anti-HPA-1a levels in DRB3*01:01-negative women, the carrier status of this particular allele could not be used to confirm or rule out FNAIT in the absence of detectable antibodies. In the haplotype analysis, the DRB3*01:01 allele was the actual factor associated with FNAIT. No other HLA allele was shown to be of additional value as a predictor of severe FNAIT or non-responsiveness to IVIG treatment. Thus, HLA genotyping was not found useful in differentiating high- and low-risk pregnancies or in guiding antenatal treatment in affected families.

Great discrepancy in antithrombin activity measured using five commercially available functional assays.

INTRODUCTION: Congenital antithrombin (AT) deficiency is an inherited thrombophilia with high thrombosis prevalence. It has been reported that functional laboratory tests have varying potential in recognizing type II defects, and that there is discrepancy between thrombin inhibition based and factor Xa inhibition based methods. MATERIALS AND METHODS: Patients with known AT deficiency (n=374) were interviewed and their current AT status was tested in a new blood sample (n=214). The samples were analyzed using five different commercial methods (either thrombin or FXa based and one thrombin based method using two different incubation times). Antigen assay was used for typing the deficiency. RESULTS: In 101 of 214 (47.2%) samples the results obtained by different methods were congruent: 91 low and 10 normal by all assays. All other 113 (52.8%) samples showed discrepant values between the assays: most of them had abnormal results by two methods and normal by other methods. The discrepancies were observed mainly in type II deficiency. The best correlation of results was observed between one thrombin based and one FXa based assay. CONCLUSIONS: There was great inter-assay variability especially in type II deficient patients, but also in patients with type I deficiency. However, most of the patients defined as having normal AT activity by some methods had thrombotic symptoms. Most tested assays find type I AT deficient patients accurately. In our study population only methods A1 and C could find most patients with type II AT deficiency, whereas methods A2, B and D misdiagnosed the majority of patients as non-deficient.

Fetal intracranial haemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry.

OBJECTIVE: To characterise pregnancies where the fetus or neonate was diagnosed with fetal and neonatal alloimmune thrombocytopenia (FNAIT) and suffered from intracranial haemorrhage (ICH), with special focus on time of bleeding onset. DESIGN: Observational cohort study of all recorded cases of ICH caused by FNAIT from the international No IntraCranial Haemorrhage (NOICH) registry during the period 2001-2010. SETTING: 13 tertiary referral centres from nine countries across the world. PARTICIPANTS: 37 mothers and 43 children of FNAIT pregnancies complicated by fetal or neonatal ICH identified from the NOICH registry was included if FNAIT diagnosis and ICH was confirmed. PRIMARY AND SECONDARY OUTCOME MEASURES: Gestational age at onset of ICH, type of ICH and clinical outcome of ICH were the primary outcome measures. General maternal and neonatal characteristics of pregnancies complicated by fetal/neonatal ICH were secondary outcome measures. RESULTS: From a total of 592 FNAIT cases in the registry, 43 confirmed cases of ICH due to FNAIT were included in the study. The majority of bleedings (23/43, 54%) occurred before 28 gestational weeks and often affected the first born child (27/43, 63%). One-third (35%) of the children died within 4 days after delivery. 23 (53%) children survived with severe neurological disabilities and only 5 (12%) were alive and well at time of discharge. Antenatal treatment was not given in most (91%) cases of fetal/neonatal ICH. CONCLUSIONS: ICH caused by FNAIT often occurs during second trimester and the clinical outcome is poor. In order to prevent ICH caused by FNAIT, at-risk pregnancies must be identified and prevention and/or interventions should start early in the second trimester.

Significance of quantitative measurement of heparin-induced platelet antibodies.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune-mediated adverse drug reaction. Antigen and platelet activation assays are used for detection of antibodies. Quantitative results from platelet factor 4 (PF4)-dependent immunoassays may lead to inter-laboratory standardization of measurements. OBJECTIVES: The aim was to modify a PF4-dependent immunoassay to measure PF4/heparin antibodies quantitatively. METHODS: Over five consecutive years, 1070 samples from thrombocytopenic, heparin-treated patients were analyzed by a PF4/heparin ELISA and the heparin-induced platelet activation assay (HIPA). Results of ELISA assay were expressed as arbitrary units per liter (AU/L). RESULTS: Precision of ELISA at the concentration of 50 AU/L was 3.6%. Of 1070 samples, 117 were positive for antibodies by ELISA and/or HIPA assay. The higher the antibody concentration was, the higher was the proportion of HIPA positive cases (>140 AU/L, 100%, n = 26; 100-140 AU/L, 55%, n = 20; 50-99 AU/L, 38%, n = 29; 30-49 AU/L, 17%, n = 36). CONCLUSIONS: The measurement of anti-PF4/heparin antibody concentration is a new parameter that may improve the diagnosis of HIT. All samples with extremely strong antibody concentration were positive also by HIPA. For accuracy, antibody concentrations must be in the linear range of the assay and an international standard is needed.

Alloantibodies against low-frequency human platelet antigens do not account for a significant proportion of cases of fetomaternal alloimmune thrombocytopenia: evidence from 1054 cases.

BACKGROUND: Maternal alloantibodies against the five common human platelet antigen (HPA) systems (HPA-1 to -3, -5, and -15) are found in only 20% of cases referred for fetal and neonatal thrombocytopenia (FMAIT) investigations. The question asked was whether mismatches for the remaining 11 low-frequency HPAs (HPA-4 and -6bw to -17bw) might in part explain the remaining 80% of cases. STUDY DESIGN AND METHODS: A total of 1054 paternal DNA samples from referred FMAIT cases (among which 223 cases where antibodies against a common HPA were found) were genotyped for 11 low-frequency HPAs as well as a recently discovered polymorphism (ITGA2B-C2320T). The initial genotyping was carried out by TaqMan and potential heterozygotes were confirmed by DNA sequencing. Clinical and serologic data were collected for each case with a heterozygote father. RESULTS: In total, eight heterozygous fathers were identified: four for HPA-6w, one each for HPA-10w and -11w, and two for HPA-12w. Maternal antibodies against the corresponding antigen were identified in four of the eight cases. In two of these cases, antibodies against HPA-1a and HPA-1b were also found. CONCLUSION: It was concluded that the minor alleles of HPA-4 and -6bw to -17bw are exceptionally rare in the Caucasian population and therefore do not explain the large number of FMAIT referrals which test negative for the common HPA antibodies.

Soluble glycoprotein V as a quality marker of platelet concentrates stressed by transportation.

BACKGROUND: Despite ongoing improvements in storage conditions for platelet concentrates (PCs) for clinical use, leukoreduced platelets (PLTs) undergo subtle changes that are partly due to PLT activation. As PLTs are activated, the expression of P-selectin (CD62P) increases, and soluble glycoprotein V (sGPV) is released. GPV, part of the GPIbIXV complex, has been suggested as a marker of PLT activation. STUDY DESIGN AND METHODS: An array of assays, used for quality control of PCs, was performed and the results were compared. The tests included PLT count, swirling, mean PLT volume, extent of shape change (ESC), hypotonic shock response (HSR), CD62P, lysosomal membrane protein (CD63), sGPV, and the metabolic tests (pH, pO(2), pCO(2), lactate, glucose). The performance of the assays was evaluated during the storage period by comparing buffy coat-derived PCs (24 PCs of 4 units) stored on flatbed agitator or stressed twice by overnight transportation. RESULTS: The repeatability of all tests was good. ESC and HSR correlated with each other (r = 0.559). Importantly, there were also associations between sGPV and ESC (r = -0.564) and HSR (r = -0.389). The correlations of sGPV with lactate and glucose concentrations and with expression of CD62P and CD63 were also good. No significant changes were induced by two overnight transportations. CONCLUSION: sGPV might be applicable for statistical process control of the quality of PCs, in addition to metabolic tests. It may also be helpful in analyzing potential improvements in blood component processing. Repeat transportation of PCs may cause minimal changes on PLT in vitro properties, if any.