Severe intracranial haemorrhage in neonatal alloimmune thrombocytopenia due to antibodies against human platelet antigen 1b: Case report and literature review.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a rare life-threatening disorder, leading to severe thrombocytopenia and potentially bleeding, with intracranial haemorrhage (ICH) being the most serious complication. We report on a FNAIT case with fourth-degree ICH that arose due to antibodies against human platelet antigen (HPA)-1b. The male infant, born to an otherwise healthy mother, presented with severe signs of ICH soon after delivery. Since only moderate thrombocytopenia was noted and there were no active signs of bleeding, the infant did not receive intravenous immunoglobulins (IVIg) or platelet transfusion. Spontaneous recovery of platelets was noted on the eighth day of life, but permanent neurological impairment remained as a consequence of ICH. We report the results of HPA and human leukocyte antigen (HLA) antibodies in the mother's and the infant's sera, the family's HPA genotype and the mother's HLA genotype, and summarise previously described cases of FNAIT due to anti-HPA-1b antibodies in the literature. FNAIT with severe ICH due to anti-HPA-1b antibodies is rarely diagnosed. An association between HLA genes and sensitization to HPA-1b antibodies was not demonstrated. The severity of FNAIT and the occurrence of ICH is often difficult to predict. In this case, the infant presented with moderate thrombocytopenia and ICH, with subsequent permanent consequences.

Evaluation of the BD Stem Cell Enumeration Kit on the BD FACSCanto II flow cytometer using bd facscanto clinical and bd facsdiva software.

INTRODUCTION: CD34+ hematopoietic stem cell (HSC) enumeration by cell flow cytometry is routinely used in clinical laboratories for monitoring of HSC mobilization into peripheral blood and assessment of the quality of HSC products. The modified ISHAGE protocol is the most often used procedure for determination of CD34+ cells using flow cytometry. The aim of this study was to evaluate BD Enumeration stem cell kit on flow cytometer BD facscanto II, using facscanto clinical and facsdiva softwares. METHODS: Validation study included determination of within-run and between-run precision, trueness (bias), comparison of the test results analyzed on facscanto clinical and facsdiva softwares, assessment of linearity, specimen stability, and carryover. RESULTS: For between-run precision, coefficients of variation (CVs) were all <10%, except for low control level on facsdiva software. CVs for within-run precision were <10%, except for high absolute count of CD34+ cells on facsdiva software. Comparison of data showed no statistically significant differences between facscanto clinical and facsdiva software (Spearman's rank correlation coefficients were .993 for % of CD34+ cells and 0.983 for absolute count of CD34+ cells). In linearity study, bias for all dilutions was < 20%, and carryover assessment cannot be considered significant on both softwares. There was a statistically significant difference (P = .044) in absolute count of CD34+ cells after 24 hours of storage, when using facscanto clinical software. CONCLUSION: BD Stem Cell Enumeration Kit can be used in routine laboratory work on BD FACSCanto II instrument, whereas facscanto clinical and facsdiva software were used for acquisition and data analysis.

Influence of flavonoids' lipophilicity on platelet aggregation.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbß3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

Susceptible and protective HLA-DR and HLA-DQ alleles for Fya alloimmunization in the Croatian population.

BACKGROUND: Alloimmunization is a known risk of transfusion therapy caused by exposure to foreign RBC antigens. However, alloimmunization is not observed in all transfused patients. Human leukocyte antigen (HLA) molecules may contribute to the recognition and presentation of foreign antigens and to the potency of immune responses that result in the production of antibodies. The aim of this study was to determine the association of HLA-DR and HLA-DQ polymorphisms with alloimunization to Fya antigen in Croatian patients. STUDY DESIGN AND METHODS: The study was conducted on 70 alloimmunized patients to Fya antigen and two control groups: 165 healthy Croatian individuals (Control 1) and 45 Fya antigen-negative nonimmunized patients exposed to Fya antigen (Control 2). Phenotype frequencies for HLA-DRB1 and HLA-DQB1 alleles were compared between the cases and control groups. RESULTS: Statistically significant differences in phenotype frequencies between cases and controls were found for DRB1*04 (odds ratios [ORs], 10.5 and 18.7 for Control 1 and Control 2, respectively), DRB1*15 (ORs, 8.0 and 6.9), and DQB1*02 alleles (ORs, 0.2 and 0.03); and DRB1*04-DQB1*03:01 (ORs, 7.9 and 17.6), DRB1*04-DQB1*03:02 (ORs, 5.5 and 7.6), DRB1*15-DQB1*06:02 (ORs, 7.3 and 5.5), DRB1*03-DQB1*02:01 (OR, 0.1), and DRB1*07-DQB1*02:02 (OR, 0.3) haplotypes. CONCLUSION: Several HLA-DRB1 and HLA-DQB1 alleles and haplotypes were proved to contribute to and protect from alloimmunization to Fya antigens. Alleles DRB1*04 and DRB1*15, as well as haplotypes DRB1*04-DQB1*03:02 and DRB1*15-DQB1*06:02 can be considered as risk factors, while allele DQB1*02 and haplotype DRB1*03-DQB1*02:01 have a protective role in Fya alloimmunization.

[Croatian Society for Haematology and Transfusion Medicine Guidelines on the diagnosis and management of heparin induced thrombocytopenia (HIT)]. / Smjernice Hrvatskog drustva za hematologiju i transfuzijsku medicinu u dijagnosticko-terapijskom postupku za trombocitopeniju izazvanu heparinom (HIT).

Heparin induced thrombocytopenia (HIT) is a serious complication of heparin administration. In the last decade, this clinical syndrome has come into the focus of interest, primarily because of the severe thromboembolic complications that may lead to lethal outcome. In addition, great improvements have been made in the treatment with direct thrombin inhibitors and in laboratory diagnosis of HIT. As guidelines for diagnostic and management of HIT upgrade the quality of patient treatment, activities for their development have been launched in the Republic of Croatia. Based on British Committee for Standards in Haematology (BCSH) recommendations on diagnostic and treatment of HIT from 2006, activities for the introduction of new assays for anti-heparin antibodies were launched in 2008 and 2009, including algorithm of laboratory testing for HIT, sheet for clinical assessment of HIT (4T score), and education oftransfusiologists and clinicians. Upon evaluation of the results collected during one-year period, the Croatian Society of Haematology and Transfusion Medicine nominated a task force for the development of guidelines for HIT in January 2010. Following wide-ranging discussion, the guidelines were adopted in May 2011.

[Neonatal alloimmune thrombocytopenia in Croatia in 1997-2007]. / Neonatalna aloimuna trombocitopenija u Hrvatskoj od 1997. do 2007. godine.

Neonatal alloimmune thrombocytopenia (NATP) is caused by maternal sensitization to paternal alloantigens on fetal platelets during pregnancy. Although the disease is rare, the severity of clinical picture and its sequels associated with central nervous system hemorrhage impose the need of an early diagnosis, and timely and specific treatment of the disease. Based on these and literature data on the prevalence of NATP in Caucasians of 1-2 cases per 1000-5000 live births, it is estimated that 10 to 50 serologically verified cases of NATP and approximately a twofold number of requests for serologic testing for suspected NATP could be expected in Croatia per year. In the present study, results of serology testing and clinical laboratory data of twenty five cases of NATP in Croatia during the 1997-2007 period are evaluated. In 20/25 cases platelet antibody screening was positive. Specific platelet antibodies were confirmed in 14/20 (70%) cases with positive screening (anti-HPA-la in 7/14, anti-HPA-5b in 5/14, and anti GP Ib-IX in 2/14 cases). Only fourteen serologically confirmed cases of NATP per -45,000 live births per year in Croatia indicate the prevalence of the disease to be considerably lower than expected.