The use of next-generation sequencing for the determination of rare blood group genotypes.

OBJECTIVES: Next-generation sequencing (NGS) for the determination of rare blood group genotypes was tested in 72 individuals from different ethnicities. BACKGROUND: Traditional serological-based antigen detection methods, as well as genotyping based on specific single nucleotide polymorphisms (SNPs) or single nucleotide variants (SNVs), are limited to detecting only a limited number of known antigens or alleles. NGS methods do not have this limitation. METHODS: NGS using Ion torrent Personal Genome Machine (PGM) was performed with a customised Ampliseq panel targeting 15 different blood group systems on 72 blood donors of various ethnicities (Caucasian, Hispanic, Asian, Middle Eastern and Black). RESULTS: Blood group genotypes for 70 of 72 samples could be obtained for 15 blood group systems in one step using the NGS assay and, for common SNPs, are consistent with previously determined genotypes using commercial SNP assays. However, particularly for the Kidd, Duffy and Lutheran blood group systems, several SNVs were detected by the NGS assay that revealed additional coding information compared to other methods. Furthermore, the NGS assay allowed for the detection of genotypes related to VEL, Knops, Gerbich, Globoside, P1PK and Landsteiner-Wiener blood group systems. CONCLUSIONS: The NGS assay enables a comprehensive genotype analysis of many blood group systems and is capable of detecting common and rare alleles, including alleles not currently detected by commercial assays.

Measurement of platelet aggregation, independently of patient platelet count: a flow-cytometric approach.

Essentials Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. A flow cytometric platelet aggregation test independent of the patient platelet count was made. Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer. High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. SUMMARY: Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count, and examine the association of aggregation with a bleeding history in thrombocytopenic patients. Patients/methods We established a flow-cytometric assay of platelet aggregation, and measured samples from healthy individuals preincubated with antiplatelet drugs, and samples from two patients with inherited platelet disorders. Then, we included 19 healthy individuals and 20 patients with platelet counts of ≤ 50 × 109 L-1 , diagnosed with acute myeloid leukemia or myelodysplastic syndrome. We measured platelet aggregation and platelet activation by platelet surface expression of activated glycoprotein IIb-IIIa, P-selectin and CD63 after addition of agonists: collagen-related peptide, thrombin receptor-activating peptide (TRAP), and ADP. Results The platelet aggregation assay showed a low intraserial coefficient of variation of ≤ 3%. Similar results were obtained for platelet-rich plasma and isolated platelets at platelet counts of > 10 × 109 L-1 ; otherwise, platelet isolation was required. The platelet aggregation percentage decreased with increasing antiplatelet drug concentration. Platelet aggregation in patients was reduced as compared with healthy individuals: 42% (interquartile range [IQR] 27-58) versus 66% (IQR 60-67) for TRAP; 41% (IQR 25-48) versus 70% (IQR 69-72) for collagen-related peptide; and 44% (IQR 30-53) versus 65% (IQR 46-72) for ADP. Platelet activation after stimulation was reduced in patients and correlated with platelet aggregation (e.g. r = 0.78-0.81 when stimulated with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.

Routine noninvasive prenatal screening for fetal RHD in plasma of RhD-negative pregnant women-2 years of screening experience from Denmark.

OBJECTIVE: Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhD-negative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening. METHODS: Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies. RESULTS: The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7-99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%. CONCLUSION: The high sensitivity, maintained over 2 years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25 weeks of gestation. The remaining challenges are logistical and are related to program compliance.

Novel assays to assess the functional capacity of the classical, the alternative and the lectin pathways of the complement system.

Deficiencies in many of the complement proteins and their regulatory molecules have been described and a variety of diseases, such as recurrent infections, systemic lupus erythematosus (SLE) and renal diseases, may be linked to deficiency in the complement system. Screening for complement defects is therefore of great importance. In this study, we present novel improved enzyme-linked immunosorbent assays for the functional assessment of the three individual pathways of the complement system. The method is applicable at high serum concentrations and we demonstrate that it minimizes both false negative as well as false positive results. In particular, for the functional mannose-binding lectin activity it represents an improvement on the existing assays. In this respect, the present assays represent novel improved diagnostic protocols for patients with suspected immunodeficiencies related to the complement system.

Monitoring and treatment of anti-D in pregnancy.

Prophylactic anti-D is a very safe and effective therapy for the suppression of anti-D immunization and thus prevention of haemolytic disease of the foetus and newborn. However, migration from countries with low health standards and substantial cuts in public health expenses have increased the incidence of anti-D immunization in many "developed" countries. Therefore, this forum focuses on prenatal monitoring standards and treatment strategies in pregnancies with anti-D alloimmunization. The following questions were addressed, and a response was obtained from 12 centres, mainly from Europe.

One-year period prevalence of blood transfusion.

Transfusion practice is reported to differ considerably between countries. Comparisons often rely on transfusion rates, incidence - or prevalence rates. In this paper, the one-year period prevalence rate (1-YPPR) of transfusion of red cells (RBC) is presented. Transfusion data, demographic data and patient data were retrospectively combined to calculate sex and diagnosis specific and age standardized 1-YPPR s of RBC transfusion for the complete population in a Danish county. During the calendar year of 2006, 4427 patients received RBC transfusion in Funen County. The crude 1-YPPR of RBC transfusion was 9.2/1000 citizens. Most of the transfused patients had a main diagnosis of neoplasm (22% of recipients), diseases of the circulatory system (15%), the digestive system (15%), injuries (13%) and diseases of the blood (8%). Age standardization reversed the relation between sex specific 1-YPPRs, yielding a statistically significantly higher 1-YPPR in males (6.8/1000) vs. females (6.3/1000). The method for calculating a diagnosis specific 1-YPPR of transfusion of RBC is presented and proposed as an option for comparison of transfusion practice with other transfusion centres. The crude and the diagnosis specific 1-YPPR s are found to increase with age. Thus, age standardization of crude prevalence rates is necessary for meaningful comparisons between populations.