High-Throughput Screening of Blood Donors for Twelve Human Platelet Antigen Systems Using Next-Generation Sequencing Reveals Detection of Rare Polymorphisms and Two Novel Protein-Changing Variants.

BACKGROUND: Exposure to non-matching human platelet alloantigens (HPA) may result in alloimmunization. Antibodies to HPA can be responsible for post-transfusion purpura, refractoriness to donor platelets, and fetal and neonatal alloimmune thrombocytopenia. For the supply of compatible apheresis platelet concentrates, the HPA genotypes are determined in a routine manner. METHODS: Here, we describe a novel method for genotyping twelve different HPA systems simultaneously, including HPA-1 to HPA-5, HPA-9w, HPA-10w, HPA-16w, HPA-19w, HPA-27w, and the novel HPA-34w by means of amplicon-based next-generation sequencing (NGS). Blood donor samples of 757 individuals with a migration background and 547 of Western European ancestry were genotyped in a mass-screening setup. An in-house software was developed for fast and automatic analysis. TaqMan assay and Sanger sequencing results served for validation of the NGS workflow. Finally, blood donors were divided in several groups based on their country of origin and the allele frequencies were compared. RESULTS: For 1,299 of 1,304 samples (99.6%) NGS was successfully performed. The concordance with TaqMan assay and Sanger sequencing results was 99.8%. Allele-calling dropouts that were observed for two samples with the TaqMan assay caused by rare single nucleotide polymorphisms were resolved by NGS. Additionally, twenty rare and two novel variants in the coding regions of the genes ITGB3, GPB1A, ITGBA2, and CD109 were detected. The determined allele frequencies were similar to those published in the gnomAD database. CONCLUSIONS: No significant differences were observed in the distribution of allele frequencies of HPA-1 through HPA-5 and HPA-15 throughout the analyzed groups except for a lower allele frequency for the HPA-1b allele in the group of donors with Southern Asian ancestry. In contrast, other nucleotide variants that have not yet been phenotypically characterized occurred three times more often in blood donors with a migration background. High-throughput amplicon-based NGS is a reliable method for screening HPA genotypes in a large sample cohort simultaneously. It is easily upgradeable for genotyping additional targets without changing the setup or the analysis pipeline. Mass-screening methods will help building up blood donor registries to provide matched blood products.

Determination of nitrotyrosine concentrations in plasma samples of diabetes mellitus patients by four different immunoassays leads to contradictive results and disqualifies the majority of the tests.

BACKGROUND: In the course of type 2 diabetes mellitus, insulin resistance has a severe impact on endothelial function leading to decreased synthesis of nitric oxide (NO). Postprandial hyperglycemia leads to the generation of reactive oxygen species, which counteracts the beneficial NO effects. NO and superoxide combine very fast in solution to form peroxynitrite, which is a potent protein-oxidizing agent. The peroxynitrite concentrations can be indirectly monitored by the detection of nitrotyrosine residues in proteins, reflecting the extent of damage caused by oxidative stress. METHODS: Four commercially available nitrotyrosine-specific immunoassays were evaluated by parallel measurement of nitrotyrosine in 224 serum samples derived from 16 patients with type 2 diabetes and 12 healthy controls (13 male and 15 female, age: 33+/-11 years) following a standardized meal. RESULTS: The available ELISA tests were not applicable for nitrotyrosine determination in human plasma samples due to technical issues and implausible results. However, a competitive luminescence assay was able to provide sufficient sensitivity and lead to clinically meaningful results in our test samples. CONCLUSIONS: All three ELISA methods were disqualified and conclusions previously derived from clinical experiments using these tests should be carefully reconsidered or reconfirmed. In the absence of a liquid tandem chromatography-mass spectrometry reference method, the luminescence test appears to be the method of choice for determination of nitrotyrosine in human plasma.

Intuitiveness, instruction time, and patient acceptance of a prefilled insulin delivery device and a reusable insulin delivery device in a randomized, open-label, crossover handling study in patients with type 2 diabetes.

BACKGROUND: Because the use of insulin therapy can place a substantial burden on patients with diabetes, insulin administration should be as simple as possible. OBJECTIVES: The primary aim of this trial was to compare the use of 2 insulin delivery devices-one prefilled (NovoMix 30 FlexPen [FP]; Novo Nordisk, Copenhagen, Denmark) and the other reusable (HumaPen Luxura [HL]; Eli Lilly and Company, Indianapolis, Indiana)-in patients with type 2 diabetes in terms of intuitiveness and training time. A secondary aim was to evaluate the ease of use and overall acceptance of the 2 devices. METHODS: This was a randomized, open-label, comparative, crossover handling study in adult patients with type 2 diabetes who had been treated with oral antidiabetic drugs for >or=2 years and had no previous experience with insulin injection devices. Patients were randomly allocated to the intuitiveness group (no instruction in the use of the devices provided) or the instruction group (instruction provided). The time taken to deliver an injection into a cushion was measured for each device in both groups. Patients answered questionnaires concerning the intuitiveness and ease of use of the 2 devices, their trust and confidence in the devices to deliver the insulin dose, and their overall pen preference. RESULTS: Sixty-one patients were enrolled in the study (70.5% male; mean [SD] age, 61.80 [7.60] years), 30 in the intuitiveness group and 31 in the instruction group. When all handling steps for the HL device were included, the mean (SD) injection time was significantly shorter for the FP device compared with the HL device in the intuitiveness group (1.21 [1.04] vs 1.74 [0.79] minutes, respectively; P = 0.035). The outcome was similar in the instruction group (0.71 [0.29] vs 1.09 [0.49] minutes; P < 0.001). When the time for cartridge insertion in the HL device was excluded, there was no significant difference in injection time for the respective devices in either group (intuitiveness group: 1.21 [1.04] and 1.07 [0.91] minutes; instruction group: 0.63 [0.35] and 0.71 [0.29] minutes). Twenty-two patients preferred the FP device in terms of ease of learning, compared with 8 patients preferring the HL device (P = 0.007). CONCLUSIONS: In this study, when all handling steps were included, the FP device was associated with significantly greater intuitiveness and a shorter injection time compared with the HL device. Further research is needed to determine whether these differences between devices are clinically meaningful.