BACKGROUND: RHD blood group alleles with reduced or absent antigen expression are a clinically significant and heterogeneous group.
Study design and methods: To detail population genetics data on ...apparently D– individuals in central Europe, a six‐center study was performed with participants from Austria, Germany, Slovenia, Switzerland, and Russia. A total of 1700 serologically D– samples, positive for C and/or E, were investigated.
RESULTS: Observed unexpressed RHD alleles were 59 RHD‐CE‐D+ hybrid alleles, 9 apparently regular RHD, 1 new RHD(Y401X); DELs were 8 RHD(M295I), 6 RHD(IVS3+1G>A), and 1 new RHD(X418L); and weakly expressed RHDs were 2 weak D type 5, 1 weak D type 1, 1 RHD category VI type 1, and 1 novel weak D type 26. Although weak D type 26 was shown to have one of the lowest D antigen densities ever observed, it gave rise to anti‐D immunization in a transfused D– individual.
CONCLUSION: The relative occurrence of RHD among serologically D– samples, positive for C and/or E, differed significantly in the investigated central European regions. Considering the growing use of molecular typing techniques, correct identification of blood group alleles with scarce or missing antigen expression is of utmost clinical importance and requires reliable population‐based frequency data.
BACKGROUND: One branch of the RHD phylogenetic tree is represented by the weak D type 4 cluster of alleles with F223V as the primordial amino acid substitution. F223V as well as a large number of ...further substitutions causing D variants are located at the extracellular RhD protein vestibule, which represents the entrance to the transmembraneous channel of the RhD protein.
STUDY DESIGN AND METHODS: RHD and RHCE nucleotide sequences were determined from genomic DNA and cDNA. D epitope patterns were established with commercial monoclonal anti‐D panels.
RESULTS: The RHD alleles DOL‐1 and DOL‐2 had the two amino acid substitutions M170T (509T>C) and F223V (667T>G) in common. DOL‐2 harbored the additional substitution L378V (1132C>G). Both alleles were observed in Africans and are probably evolutionary related. DMI carried M170I (510G>A), which differed from the DOL‐typical substitution. DFW and DFL harbored the substitutions H166P (497A>C) and Y165C (494A>G). The antigen densities of DOL‐1, DFL, and DFW were only moderately reduced.
CONCLUSION: DOL‐1 and DOL‐2 belong to the weak D type 4 cluster of RHD alleles. Together with DMI, DFL, and DFW they represent D variants with amino acid substitutions located at extracellular loops 3 or 4 lining the RhD protein vestibule. These substitutions were of minor influence on antigen density while adjacent substitutions in the transmembraneous section caused weak D antigen expression. All these D variants were partial D and alloanti‐D immunizations have been observed in DOL‐1, DMI, and DFL carriers. The substitution at position 170 causes partial D although located deep in the vestibule.
BACKGROUND: Noninvasive pH measurement of platelet concentrates (PCs) was evaluated as a tool for the quality control of PC storage by simulating worst‐case conditions.
STUDY DESIGN AND METHODS: PCs ...from pooling four buffy coats in 70% PAS‐3M were both stored in bags wrapped to impair gas permeability and agitated or not until Day 9 of storage. pH values measured both in samples (electrode, blood gas analyzer) and noninvasively by fluorimetry (BCSI pH1000, Blood Cell Storage, Inc.) were compared groupwise and to changes in platelet (PLT) size and biochemical variables.
RESULTS: The noninvasive pH measurements agreed well with the results from each of the two reference methods (R2 > 0.9) in a wide range of pH values between 6.4 and 7.5. Changes of the pH of PCs (n = 64) by all interventions (agitation or resting plus occlusion by 0, 25, 50, or 100%; n = 8/group) were subtle but already significant after 20 to 24 hours of treatment in comparison to the controls. A steady state after Day 6 and reductions up to a mean pH of approximately 6.5 were observed. The extent of manipulation determined both the absolute pH differences to the controls and the interindividual variation of pH changes. Termination of the agitation significantly enhanced pH reduction by surface blockade. Significant changes were also observed for the mean PLT volume, β‐thromboglobulin, and soluble P‐selectin.
CONCLUSION: Noninvasive pH measurement in PCs using this technique reliably detects pH changes of 0.1 or more. Storage of PLTs in buffered additive solution requires profound impairment of gas exchange to trigger a substantial decline in pH.
BACKGROUNDUVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in ...platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology.STUDY DESIGN AND METHODSSamples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out‐of‐specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross‐validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed.RESULTSApheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single‐nucleotide polymorphisms in the primer‐ and probe‐binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra‐ and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment.CONCLUSIONThe ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC‐treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.
Background and Objectives
Corrected count increment (CCI) measurements monitor the effectiveness of platelet transfusions in haemato‐oncology, but they usually fail in patients undergoing cardiac ...surgery. We investigated whether polymerase chain reaction (PCR) of mitochondrial single‐nucleotide polymorphisms (SNPs) is able to monitor the survival of transfused platelets in these patients.
Materials and Methods
Leukocyte‐free, platelet‐rich plasma was prepared from patients' blood to measure platelet counts based on patient‐/donor‐specific SNPs by digital PCR after DNA extraction. Platelet counts in samples from patients with severe thrombocytopenia were analysed by both PCR and flow cytometry. Ten patients undergoing cardiac surgery with the use of heart lung machine and without overt bleeding received a single apheresis platelet concentrate because of either dual platelet inhibition during a non‐elective intervention or a complex procedure. Blood samples were collected at nine defined intervals (0–120 h) post transfusion.
Results
The digital PCR of the seven SNPs reliably quantified levels ≥0.6 G/L platelets, in good agreement with flow cytometry and without interference by other SNPs or by platelet activation. A mean 24‐h CCI of 11.8 (range: 5.6–19.8) and a mean 120‐h area under the curve (AUC) of 1386 (915–1821) hxG/L were observed for the transfused platelets. The mean AUC of 14,103 (3415–27,305) hxG/L for the patients' endogenous platelets indicates that transfused platelets represented only 11% (5–25) of the total platelet counts during 120 h post transfusion.
Conclusion
PCR of mitochondrial SNPs offers a tool to assess the survival of platelets from apheresis concentrates in cardiac surgery patients to facilitate the implementation of improved transfusion strategies.
Background
Neonatal alloimmune thrombocytopenia is a rare but potentially severe postnatal complication caused by maternal allo‐antibodies against platelet antigens of the newborn. In relatively few ...cases, immunisation against low‐frequency antigens has been reported.
Methods
Platelet antigens of a newborn with severe thrombocytopenia and his family members were investigated by serological and molecular biological methods. A real‐time PCR assay was developed to reliably detect this mutation in pools of DNA from up to seven individuals.
Results
Serological testing showed positive reactions of maternal plasma with paternal platelets but not with conventional platelet donor panels. Sequencing of the ITGB3 gene revealed a G > A polymorphism in position c.1915 of exon 12 for the father, the newborn and three of four paternal relatives. Screening of samples from a local population of 1575 Caucasian blood donors identified only a single individual with this mutation.
Conclusion
This finding of a previously unreported private platelet antigen demonstrates that the identification of the target glycoprotein by MAIPA assay followed by sequencing of the affected gene can be combined with an efficient population screening by real‐time PCR with pooling of DNA samples.
BACKGROUND
UVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) ...in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology.
STUDY DESIGN AND METHODS
Samples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out‐of‐specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross‐validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed.
RESULTS
Apheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single‐nucleotide polymorphisms in the primer‐ and probe‐binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra‐ and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment.
CONCLUSION
The ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC‐treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.
Summary
Determining blood group antigens by serological methods may be unreliable in certain situations, such as in patients after chronic or massive transfusion. Red cell genotyping offers a ...complementary approach, but current methods may take much longer than conventional serological typing, limiting their utility in urgent situations. To narrow this gap, we devised a rapid method using direct polymerase chain reaction (PCR) amplification while avoiding the DNA extraction step. DNA was amplified by PCR directly from plasma or serum of blood donors followed by a melting curve analysis in a capillary rapid‐cycle PCR assay. We evaluated the single nucleotide polymorphisms underlying the clinically relevant Fya, Fyb, Jka and Jkb antigens, with our analysis being completed within 40 min of receiving a plasma or serum sample. The positive predictive value was 100% and the negative predictive value at least 84%. Direct PCR with melting point analysis allowed faster red cell genotyping to predict blood group antigens than any previous molecular method. Our assay may be used as a screening tool with subsequent confirmatory testing, within the limitations of the false‐negative rate. With fast turnaround times, the rapid‐cycle PCR assay may eventually be developed and applied to red cell genotyping in the hospital setting.
PDF