Digital polymerase chain reaction to monitor platelet transfusions in cardiac surgery patients.

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.

Intensity of endogenous thrombocytopenia after autologous stem cell transplantation in patients prophylactically transfused with platelets.

BACKGROUND AND OBJECTIVES: Large clinical trials have demonstrated that some patient groups with hypoproliferative thrombocytopenia benefit from prophylactic platelet transfusions, while in others, a therapeutic transfusion regimen might be sufficient. The remaining capacity to generate endogenous platelets might be helpful to select the platelet transfusion regimen. We assessed whether the recently described method of digital droplet polymerase chain reaction (PCR) can be used to assess the endogenous platelet levels in two groups of patients undergoing high-dose chemotherapy with autologous stem cell transplantation (ASCT). MATERIALS AND METHODS: Multiple myeloma (n = 22) patients received high-dose melphalan alone (HDMA); lymphoma patients (n = 15) received BEAM or TEAM (B/TEAM) conditioning. Patients with a total platelet count <10 G/L received prophylactic apheresis platelet concentrates. Daily endogenous platelet counts were measured by digital droplet PCR for at least 10 days post-ASCT. RESULTS: Post-transplantation B/TEAM patients received their first platelet transfusion on average 3 days earlier than HDMA patients (p < 0.001) and required about twofold more platelet concentrates (p < 0.001). The endogenous platelet count fell ≤5 G/L for a median of 115 h (91-159; 95% confidence interval) in B/TEAM-treated patients compared to 12.6 h (0-24) (p < 0.0001) in HDMA-treated patients. Multivariate analysis confirmed this profound effect of the high-dose regimen (p < 0.001). The CD-34+ -cell dose in the graft was inversely correlated with the intensity of endogenous thrombocytopenia in B/TEAM-treated patients. CONCLUSION: Monitoring endogenous platelet counts detects the direct effects of myelosuppressive chemotherapies on platelet regeneration. This approach may help to develop a platelet transfusion regimen tailored to specific patient groups.

A nait-associated and previously unreported mutation in the ITGB3 gene with a low frequency in the local population.

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.

Digital droplet polymerase chain reaction to monitor ultraviolet C treatment of single-donor and buffy coat platelet units.

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.

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting.

BACKGROUND: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. METHODS: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. RESULTS: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. CONCLUSIONS: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

Platelet recovery and survival measured in patients by quantitative polymerase chain reaction of mitochondrial DNA.

BACKGROUND: Mitochondrial (mt) DNA markers have been identified as potential targets for the quantification of endogenous and allogeneic platelets (PLTs) in the blood of individuals who received transfusions. Our goal was to develop a routine polymerase chain reaction (PCR) assay for ex vivo monitoring of PLT survival in patients after transfusion. STUDY DESIGN AND METHODS: Targets were selected for real-time (RT)-PCR of mt DNA based on the frequency distribution of nucleotide polymorphisms and assay sensitivity in vitro. The assays were then evaluated with ex vivo samples to measure PLT survival and recovery of therapeutic doses of apheresis PLTs in hematooncologic patients with thrombocytopenia. RESULTS: Nucleotides in two positions (73/310 hypervariable region [HVR] 2) and three positions (295 HVR 2, 16069/16311 HVR 1) had allele frequencies of approximately 0.5 and 0.85, respectively, in a population of 960 Caucasian PLT donors. They provided targets for sensitive assays detecting at least 1 × 10(3) PLTs per whole blood sample with adequate reproducibility (interassay coefficient of variation <4.0%). Transfusions of single-donor PLT concentrates in patients with thrombocytopenia (n = 30) were monitored with these markers. The mean 24-hour corrected count increment was 8.3 and the mean calculated survival time was 3.3 days. Results for a second marker were available for 13 transfusions. The survival time values derived from both markers for the same transfusion were almost identical (linear regression: r(2) = 0.957, slope = 0.87). CONCLUSION: This RT-PCR method detects mt DNA polymorphisms in Caucasians for a highly sensitive and reproducible quantification of endogenous and allogeneic PLT numbers in blood samples from transfused patients with thrombocytopenia.

The Glass Slide Extraction System Snap Card Improves Non-Invasive Prenatal Genotyping in Pregnancies with Antibodies.

BACKGROUND: Determination of fetal blood groups in maternal plasma samples critically depends on adequate pre-analytical steps for optimal amplification of fetal DNA. We compared the extraction of cell-free DNA by binding on a glass surface (BCSI SNAP™ Card) with an automated system based on bead technology (MagnaPure compact™). METHODS: Maternal blood samples from 281 pregnancies (7th-39th week of gestation) with known antibodies were evaluated in this study. Both the SNAP card and the MagnaPure method were applied to isolate DNA in order to directly compare the amplification in a single base extension assay and/or real-time PCR. RESULTS: The mean concentration of total DNA obtained by the SNAP card (33.8 ng/µl) exceeded more than twofold that of MagnaPure extraction (15.7 ng/µl). SNAP card-extracted samples allowed to detect 3.7 single nucleotide polymorphisms (SNPs) versus 2.5 SNPs in MagnaPure extracts to control for traces of fetal DNA. This difference is highest for samples from 7th-13th week of gestation. CONCLUSION: The SNAP card system improves DNA extraction efficacy for prenatal diagnosis in maternal blood samples and provides an at least eightfold higher total amount of DNA for the ensuing analysis. Its advantage is most evident for samples from early stages of pregnancy and thus especially valuable for pregnancies with antibodies.

Evaluation of single-nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups.

BACKGROUND: Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. STUDY DESIGN AND METHODS: DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma. RESULTS: The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. CONCLUSION: Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples.

Noninvasive pH Monitoring in Platelet Concentrates.

BACKGROUND: New technology introduces the option to measure pH in platelet concentrates noninvasively. Findings that in one out of 25 apheresis concentrates the pH decreased significantly during storage motivated us to evaluate apheresis concentrates from 307 platelet donors. METHODS: Small (15 ml) BSCI storage containers for noninvasive pH measurement were used in parallel with Fenwal PL2400 bags for storage of 5 days. Noninvasive pH measurement was compared to pH determination in samples from the storage container. Decrease of pH during storage was calculated as ΔpH d2-d5. RESULTS: The coefficient of correlation for noninvasive pH measurement (n = 256) versus standard methods was R(2) = 0.964 (pH electrode) or 0.952 (ABL 510). pH values in BSCI bags were slightly lower than in Fenwal PL2400 containers. Concentrates collected from 8 of the 307 donors showed a significant drop in pH. Repeated collection did not confirm these findings with a single exception. CONCLUSION: Noninvasive pH measurement demonstrates a high reproducibility. Our study could not confirm the frequency of 1 out of 25 plateletpheresis donors with concentrates developing a significant drop in pH during storage. This may be due to a substantially lower platelet concentration in our preparations.

Noninvasive pH measurement to monitor changes during suboptimal storage of platelet concentrates.

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 (R(2) >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, ß-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.

D variants at the RhD vestibule in the weak D type 4 and Eurasian D clusters.

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.

Sequence-Based Typing of Human Blood Groups.

In the last two decades, all but one of the genes encoding the 30 blood group systems present on red blood cells have been identified. This body of knowledge has permitted the application of molecular techniques to characterize the common blood group antigens and to elucidate the background for some of the variant phenotypes. DNA sequencing methodology was developed in the late 1970s and has become one of the most widely used techniques in molecular biology. In the field of immunohematology, this method is currently used by specialized laboratories to elucidate the molecular basis of unusual blood group phenotypes that cannot be defined by serology and genotyping. Because of the heterogeneity of the blood groups on both the antigen and the genetic level, special knowledge of the biology of blood group systems is needed to design sequencing strategies and interpret sequence data. This review summarizes the technical and immunohematologic expertise that is required when applying sequence-based typing for characterization of human blood groups.

Platelet engraftment after allogenic stem cell transplantation is monitored by digital polymerase chain reaction without interference by platelet support.

Platelet engraftment after allogeneic hematopoietic stem cell transplantation is conventionally monitored by daily platelet counts. Platelet transfusions are frequently required and obscure the detection of platelet engraftment. Digital polymerase chain reaction (ddPCR) of mitochondrial DNA isolated from platelets reliably quantifies circulating platelets derived from the stem cell graft and allows us to distinguish them from transfused single-donor apheresis platelets. In a feasibility study, consecutive daily peripheral blood samples from day 7 to day 20 after transplantation were analyzed by ddPCR in 22 patients after allogeneic transplantation. Platelet engraftment according to ddPCR was defined as the third of at least 3 consecutive days of increasing levels exceeding 1000/µL endogenous platelets. Platelet counts were also assessed according to the engraftment cri`teria of the Center for International Blood & Marrow Transplant Research (CIBMTR) and the European Society for Blood and Marrow Transplantation (EBMT). Out of the 22 patients, five did not achieve platelet engraftment within 20 days by any of the predefined criteria. A subgroup of nine patients did show platelet engraftment by all three definitions. In five patients, engraftment was detectable according to ddPCR and EBMT, whereas in three patients, platelet engraftment within 20 days was only confirmed by ddPCR. The detailed findings suggest that the day of platelet engraftment according to the EBMT criteria closely reflected the ddPCR detection of transplantation-derived platelets. The results from this feasibility study demonstrate that ddPCR offers a sensitive approach to detect platelet engraftment reliably and without interference from the individual noise of platelet counts due to platelet transfusions.

Noninvasive prenatal blood group genotyping.

Determination of fetal RHD from maternal plasma is increasingly used as a valuable tool for prenatal diagnosis. A remaining pitfall which hampers its use in situations with severe consequences is the following: (a) The reliability of negative results, however, is limited by difficulties to distinguish true negative results from false negative results due to insufficient amounts of free fetal DNA (ffDNA). False negative results can result in severe complications for the fetus and have to be reliably excluded. Large studies were performed in the last 10 years to investigate the reliability of noninvasive fetal RHD typing with real-time PCR. The majority of the assays were performed without internal controls. We present a protocol for inclusion of standards to assess the presence of adequate amounts of ffDNA for prenatal genotyping in maternal blood.

Clinical effects of phosphodiesterase 3A mutations in inherited hypertension with brachydactyly.

Autosomal-dominant hypertension with brachydactyly is a salt-independent Mendelian syndrome caused by activating mutations in the gene encoding phosphodiesterase 3A. These mutations increase the protein kinase A-mediated phosphorylation of phosphodiesterase 3A resulting in enhanced cAMP-hydrolytic affinity and accelerated cell proliferation. The phosphorylated vasodilator-stimulated phosphoprotein is diminished, and parathyroid hormone-related peptide is dysregulated, potentially accounting for all phenotypic features. Untreated patients die prematurely of stroke; however, hypertension-induced target-organ damage is otherwise hardly apparent. We conducted clinical studies of vascular function, cardiac functional imaging, platelet function in affected and nonaffected persons, and cell-based assays. Large-vessel and cardiac functions indeed seem to be preserved. The platelet studies showed normal platelet function. Cell-based studies demonstrated that available phosphodiesterase 3A inhibitors suppress the mutant isoforms. However, increasing cGMP to indirectly inhibit the enzyme seemed to have particular use. Our results shed more light on phosphodiesterase 3A activation and could be relevant to the treatment of severe hypertension in the general population.

Cost-efficient sequence-specific priming-polymerase chain reaction screening for blood donors with rare phenotypes.

BACKGROUND: Transfusion support for patients with irregular antibodies to red blood cell (RBC) antigens of high frequency may be hampered by lack of appropriate antigen-negative RBC units. Often, this perceived lack is due to the low number of typed donors. We developed a simple multiplex polymerase chain reaction (PCR) method to screen for donors with rare blood group phenotypes. STUDY DESIGN AND METHODS: A multiplex PCR with sequence-specific priming predicting Yt(a), Co(a), Lu(b), and Kp(b) antigens was developed based on a commercially available system (Extract-N-Amp, Sigma-Aldrich) that obviates the DNA purification step. PCR amplicons were analyzed by size fractionation in a 2 percent agarose gel. Samples representing rare phenotypes were identified by the lack of one of the four visible bands. Donors of blood phenotype O D- ccddee were screened. RESULTS: Excluding the preparation of the reaction mixture and the gel, the whole procedure consisted of five pipetting steps. Hands-on time was 102 minutes for 91 donors. After optimization, interpretable results were obtained in 85 percent of samples without repetition. Among 3422 donors tested, 1 Kp(b-), 6 Co(a-), 10 Yt(a-), and 5 Lu(b-) donors were detected. CONCLUSIONS: Multiplex PCR is a simple, versatile, and cost-efficient method for the screening for donors with rare phenotypes who may be identified by their genotype. If such donor screening is introduced on a broad basis, transfusion support for patients with anti-Co(a), anti-Yt(a), or anti-Lu(b) will be considerably improved.

DCS-1, DCS-2, and DFV share amino acid substitutions at the extracellular RhD protein vestibule.

BACKGROUND: RhD and RhCE are structurally related to ammonium transporter proteins, yet their physiologic function remains unclear. Recent three-dimensional homology modeling with Escherichia coli AmtB as a template defined a putative transmembraneous channel. Three RhD variants with amino acid substitutions located at the extracellular channel aperture are described. STUDY DESIGN AND METHODS: Blood samples were selected because of serologic abnormalities. RHD, RHCE, and LW nucleotide sequences were determined from genomic DNA. D epitope patterns were established with monoclonal anti-D panels. Three-dimensional Rh structures were calculated by alignment to AmtB. RESULTS: The RHD allele DCS-1 was found to carry the two amino acid substitutions F223V (667T > G) and A226P (676G > C) caused by missense mutations in RHD exon 5. This study compared DCS-1 with the D variants DFV (F223V) and DCS-2 (A226P), harboring solely one or the other of the two substitutions. All three D variants were associated with a cDE haplotype. The antigen densities were approximately 3,000 D antigens per red blood cell for DCS-1, 800 for DCS-2, and 9,300 for DFV. DCS-1 and DCS-2 were partial D, because they lacked distinct epitopes. DFV presented as an almost normal D phenotype; the sample contained allo-anti-LW(a). The D(w) antigen was absent from DCS-1, DFV, and DAU-4, but expressed by DAU-5. CONCLUSION: DCS-1, DCS-2, and DFV carry amino acid substitutions at the extracellular vestibule, visualized by 3-dimensional modeling. Proline at position 226 greatly influenced the D antigen density and may reduce the RhD membrane integration. Although the F223V substitution is regarded as the initial event in the evolution of the weak D Type 4 cluster, the current DFV allele probably evolved independently, as evident from different RHCE haplotypes.