Andrea Doescher

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.

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.

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.

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

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.

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.

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.

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.

Digital PCR to assess platelet recovery after stem cell transplantation

Engraftment of platelets in patients after human hematopoietic stem cell (HSC) transplantation serves as an early and relevant sign of the success of the transplantation. The time to platelet engraftment appears to be a valid predictor of the probability of complications in transplanted patients and especially for allogeneic HSC transplantation [1]. At least two different criteria of platelet engraftment, i.e. definitions according to the CIBMTR and EBMT, have been widely applied. Both criteria utilize daily platelet counts to assess the time to a threshold count of more than 20,000/μL platelets in peripheral blood, provided that platelet transfusions have been terminated for either at least three [2] (EBMT) or at least seven [3] (CIBMTR) consecutive days. Here, we describe the development of a digital PCR assay to specifically quantify graft-derived platelets versus transfused single-donor platelets. The performance of this assay was then tested with samples from allogeneic HSC transplant patients. Samples of 2 mL EDTA anticoagulated blood from patients were mixed with an equal volume of 0.9% NaCl and centrifuged at 100×g for 10 min without brake (Hettich Rotina 46S, Hettich, Tuttlingen, Germany). A final centrifugation step at 1569×g for 5 min followed. The pellet was resuspended in 500 μL phosphate buffered saline (PBS, Invitrogen Karlsruhe, Germany). The number of platelets in the PRP was adjusted with PBS to 5 × 10–2.5 × 10 to extract the mt DNA from these platelet suspensions (500 μL). The MagnaPure Large Volume protocol (MagnaPure compact, Roche Diagnostics GmbH, Mannheim, Germany) was used to extract the mt DNA. Elution volume was 50 μL. The PCR strategy was based on generic amplification primers and allele-specific probes. The probes were designed to specifically detect alleles carrying SNPs at positions 73, 195, 295, 310, 16069, 16399 and 16,516 of the non-coding regions HVR 1 and HVR 2 of the mt genome. Droplet generation, amplification and detection of PCR products were performed according to the manufacturer’s instructions (QX200 ddPCR System, Bio-Rad Laboratories GmbH, München, Germany) using 5 μL extracted DNA. Specificity of the assay was evaluated in mt DNA samples pre-typed by Sanger sequencing. Sensitivity was tested by dilution series, described in detail elsewhere [4]. The reproducibility of the background signal (“false positive droplets”) was determined by calculation of mean and standard deviation of n= 50 non-template controls and n= 20 positive controls each for FAMor VIClabelled mt DNA samples. Residual white blood cells and their mt DNA content could interfere with the platelet quantification based on the mt DNA PCR. Samples prepared for mt DNA analysis were, therefore, tested for the presence of genomic DNA as an indicator for residual white blood cells as described before [4]. The absolute count of platelets was calculated from the readout of the droplet digital PCR system (copies/μL) as: concentration (copies/μL)/genome mass (pg) × dilution factor (PRP preparation; PCR assay). The cut-off value for quantification of platelets for all mt SNPs was set to 1000 platelets/μL based on dilution series. The third of three consecutive days with increasing platelet counts was defined as day of engraftment. A digital PCR for SNP detection was developed and validated to monitor the platelet engraftment in patients after allogeneic stem cell transplantation. DNA samples from 30 patients examined in our previous study for quantification of endogenous platelets with real-time PCR were used to validate the digital PCR assay [4]. The quantification of platelets in 150 samples was congruent in * Andrea Doescher doescher@bsd-nstob.de

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 R2 = 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.

D blocking phenomenon overcome: Overcoming the D blocking phenomenon

A multigravida was referred at 23 1 4 weeks’ gestation. Doppler sonography of the fetal middle cerebral artery predicted fetal anemia. Fetal blood sampling confirmed a hemoglobin concentration of 3.3 g/dL (2.05 mmol/L), leading to an immediate intrauterine transfusion of red blood cells (RBCs) of blood group O D–. The maternal blood group was B D– ccddEe. D was confirmed as negative in the indirect antiglobulin test. Maternal plasma revealed high titers of anti-D (8192, column agglutination technique) and anti-C (1024). Both antibodies had been detected in the early pregnancy antibody screen. Adsorption and elution studies found anti-G as a third specificity, but adsorption to exhaustion with donor cells C1, D– did not deplete apparent anti-D, indicating only a minor proportion of anti-G. The direct antiglobulin test (DAT) of fetal RBCs was strongly positive. Serology of fetal RBCs indicated blood group O D– (see figure, top right, standard pipetting and immediate centrifugation, DiaClon ABO/D 1 DAT, DiaMed GmbH). The complete Rh typing result was Ccddee (DiaClon Rh-Subgroups 1 K and DiaClon Rh 1 K Pheno II, DiaMed GmbH), but anti-C alone seemed insufficient to explain the severity of the hemolytic disease. Thus, antibody blocking of fetal antigen D was suspected.