Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Extreme Reticulocytosis After Splenectomy in a Patient With Hemoglobin Mizuho.

BACKGROUND: Patients with Hb Mizuho may be splenectomized at a young age to decrease their need for blood transfusions. OBSERVATIONS: Transfusion-dependency decreased dramatically in a 4-year-old white boy with Hb Mizuho after splenectomy. Surprisingly, he developed reticulocytosis (>1000×10 9 /L) with a peak reticulocyte percentage of 49%, and erythrocyte abnormalities, including Heinz bodies, Howell-Jolly bodies, and basophilic stippling. Manual reticulocyte counting and flow cytometric measurement with anti-CD71 antibodies supported a truly elevated reticulocyte count. CONCLUSIONS: We propose possible explanations for the extreme reticulocytosis that arose postsplenectomy and compare the reticulocyte count in the present case with previously published cases.

Classification of fetal and adult red blood cells based on hydrodynamic deformation and deep video recognition.

Flow based deformation cytometry has shown potential for cell classification. We demonstrate the principle with an injection moulded microfluidic chip from which we capture videos of adult and fetal red blood cells, as they are being deformed in a microfluidic chip. Using a deep neural network - SlowFast - that takes the temporal behavior into account, we are able to discriminate between the cells with high accuracy. The accuracy was larger for adult blood cells than for fetal blood cells. However, no significant difference was observed between donors of the two types.

Secretor status of blood group O mothers is associated with development of ABO haemolytic disease in the newborn.

BACKGROUND AND OBJECTIVES: Identification of antibody characteristics and genetics underlying the development of maternal anti-A/B linked to inducing haemolytic disease of the foetus and newborn could contribute to the development of screening methods predicting pregnancies at risk with high diagnostic accuracy. MATERIALS AND METHODS: We examined 73 samples from mothers to 37 newborns with haemolysis (cases) and 36 without (controls). The secretor status was determined by genotyping a single nucleotide polymorphism in FUT2, rs601338 (c.428G>A). RESULTS: We found a significant association between secretor mothers and newborns developing haemolysis (p = 0.028). However, stratifying by the newborn's blood group, the association was found only in secretor mothers to blood group B newborns (p = 0.032). In fact, only secretor mothers were found in this group. By including antibody data from a previous study, we found higher median semi-quantitative levels of IgG1 and IgG3 among secretor mothers than non-secretor mothers to newborns with and without haemolysis. CONCLUSION: We found that the maternal secretor status is associated with the production of anti-A/B, pathogenic to ABO-incompatible newborns. We suggest that secretors experience hyper-immunizing events more frequently than non-secretors, leading to the production of pathogenic ABO antibodies, especially anti-B.

Blood donor genotyping for prediction of blood group antigens: Results from 5 years' experience (2017-2022).

BACKGROUND AND OBJECTIVES: For 5 years, routine genotyping has been performed for selected blood groups of blood donors in the Copenhagen Capital Region, Denmark. The result is summarized in the following. MATERIALS AND METHODS: Genotyping was carried out by an external service provider using the competitive allele specific PCR (KASP) technology. The genotypes were returned to the blood bank and translated into phenotypes by a proprietary IT application. RESULTS: In total, 65 alleles from 16 blood group systems (ABO, MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, Colton, Landsteiner-Wiener, Cromer, Knops, Vel, secretor status) and the HPA1, HPA5 and HPA15 antigens were interrogated. After translation, phenotypes were imported into the laboratory information management system of the blood bank. The results from 31,538 genotyped blood donors were used to calculate the allele frequencies for a Danish blood donor population. ABO genotyping was done for sample ID purposes. Determination of the 1061delC single nucleotide polymorphism (SNP) (NM_020469.2), most frequently characteristic of ABO*A2, was validated against a series of 1287 samples with Dolichos biflorus lectin determination of the A1 phenotype. CONCLUSION: We report allele frequencies and phenotype frequencies for 16 blood groups from a total of 31,538 genotyped blood donors. Blood products were supplied from a total of 64,312 active blood donors, and of these active blood donors 25,396 (39.5%) were genotyped. These donors represent a valuable resource for patient treatment. This genotyping has enabled the provision of rare genotyped donor blood for patients with alloantibodies and rare reagent cells for serology.

ABO haemolytic disease of the newborn: Improved prediction by novel integration of causative and protective factors in newborn and mother.

BACKGROUND AND OBJECTIVES: Prediction of haemolytic disease of the foetus and newborn (HDFN) caused by maternal anti-A/-B enables timely therapy, thereby preventing the development of kernicterus spectrum disorder. However, previous efforts to establish accurate prediction methods have been only modestly successful. MATERIALS AND METHODS: In a case-control study, we examined 76 samples from mothers and 76 samples from their newborns; 38 with and 38 without haemolysis. The IgG subclass profile of maternal anti-A and anti-B was determined by flow cytometry. Samples from newborns were genetically analysed for the A2 subgroup, secretor and FcγRIIa receptor alleles. RESULTS: Surprisingly, we found a correlation between the newborn secretor allele and haemolysis (p = 0.034). No correlation was found for FcγRIIa alleles. The A2 subgroup was found only in newborns without haemolysis. Unexpectedly, different reaction patterns were found for maternal anti-A and anti-B; consequently, the results were treated separately. For the prediction of haemolysis in A-newborns, the maternal IgG1 subclass determination resulted in an accuracy of 83% at birth. For B-newborns, an accuracy of 91% was achieved by the maternal IgG2 subclass determination. CONCLUSION: We improved the prediction of ABO-HDFN by characterizing maternal anti-A and anti-B by flow cytometry and we presented genetic traits in newborns with correlation to haemolysis. We propose a new understanding of A- and B-substances as immunogens that enhance the maternal immune response and protect the newborn, and we suggest that the development of ABO-HDFN is different when caused by maternal anti-A compared to maternal anti-B.

Non-Invasive Fetal K Status Prediction: 7 Years of Experience.

Introduction: In the Kell blood group system, the K and k antigens are the clinically most important ones. Maternal anti-K IgG antibodies can lead to the demise of a K-positive fetus in early pregnancy. Intervention can save the fetus. Prenatal K status prediction of the fetus in early pregnancy is desirable and gives a good basis for pregnancy risk management. We present the results from 7 years of clinical experience in predicting fetal K status as well as some theoretical considerations relevant for design of the assay and evaluation of results. Methods: Blood was collected from 43 women, all immunized against K, at a mean gestational age of 18 weeks (range 10-38). A total of 56 consecutive samples were tested. The KEL *01.01 /KEL *02 single nucleotide variant that determines K status was amplified from maternal plasma DNA by PCR without allele specificity. The PCR product was sequenced by NGS technology, and the number of sequenced KEL *01.01 and KEL *02 reads were counted. Prediction of the fetal K status was based on this count and was compared with the serologically determined K status of the newborns. Results: All fetal K predictions were in accordance with postnatal serology where available (n = 34), using our current data analysis. Conclusion: We have developed an NGS-based method for the non-invasive prediction of fetal K status. This approach requires special considerations in terms of primer design, stringent preanalytical sample handling, and careful analytical procedures. We analyzed samples starting at GA 10 weeks and demonstrated the correct prediction of fetal K status. This assay enables timely clinical intervention in pregnancies at risk of hemolytic disease of the fetus and newborn caused by maternal anti-K IgG antibodies.

Exome-Based Trio Analysis for Diagnosis of the Cause of Congenital Severe Hemolytic Anemia in a Child.

Inborn hemolytic anemia requiring frequent blood transfusions can be a life-threatening disease. Treatment, besides blood transfusion, includes iron chelation for prevention of iron accumulation due to frequent blood transfusions. We present the results of a clinical investigation where the proband was diagnosed with severe hemolytic anemia of unknown origin soon after birth. Transfusion was required every 4-6 weeks. After whole exome sequencing of the proband and his parents as well as a healthy sibling, we established that the proband had a compound heterozygous state carrying two rare variants in the erythrocytic spectrin gene, SPTA1. The maternal allele was a stop mutation (rs755630903) and the paternal allele was a missense mutation (rs375506528). The healthy sibling had the paternal variant but not the maternal variant. These rare variants of SPTA1 most likely account for the hemolytic anemia. A severely reduced osmotic resistance in the erythrocytes from the proband was demonstrated. Splenectomy considerably improved the hemolytic anemia and obviated the need for blood transfusion despite the severe clinical presentation.

Prenatal prediction of fetal Rh C, c and E status by amplification of maternal cfDNA and deep sequencing.

BACKGROUND: The Rh blood group system has considerable clinical importance. The C, c, and E antigens are targets of alloantibodies. Anti-C, anti-c or anti-E alloreactive antibodies produced in pregnant women can cause anemia of a fetus carrying the corresponding antigens. AIMS: Based on NGS technology, we have developed a noninvasive diagnostic assay to predict the fetal blood group of C, c or E antigens by sequencing cell-free DNA (cfDNA) during pregnancy. MATERIALS AND METHODS: The SNVs underlying either the C, c or E antigens were PCR amplified and sequenced using NGS on a MiSeq instrument. The DNA sequences encoding the C, c or E antigen were counted, as were the number of total sequences. Based on the percentage of fetally derived target SNVs inherited from the father, the fetal blood group could be predicted. RESULTS: The results of 55 consecutive RHCE prenatal analyses with postnatal serological blood group determination of 30 newborns showed no discordant results. A threshold discerning positive from negative samples was set at 0.05% specific reads. DISCUSSION: Noninvasive, prenatal prediction of fetal blood groups by sequencing cfDNA for the detection of low-level RHCE*C, RHCE*c and RHCE*E sequences was established as an accurate and robust assay applicable for use in clinical settings.

Laboratory Monitoring of Mother, Fetus, and Newborn in Hemolytic Disease of Fetus and Newborn.

BACKGROUND: Laboratory monitoring of mother, fetus, and newborn in hemolytic disease of fetus and newborn (HDFN) aims to guide clinicians and the immunized women to focus on the most serious problems of alloimmunization and thus minimize the consequences of HDFN in general and of anti-D in particular. Here, we present the current approach of laboratory screening and testing for prevention and monitoring of HDFN at the Copenhagen University Hospital in Denmark. SUMMARY: All pregnant women are typed and screened in the 1st trimester. This serves to identify the RhD-negative pregnant women who at gestational age (GA) of 25 weeks are offered a second screen test and a non-invasive fetal RhD prediction. At GA 29 weeks, and again after delivery, non-immunized RhD-negative women carrying an RhD-positive fetus are offered Rh immunoglobulin. If the 1st trimester screen reveals an alloantibody, antenatal investigation is initiated. This also includes RhD-positive women with alloantibodies. Specificity and titer are determined, the fetal phenotype is predicted by non-invasive genotyping based on cell-free DNA (RhD, K, Rhc, RhC, RhE, ABO), and serial monitoring of titer commences. Based on titers and specificity, monitoring with serial peak systolic velocity measurements in the fetal middle cerebral artery to detect anemia will take place. Intrauterine transfusion is given when fetal anemia is suspected. Monitoring of the newborn by titer and survival of fetal red blood cells by flow cytometry will help predict the length of the recovery of the newborn.

Impact of long-term storage of plasma and cell-free DNA on measured DNA quantity and fetal fraction.

BACKGROUND AND OBJECTIVE: Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR. MATERIALS AND METHODS: Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6 years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18 months and 3 years of storage at -25°C. RESULTS: We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3 years, and fetal fraction was slightly decreased after 3 years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2 years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA. CONCLUSION: Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes.

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma.

INTRODUCTION: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. OBJECTIVE: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. MATERIALS AND METHODS: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. RESULTS: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. CONCLUSIONS: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

Blood group genotyping of blood donors: validation of a highly accurate routine method.

BACKGROUND: In the past century, blood group determination using serology has been the standard method. Now, molecular methods are gaining traction, which provide additional and easily accessible information. Here we designed and validated a high-throughput extended genotyping setup. STUDY DESIGN AND METHODS: We developed 35 competitive allele-specific polymerase chain reaction assays for genotyping of blood donors. Samples from 1034 Danish blood donors were genotyped, and 45,314 red blood cell antigens and 6148 platelet antigens were predicted. Predicted phenotypes were compared with 16,119 serologic phenotypes. RESULTS: We found 62 discrepancies of which 43 were due to serology. After exclusion of the discrepancies caused by serology, the accuracy of genotyping was 99.9%. Of 17 discrepancies caused by the genotype, three were incorrect antigen-negative predictions and could potentially, as the solitary analysis, have caused an adverse transfusion reaction. CONCLUSION: We have established a robust and highly accurate blood group genotyping system with a very high capacity for screening blood donors. The system represents a significant improvement over the former serotyping-only procedure. Almost all new technology in medicine incurs increased costs, but the presented efficient genotyping system is a rare example of a significant qualitative and quantitative technologic progress that is also more cost-efficient than previous technologies.

The VICM biomarker is released from activated macrophages and inhibited by anti-GM-CSFRα-mAb treatment in rheumatoid arthritis patients.

OBJECTIVES: Macrophages possess widespread pro-inflammatory, destructive, and remodelling capabilities that can critically contribute to acute and chronic diseases, such as rheumatoid arthritis (RA). Continuous monitoring and measurement of selective counteraction of macrophage activity in patients require a sensitivity and non-invasive marker. We characterised the VICM (citrullinated and MMP degraded vimentin fragment) biomarker by investigating the release from in vitro activated macrophages and by monitoring the change in serum levels after treatment with the anti-GM-CSFRα-mAb (mavrilimumab). METHODS: Peripheral blood mononuclear cells were isolated, and lipopolysaccharide (LPS) was used to activate the macrophages and calcium chloride (CaCl2) was used to facilitate the citrullination process of vimentin. Supernatants, cell lysates, was collected and analysed by ELISA, and western blotting. RA patients were treated with mavrilimumab+methotrexate or methotrexate alone in a phase 2b study (NCT01706926) once every two weeks for 24 weeks. Serum levels of VICM were measured at baseline and multiple time points post-treatment. In addition, whole blood expression of peptidylarginine deiminase-2 (PAD-2) and matrix metalloproteinase-9 (MMP-9) transcripts were tested by quantitative reverse transcriptase PCR assays at day 0 and day 169 post-treatment. RESULTS: VICM levels were significantly higher at day 5 and 8 in supernatants of activated macrophages compared to controls (p<0.01), which was confirmed by Western blot. In RA patients, VICM correlated with disease activity (DAS28), modified total sharp score (mTSS), joint space narrowing (JSN), joint erosions and CRP at baseline. VICM was dose-dependently and significantly (p<0.01) inhibited by mavrilimumab. This suppression of VICM serum levels was supported by a decreased expression of PAD2 and MMP9 transcripts in patients treated with mavrilimumab. CONCLUSIONS: These data verified that VICM is released by activated macrophages. Treatment of RA patients with mavrilimumab significantly reduced release of VICM and peptidylarginine deiminases-2 (PAD-2) gene expression indicating that mavrilimumab indeed is targeting activated macrophages and that VICM may be a novel blood-based marker of anti-GM-CSF response.

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>>>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.

Noninvasive fetal RhD genotyping.

Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10-11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women.

Identifying mild and severe preeclampsia in asymptomatic pregnant women by levels of cell-free fetal DNA.

BACKGROUND: The objective was to investigate whether women who develop preeclampsia can be identified in a routine analysis when determining fetal RHD status at 25 weeks' gestation in combination with PAPP-A levels at the first-trimester combined risk assessment for Trisomy 21. STUDY DESIGN AND METHODS: D- women participating in the routine antenatal RHD screening program in the capital region of Denmark were retrospectively studied. We used a standard dilution curve to quantify the amounts of cell-free fetal DNA (cffDNA) and divided women into groups according to cffDNA levels. PAPP-A was measured at 11 to 14 weeks. Information about pregnancy outcome and complications was obtained from the National Fetal Medicine Database, medical charts, and discharge letters. RESULTS: The odds ratio (OR) of developing severe preeclampsia given a cffDNA level above the 90th percentile compared to cffDNA below the 90th percentile was 8.1 (95% confidence interval [CI], 2.6-25.5). The OR of developing mild preeclampsia given a cffDNA level below the 5th percentile compared to cffDNA levels above the 5th percentile was 3.6 (95% CI, 1.1-11.7). PAPP-A levels below the 5th percentile were associated with mild preeclampsia, but adding it to the analysis did not increase the detection rate (DR). CONCLUSION: Women with cffDNA levels below the 5th percentile and above the 90th percentile quantified at 25 weeks' gestation are at increased risk of developing preeclampsia. Adding PAPP-A levels to the analysis did not increase the DR of preeclampsia.

Next-generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell-free fetal DNA in maternal plasma.

BACKGROUND: Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next-generation sequencing (NGS) technology. STUDY DESIGN AND METHODS: The KEL1/2 single-nucleotide polymorphism was polymerase chain reaction (PCR) amplified with one adjoining base, and the PCR product was sequenced using a genome analyzer (GAIIx, Illumina); several millions of PCR sequences were analyzed. RESULTS: The results demonstrated the feasibility of diagnosing the fetal KEL1 or KEL2 blood group from cell-free DNA purified from maternal plasma. CONCLUSION: This method requires only one primer pair, and the large amount of sequence information obtained allows well for statistical analysis of the data. This general approach can be integrated into current laboratory practice and has numerous applications. Besides DNA-based predictions of blood group phenotypes, platelet phenotypes, or sickle cell anemia, and the determination of zygosity, various conditions of chimerism could also be examined using this approach. To our knowledge, this is the first report focused on antenatal blood group determination using NGS.

Urea and water are transported through different pathways in the red blood cell membrane.

Several studies of the urea transporter UT-B expressed in Xenopus oocytes and in genetically modified red blood cells (RBC) have concluded that UT-B also transports water. In the present study, we use unmodified RBC to test that conclusion. We find that the permeability of urea, Pu (cm/s), has a 10-fold donor variation, while the diffusional water permeability, Pd (cm/s), remains unchanged. Additionally, we observe that phloretin inhibits Pu but not Pd, and that the time course of maximum p-chloromercuribenzosulfonate inhibition of Pu and Pd differs-Pu inhibition takes <2 min, whereas Pd inhibition requires ≥1 h of incubation. The findings in the present study are in line with a previous comparative study using unmodified RBC from four animals and a solvent drag study using human RBC, and they lead us to reject the conclusion that the UT-B transporter represents a common pathway for both solutes.

Urea transport in human red blood cells: Donor variation compared to chloride, glucose, and water transport.

We determined the permeability (P, cm/s) of unmodified human red blood cells (HRBC) to urea (Pu), chloride (PCl), glucose (Pglu), and water diffusion (Pd) under conditions of self-exchange (SE) with the continuous flow tube method at pH 7.2, 25°C. Among 24 donors, Pu at 1 mM varied >100%. Two of the donors were also tested in 1983. Their Pu had decreased by 77 and 90%. High age in males and Kidd genotype Jk(a+,b+), but not blood types AB0, appear related to low Pu. For one of the two donors, PCl (150 mM, 38°C, pH 7.2), Pglu (1 mM, 38°C, pH 7.2), and Pd (55.5 M, 25°C, pH 7.2) were determined then and now and showed no significant changes with age. The results from six more donors show donor PCl, Pglu, and Pd in the range of ≈1%. PCl and Pglu are vital for the metabolism of cells and tissues, and we see but little donor variation, and so far, no phenotypes without glucose (GLUT1) and anion (AE1) transporters in HRBC. Phenotypes with no urea transporter (UT-B) or no water transporters (aquaporin, AQP1) are registered and are compatible with life. Our results are in line with the concept that the solutes do not share pathways in common. The great donor variation in Pu must be considered in comparative transport physiological studies.