Tobias J. Legler

Presence of RHD in serologically D–, C/E+ individuals: a European multicenter study

BACKGROUND: RHD blood group alleles with reduced or absent antigen expression are a clinically significant and heterogeneous group.

Molecular background of Rh D-positive, D-negative, Del and weak D phenotypes in Chinese

Background and Objectives The aim of this study was to elucidate the genetic background of D‐negative and Del in the Chinese population.

Targeted deletion of murine coagulation factor XII gene-a model for contact phase activation in vivo

To analyze the biological role of factor XII (FXII, Hageman Factor) in vivo, we generated mice deficient for FXII using a gene targeting approach on two distinct genetic backgrounds, i.e. mixed C57Bl/6J X 129X1/SvJ and inbred 129X1/SvJ. Homozygous FXII knockout (FXII(-)/(-)) mice showed no FXII plasma activity and had a markedly prolonged activated partial thromboplastin time (aPTT). In contrast, coagulation factors XI, VIII, IX, X,VII, V, II and fibrinogen did not differ between FXII(-/-) mice and their wild-type littermates. Heterozygous matings segregated according to the Mendelian inheritance indicating that FXII deficiency does not increase fetal loss. Furthermore, matings of FXII(-/-) males and FXII(-/-) females resulted in normal litter sizes demonstrating that total FXII deficiency in FXII(-/-) females does not affect pregnancy outcome. Also, gross and histological anatomy of FXII(-/-) mice was indistinguishable from that of their wild-type littermates on both genetic backgrounds. Thus it appears that deficiency of murine FXII does not cause thrombophilia or impaired fibrinolysis in vivo. These results indicate that FXII deficiency does not affect hemostasis in vivo and we anticipate that the FXII(-/-) mice will be helpful to elucidate the biological role(s) of FXII in health and disease.

RHD sequencing: a new tool for decision making on transfusion therapy and provision of Rh prophylaxis

. The serological differentiation of weak D from partial D, D‐negative and D‐positive is not always unequivocal. Therefore, sequencing of the RHD gene is required in some cases. Very recently, several new differences between RHD and RHCE have been identified which permitted us to design primers close to the exon/intron boundaries of the RHD‐exons. We evaluated these primers in 83 D‐positive and 18 D‐negative blood donors and applied the new method for the characterization of the RHD gene in six individuals with weak D phenotype. The amplification reactions were concordant with serological findings in 100 of 101 donors (99·0%). In one D‐positive donor the PCR for exons 2 and 5 gave a negative result, while the sequence of the remaining eight exons was unchanged. By sequencing samples with very weak D serological reactions, we identified weak D type 4.2.2 and weak D type 15, both previously reported to be associated with anti‐D‐alloimmunization. Consequently, we recommended the selection of D‐negative blood in the weak D type 4.2.2 patient, and the provision of Rh prophylaxis for pregnant women with weak D type 15. In summary, a new RHD sequencing method was developed which can be applied if serological reactions are inconclusive.

A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization

BACKGROUND: The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion‐ and pregnancy‐induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption‐elution techniques, with so far only poorly defined antigenic properties.

The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible

BACKGROUND: Noninvasive fetal RHD genotyping might become a valuable tool in decision making on antenatal Rh prophylaxis, which is currently in routine practice for all D− pregnancies in several countries. This study provides a large‐scale validation study of this technology to address questions concerning feasibility and applicability of its introduction into clinical routine.

Prediction of fetal Rh D and Rh CcEe phenotype from maternal plasma with real-time polymerase chain reaction

Real-time PCR methods for the detection of RHD and the C, c, and E allele of RHCE were applied for the prediction of fetal Rh phenotype using maternal plasma. In one of 36 samples investigated the DNA extraction failed. When we tested the remaining 35 samples for Rh antigens which were absent on the mothers red cells, the fetal D-status was correctly determined in 26 of 27 cases (1 false negative). Fetal C was tested correctly in 23 samples, c was true positive in the only c-negative woman and the fetal E-status was correctly determined in 35 cases. In conclusion real-time PCR of maternal plasma is a non-invasive method to determine fetal RH genotype. However, more studies are required for routine applications because the method is not 100% sensitive.

Frequency and causes of refractoriness in multiply transfused patients

Abstract The use of leukocyte-depleted blood components has become the standard therapy for multiply transfused patients during the past few years, as a measure to reduce the frequency of alloimmunization and refractoriness. We assessed frequency and causes of refractoriness, defined as a repeated 24-h post-transfusion platelet count below 20 000/μl, in 145 consecutive patients who received three or more single-donor platelet concentrates during a 1-year period. Flow-cytometric detection of anti-platelet antibodies and a glycoprotein-specific ELISA were applied for the diagnosis of alloimmunization. Forty patients (27.6%) had at least one episode of refractoriness. In 25 of these 40 patients (62.5%), nonimmune factors (fever, sepsis, coagulopathy, splenomegaly) alone were the cause. In 15 refractory patients alloantibodies were detected. In seven patients (17.5%), alloimmunization alone caused an inadequate transfusion response, while in eight refractory patients (20.0%) alloimmunization and fever or sepsis were present. HLA antibodies were detected in 17 patients (11.7%); three patients (2%) had platelet-specific antibodies in addition to HLA antibodies; in two patients panreactive platelet antibodies were detectable. All 17 patients had a history of previous transfusions or pregnancy. We did not observe primary immunization in patients transfused exclusively with filtered (leukodepleted) blood products. Our data suggest that alloimmunization in patients with a negative risk history can be prevented by the exclusive use of leukodepleted blood components.

Testing for the D zygosity with three different methods revealed altered Rhesus boxes and a new weak D type.

BACKGROUND : The discrimination of D+/D+ from D+/D– partners of D– mothers with anti‐D is important to estimate the risk for HDN. This may be achieved if the presence or absence of the hybrid Rhesus box in the father can be demonstrated.

Recurrent Pregnancy Loss and Its Relation to FV Leiden, FII G20210A and Polymorphisms of Plasminogen Activator and Plasminogen Activator Inhibitor

Thrombophilic disorders and hypofibrinolysis were demonstrated to be risk factors in a majority of women with recurrent pregnancy loss (RPL) and infertility. We investigated the association of FV G1691A mutation, F II G20210A gene polymorphism (PM), 4G/5G PAI-1 and Alu I/D tPA PM in 32 women with infertility and 49 women with at least 2 unexplained early abortions. FV Leiden mutation was significantly more common in women with RPL (10%, p = 0.02) and infertility (19%, p = 0.0005) compared with controls (2%). PAI-1 4G PM and t-PA Alu I PM, alone or in combination, were not associated with RPL or infertility. 9/49 women with RPL showed coagulation disorders with heterozygous FV Leiden mutation (5), FXII (1), protein C (1) or protein S (2) deficiency. However, due to the small number of patients studied, no definite conclusion can be drawn.