R. Lynen

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.

Application of RHD and RHCE genotyping for correct blood group determination in chronically transfused patients

BACKGROUND: In chronically transfused patients, conventional blood group typing may be impossible because of mixed‐field agglutination.

Flow cytometric detection of platelet‐reactive antibodies and application in platelet crossmatching

Background: Alloimmunization against HLA or platelet antigens can cause refractoriness to platelet transfusions in multiply transfused patients. Crossmatching of platelet concentrates is effective in overcoming this problem.

A dispermic chimerism in a 2-year-old Caucasian boy

Abstract Detection of two different cell populations in a child is a rare event. The following case of a dispermic chimera was diagnosed before surgery due to problems in blood group determination. A 2-year-old phenotypically male child was admitted for correction of a penoscrotal hypospadia and unilateral cryptorchism. During presurgical laboratory investigation, difficulties in blood group determination occurred. Blood group typing was performed by the DiaMed-ID Micro Typing System and by FACS. Additionally, cytogenetic analysis of lymphocytes and analysis of DNA polymorphisms in different tissues were performed. Two populations of red blood cells were detected, 0 cells accounting for 75% and B cells for 25%. Analysis of DNA-PCR polymorphisms in lymphocytes, nails, and in cells of the oral mucous membrane demonstrated a chimerism, with two alleles inherited from the father and one from the mother. A cytogenetic analysis of cultured lymphocytes showed a mosaic 46,XY/46,XX. Surgery revealed a prostatic utricle grade III, also called pseudovagina; genitography confirmed a vagina. Bilateral gonad biopsy showed a testis on one side and an ovary on the other. This case of chimerism represents a true hermaphroditism that most probably developed by double fertilization of one or more egg nuclei by two sperms.

Flow cytometric analyses of the subclasses of red cell IgG antibodies.

We report on flow cytometric IgG subclass determinations of red cell antibodies using polyclonal FITC‐labeled antibodies. The limit of detection of this method was 1 ng anti‐D per 1×107 red cells. The inter‐ and intra‐assay coefficients of variance were 8.2 and 2.3%, respectively. In 8 newborns with a positive direct antiglobulin test (DAT) in the gel centrifugation test (GCT), due to ABO antibodies, IgG1 was detected in all and IgG2 additionally in 4 of these cases. In 5 severe cases of hemolytic disease of the newborn (HDN) due to anti‐D, large amounts of IgG1 were found, and in 3 of these 5, IgG3 in combination with IgG1. In 8 mild or moderate HDN cases (4 anti‐D, 2 anti‐E, 1 anti‐Fya, 1 anti‐Jka), phototherapy sufficed, and IgG1 was the only antibody. In 7 adult patients with malignant lymphoma and a positive DAT (GCT), only small amounts of IgG1 red cell autoantibodies could be demonstrated by flow cytometry. In 5 further patients with malignant lymphoma, a positive DAT, and severe hemolytic anemia, large amounts of IgG1 autoantibodies were found and IgG3 was also present in 3 of these cases. Flow‐cytometric determination of IgG subclasses may be a useful tool in immunohematology, since subclass determinations were possible in all of these cases. This method is suited for clinical routine and offers the possibility of sufficient standardization.

A newly developed gel centrifugation test for quantification of RBC‐bound IgG antibodies and their subclasses IgG1 and IgG3: comparison with flow cytometry

BACKGROUND: Novel gel centrifugation test (GCT) cards were evaluated with respect to their ability to estimate the quantity of IgG on RBCs and the determination of the IgG subclasses IgG1 and IgG3.

RHD genotyping in weak D phenotypes by multiple polymerase chain reactions

BACKGROUND: Weak D phenotypes involve a quantitative variation of D. The genomic basis in weak D has been disputed, however.

Erste Ergebnisse einer prospektiven Studie mit automatisationsfähigen Festphasen- und Säulen-/Agglutinationstechniken beim Screening auf transfusionsrelevante IgG-Antikörper

Wir berichten uber die ersten Ergebnisse einer prospektiven Studie, in welcher wir verschiedene Methoden fur den Antikorpersuchtest zum Nachweis von erythrozytaren IgG-Antikorpern bei Transfusionsempf

Detection of Trace Amounts of Fetal RhD-Positive Red Cells with Flow Cytometry after Multiple Rh-Negative Intrauterine Transfusions and RhD-Gene Determination with PCR

In 2 cases of RhD-sensitized women, hemolytic disease of the newborns (hdn) was mitigated by intrauterine transfusions of·Rh-negative donor blood during pregnancy. In both cases preterm delivery was p