Real-Time PCR Analysis of Chimerism in T Cell Subsets as an Early Predictor of Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation.

BACKGROUND: Graft-versus-host-disease (GvHD) is the major cause of morbidity and mortality after stem cell transplantation. The development of early prediction methods is therefore of importance. Our aim was to analyze the usefulness of early donor chimerism monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in T cells and in CD4+ and CD8+ (lineage chimerism) for GvHD prediction. MATERIAL AND METHODS: Chimerism was analyzed in 76 consecutive adult patients using RQ-PCR TaqMan technology on DNA extracted from Pan T, CD4+, and CD8+ cell subsets on Day 5, 10, 15 and 30 after allo-HSCT. RESULTS: The threshold of chimerism predictive for GvHD was the same for all tested cell subsets. In acute myeloid leukemia (AML) patients treated with myeloablative conditioning (MAC), the threshold predictive for acute graft versus host disease was 95% and 99% for Day 10 and Day 15, respectively. In patients treated with reduced intensity conditioning (RIC), the threshold predictive for chronic graft versus host disease was 98% on Day 10. The differences were statistically significant. CONCLUSIONS: Chimerism analysis in T cell subsets by RQ-PCR on Day 10 and Day 15 after transplantation is useful for prediction of aGvHD (AML patients after MAC) and cGvHD (patients after RIC). However, there was no difference in the results between chimerism in the T cell subsets. Our RQ-PCR protocol was highly sensitive and proved effective for analysis of lineage chimerism.

[RHD variant in RhD/-/ mother with anti-D makes noninvasive fetal RHD genotyping impossible]. / Obecnosc "niemego" genu RHD u RhD ujemnej kobiety ciezarnej z przeciwcialami anty-RhD uniemozliwia okreslenie genotypu RHD plodu metoda nieinwazyjna.

OBJECTIVES: Noninvasive fetal RHD genotyping from maternal plasma of RhD(/-) pregnant women of Caucasian race may be used for predicting the risk of hemolytic disease because the RHD gene is usually absent in such populations. If detected in plasma of such women, the RHD gene originates from the RhD(+) fetus. The number of fetal copies of the gene in maternal plasma is extremely small. In the presented case of the RhD(/-) pregnant woman with anti-D it was impossible to give a fetal RHD result due to mother's RHD(+) genotype. The fetal RHD was determined from amniocytes. AIM: to present the difficulties related to the interpretation of results of invasive and noninvasive procedures. MATERIAL AND METHODS: whole blood, plasma and amniotic fluid of the RhD(-) woman with anti-D (14 week of pregnancy) as well as whole blood of the newborn. RHD and RHCE*c were genotyped by real-time PCR in DNA isolated from maternal plasma and amniocytes and the RHD and d-genotypes were tested by SSP methods in DNA isolated from whole blood and amniocytes. RESULTS: RHD and RHCE*c were detected in DNA isolated from plasma. The high level of RHD suggested its origin from the mother's DNA therefore it was impossible to determine the fetal RHD. The d-little test identified a RHD(IVS3+ 1G>A) variant in the mother's genome. A weak signal of real-time PCR for the RHD was obtained in amniocytes but the RHD was not detected by SSP. The RHCE*c was detected by both methods. Results were inconclusive; the fetal RHD status remained unknown. The child was RhD(-) with RHD in its DNA undetected by either method. CONCLUSIONS: 1/The RHD(IVS3+ 1G>A) variant in the RhD(-) mother precluded formal noninvasive fetal RHD genotyping. 2/Real-time PCR is too sensitive for amniocyte testing and may lead to false results as it detects trace maternal DNA in amniotic fluid. 3/The frequency of RHD(IVS3+1G>A) occurrence in Poland requires further studies.

[HCV genotypes in Polish blood donors in period 1995-2007]. / Genotypy HCV u polskich dawców krwi w okresie 1995-2007.

The results of HCV genotype distribution between 1995 and 2007 in Polish blood donors were analyzed. Special attention was drawn into the analysis of HCV polymorphism isolated from blood donors in the early phase of infection (RNA HCV positive/anti-HCV negative). Such donors identified in Poland since 2000 when the NAT for HCV was introduced are very important for molecular epidemiology analysis. The increasing frequency of 3a and 4 genotypes is observed in comparison to blood donors and patients with anti-HCV antibodies.