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C E van der Schoot

Publications and source records attributed to C E van der Schoot.

At least 19 recordsLinked to original sources
PubMed2006

The analysis and quantification of a clonal B cell response in a hyperimmunized anti-D donor.

Healthy volunteers are hyperimmunized with RhD-positive red cells in order to obtain plasma containing high titres of anti-D immunoglobulin, which is used for the prevention of haemolytic disease of the fetus and newborn. We analysed the anti-D immune response in a donor who had been hyperimmunized for 7 years and who showed declining anti-D titres despite re-immunization. A phage display library representing the complete immunorepertoire and a second library representing the IGHV3 superspecies family genes (IGHV3s) repertoire in the donor were constructed and analysed. A clonal Ig-gene rearrangement was quantified in the peripheral blood by limiting dilution polymerase chain reaction (PCR) All RhD-binding phages from both libraries, except one, had heavy chains with IGH-VDJ rearrangements of the same clonal origin, but with different patterns of somatic mutations and joined with different light chains. Limiting dilution PCR performed on mRNA and genomic DNA showed a frequency of 1 clonal B cell in 2000 IgG1/3-positive B cells. We show the presence of clonally related RhD-specific B cells in a hyperimmunized anti-D donor who had declining anti-D titres and who was unresponsive to re-immunization. Furthermore, we found a high frequency of clonal B cells. These results contribute to the understanding of the immune response against RhD in hyperimmunized anti-D donors.

Amino Acid Sequence↗
PubMed2005

Systemic analysis and zygosity determination of the RHD gene in a D-negative Chinese Han population reveals a novel D-negative RHD gene.

BACKGROUND AND OBJECTIVES: The aim of this study was to systemically analyse the genetic background of D negativity in a Chinese Han population. MATERIALS AND METHODS: DNA of 74 D-negative samples was analysed by using an RHD multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by PCR-restriction fragment length polymorphism (PCR-RFLP) for RHD zygosity determination. Sixty-five samples were additionally analysed by using real-time quantitative PCR on RHD exon 7. RHD exon-specific sequencing was performed on discrepant samples. RESULTS: Forty-six samples (62%) showed the absence of RHD-specific exons by RHD MPX PCR and homozygous RHD negativity by PCR-RFLP. Twenty-two samples (30%) showed a 1227G>A mutation, characteristic for the Del phenotype. Five (7%) samples showed all characteristics of the RHD(1-2)-CE(3-9)-D(10) hybrid gene. One sample (1.4%) showed a novel 933C>A nonsense mutation in RHD exon 6, which resulted in a premature stop codon. CONCLUSIONS: The RHD gene deletion, RHD-CE-D hybrid genes and one novel 93C>A mutation were found to be the three mechanisms that cause D negativity in our samples. The 1227G>A Del mutation was found to be the major cause of discrepant results between genotyping and phenotyping strategies, favouring genotyping of D-negative samples.

China↗
PubMed2005

Report of the First International Workshop on molecular blood group genotyping.

The use of molecular genetic technology for blood group typing is becoming routine procedure in many reference laboratories worldwide. A First International Workshop was organized on behalf of the International Society of Blood Transfusion (ISBT) and the International Council for Standardization in Haematology (ICSH). Thirty laboratories that provide a molecular diagnostic service participated in the workshop. Six samples were distributed: two represented DNA from transfusion-dependent patients for testing for multiple polymorphisms; two represented fetal DNA prepared from amniotic fluid for RhD, Rhc and K-testing; and two represented plasma from RhD-negative pregnant women for fetal RhD testing. Error rates varied from 0 to 11% for different polymorphisms. A consensus arising from discussion on the workshop results between participants at a feedback meeting and by e-mail has resulted in seven recommendations for molecular blood group genotyping. Further international workshops will take place every 2 years, with a more limited exercise being organized in the intervening years.

Blood Group Antigens↗
PubMed2005

White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods.

BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments in platelet concentrates (PCs) may induce allo-immunization in the recipient. MATERIALS AND METHODS: As the level of WBC fragments can differ between PCs produced using different methods, we compared PCs prepared by using the buffy-coat method (BC-PCs) in plasma or platelet additive solution (Composol) and PCs prepared using the platelet-rich plasma method (PRP-PCs). RESULTS: Post-filtration results revealed identical levels of WBC, but significantly higher CD62p expression and a significantly lower amount of total DNA, cell-free DNA and number of WBC fragments (0.6 vs. 4.2 WBC equivalents/microl) in PRP-PCs than in BC-PCs in either plasma or Composol. CONCLUSIONS: The number of WBC fragments is significantly higher in BC-PCs than in PRP-PCs.

DNA↗
PubMed2004

[Fetal DNA in maternal blood].

There is a certain degree of foetal-maternal transfusion in every pregnancy. The possibilities for using intact foetal cells extracted from maternal blood for prenatal diagnosis are limited. Recently real-time polymerase chain reaction (PCR) techniques have allowed the identification and quantification of foetal DNA in the maternal blood. Foetal sex determination with Y-chromosome-PCR has been found to have a specificity of 100%. The sensitivity is 96% but increases with gestational age, so that from 10 weeks onwards the sensitivity approaches 100%. In the case of X-linked diseases, this technique can reduce invasive prenatal diagnosis by 50%. In the case of a foetus with an elevated risk for congenital adrenal hyperplasia, early non-invasive foetal sexing can also shorten the period of maternal dexamethasone use so as to prevent virilisation in the female foetus. Early second trimester non-invasive foetal RhD genotyping with an RhD-PCR has a sensitivity and specificity of 100% each. Therefore in the future, anti-D immunoprophylaxis will be superfluous in RhD-negative women with an RhD-negative foetus. Theoretically all new and paternal inherited disorders with a known gene defect can be detected in maternal plasma. Some examples have already been described. Recently published small scale studies describe elevated concentrations of free foetal DNA in maternal plasma in (threatening) preterm labour, pre-eclampsia and aneuploidy. Large-scale studies are necessary to demonstrate the value of these findings.

Chromosomes, Human, X↗
PubMed2004

ICAM-3 activation modulates cell-cell contacts of human bone marrow endothelial cells.

The Ig-like cell adhesion molecule ICAM-3 is mainly expressed on human leukocytes and is involved in cell-cell interactions. Its expression on endothelium is observed during disorders such as Crohn's disease and in solid tumors. We found low but detectable expression of ICAM-3 on VE-cadherin-expressing cells from primary human bone marrow aspirates, i.e. endothelial cells, and on primary human endothelial cells from cord blood. Also, immortalized human umbilical cord endothelial cells and human bone marrow endothelial cells showed ICAM-3 expression. However, its function on human endothelium is not known. Surprisingly, activation of endothelial ICAM-3 by crosslinking with specific antibodies resulted in a drop in the electrical resistance of bone marrow endothelial monolayers. In line with this, immunocytochemical analysis showed a loss of endothelial cell-cell contacts after ICAM-3 crosslinking in HBMEC. Detailed biochemical analysis showed an association of moesin and in a later stage ezrin with ICAM-3 upon crosslinking in HBMEC. Moreover, ICAM-3 crosslinking induced the production of reactive oxygen species (ROS), which are known to be involved in the control of endothelial cell-cell contacts. In conclusion, we showed that ICAM-3 is expressed on human bone marrow endothelial cells and controls endothelial integrity via ROS-dependent signaling.

Antigens, CD↗
PubMed2004

Influence of cell-free DNA in plasma on real-time polymerase chain reaction for determination of residual leucocytes in platelet concentrates.

BACKGROUND AND OBJECTIVES: Real-time quantitative (RQ) polymerase chain reaction (PCR) can be used to determine the number of residual leucocytes in leucocyte-reduced platelet concentrates (LR-PCs), which should contain < 3.3 leucocytes/ micro l. In this study we investigated the extent to which cell-free DNA, known to be present in plasma, might interfere with this determination. In this study, RQ-PCR was employed to determine the following: the influence of filtration of platelet concentrates (PCs) on the amount of cell-free DNA; the variation in concentration of cell-free DNA between the buffy coats (BCs) of different donors; and the amount of cell-free DNA during storage and processing of whole blood. MATERIALS AND METHODS: PCs were sampled before and after filtration (n = 5), BCs were sampled (n = 100) and whole blood units were sampled < 2 h and 16-20 h after collection, and the BCs were also sampled after processing the whole blood (n = 10). Samples were centrifuged to obtain cell-free plasma in which the amount of cell-free DNA was determined using an RQ-PCR for the albumin gene. RESULTS: The amount of cell-free DNA was not influenced by filtration of the PCs [1.7 +/- 0.8 vs. 1.5 +/- 0.8 leucocyte-equivalents (eq)/ micro l]. However, the amount of cell-free DNA in plasma of the BCs varied considerably, from 0.1 to 18.2 leucocyte-eq/ micro l (median = 1.5 leucocyte-eq/ micro l; mean +/- SD: 2.2 +/- 2.4 leucocyte-eq/ micro l). In 18% of the BCs the amount cell-free DNA was > 3.3 leucocyte-eq/ micro l. The amount of cell-free DNA increased during storage, from 0.3 +/- 0.3 leucocyte-eq/ micro l (< 2 h after collection) to 0.9 +/- 0.6 leucocyte-eq/ micro l (16-20 h after collection) and, after processing the whole blood, to 2.0 +/- 2.0 leucocyte-eq/ micro l. CONCLUSIONS: Variable amounts of cell-free DNA in plasma will interfere if RQ-PCR is applied to estimate leucocyte numbers in leucocyte-reduced PCs.

Artifacts↗
PubMed2004

Development of white blood cell fragments, during the preparation and storage of platelet concentrates, as measured by using real-time polymerase chain reaction.

BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments may cause human leucocyte antigen (HLA) immunization in recipients. We investigated the occurrence and production of WBC fragments in platelet concentrates (PCs) and plasma units, during storage and filtration, by using real-time polymerase chain reaction (PCR) and flow cytometry. MATERIALS AND METHODS: To study the occurrence of WBC fragments, 'male' WBCs were spiked into double-filtered 'female' PCs in a concentration series of 0.03-100 WBCs/microl (n = 4 per level). To study the production of WBC fragments, 'male' WBCs were spiked into 'female' plasma units to 4 x 10(9) WBCs/l and stored at room temperature prior to filtration (n = 4 per storage time; t = 0, 24 or 48 h). DNA was measured by both albumin real-time PCR and Y real-time PCR. Intact WBCs were counted by using flow cytometry. The number of WBC fragments was calculated by subtracting cell-free DNA (real-time PCR on supernatant) and intact WBCs (flow cytometry) from the total DNA amount (real-time PCR). RESULTS: Spiking of 'male' WBCs into 'female' PCs showed that the Y real-time PCR is linear and has a reproducible quantitative range down to 0.03 WBC/microl, but that the albumin-PCR, in unspiked samples, revealed a total of 6-10 WBC equivalents/microl (eq/microl). After centrifugation, half of this was observed as cell-free DNA in the supernatant, suggesting that the remaining DNA is derived from WBC fragments. The number of intact WBCs, amount of cell-free DNA and number of WBC fragments after filtration increased significantly when filtration was delayed for up to 48 h, from 0.1 WBC/microl, 1.3 WBC eq/microl and 0.6 WBC eq/microl at t = 0 h to 25 WBC/microl, 38 WBC eq/microl and 57 WBC eq/microl at t = 48 h, respectively. CONCLUSIONS: WBC fragments occur in WBC-reduced PCs and increase when products are stored, prior to filtration, up to levels that are equivalent to the amounts of intact WBCs that induce HLA immunization (i.e. > 5 x 10(6)/unit).

Blood Preservation↗
PubMed2002

Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR.

Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex analysis at diagnosis, their stability at relapse, and their applicability in real-time quantitative PCR (RQ-PCR) analysis. In 77 selected children with precursor-B-ALL, Southern blotting detected 122 IGK-Kde rearrangements, 12 of which were derived from subclones in six patients (8%). PCR/heteroduplex analysis with BIOMED-1 Concerted Action primers identified 100 of the 110 major IGK-Kde rearrangements (91%). Comparison between diagnosis and relapse samples from 21 patients with PCR-detectable IGK-Kde rearrangements (using Southern blotting, PCR/heteroduplex analysis, and sequencing) demonstrated that 27 of the 32 rearrangements remained stable at relapse. When patients with oligoclonal IGK-Kde rearrangements were excluded, 25 of the 27 rearrangements remained stable at relapse and at least one stable rearrangement was present in 17 of the 18 patients. Subsequently, RQ-PCR analysis with allele-specific forward primers, a germline Kde TaqMan-probe, and a germline Kde reverse primer was evaluated for 18 IGK-Kde rearrangements. In 16 of the 18 targets (89%) a sensitivity of < or =10(-4) was reached. Analysis of MRD during follow-up of eight patients with IGK-Kde rearrangements showed comparable results between RQ-PCR data and classical dot-blot data. We conclude that the frequently occurring IGK-Kde rearrangements are generally detectable by PCR (90%) and are highly stable MRD-PCR targets, particularly where monoclonal rearrangements at diagnosis (95%) are concerned. Furthermore, most IGK-Kde rearrangements (90%) can be used for sensitive detection of MRD (< or =10(-4)) by RQ-PCR analysis.

Adolescent↗
PubMed2002

The TEL-AML1 real-time quantitative polymerase chain reaction (PCR) might replace the antigen receptor-based genomic PCR in clinical minimal residual disease studies in children with acute lymphoblastic leukaemia.

Prospective studies in children with B-precursor acute lymphoblastic leukaemia (ALL) have shown that polymerase chain reaction (PCR)-based detection of minimal residual disease (MRD) using immunoglobin (Ig) and T-cell receptor (TCR) gene rearrangements as targets can be used to identify patients with a high relapse risk. The disadvantage of this approach is that for each patient preferably two different targets have to be identified. The t(12;21)(p13;q22) with the TEL-AML1 fusion gene is present in approximately 25% of children with B-precursor ALL. In these patients, sensitive reverse transcription (RT)-PCR analysis of the TEL-AML1 fusion transcript might be a more simple and less laborious alternative approach. However, it is unknown how stable the mRNA is and whether the number of transcripts per leukaemic cell remains constant during follow-up. We investigated whether the MRD results obtained using RT-PCR of TEL-AML1 transcripts correlated with the clinically validated genomic PCR for Ig and TCR gene rearrangements. Therefore, we used real-time quantitative (RQ)-PCR analysis for both types of targets and assessed the MRD levels in 36 follow-up bone marrow samples (obtained during the first 1.5 years after diagnosis) from 13 patients with B-precursor ALL. In 34/36 bone marrow samples the Ig/TCR RQ-PCR and TEL-AML1 RQ-PCR revealed equal levels of MRD and these results had a strong correlation (P < 0.0001, R2 = 0.84). Therefore, we conclude that the TEL-AML1 RQ-PCR can, in principle, replace Ig/TCR RQ-PCR in B-precursor ALL with t(12;21).

Child↗
PubMed2001

In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation.

To investigate whether the migratory ability of peripheral blood-derived CD34+ cells of patients undergoing autologous peripheral blood stem cell transplantation is related to the homing efficiency of these cells, the migration in vitro of these cells was determined and correlated with in vivo hematopoietic recovery. Large inter-individual differences of the in vitro migratory ability of the CD34+ cells were observed, ranging from 1.1% to 16.4% for spontaneous migration and 6.2% to 40.8% for SDF-1-induced (100 ng/mL) migration. Significantly faster hematologic recovery was observed in those patients who received transplanted CD34+ cells that showed high spontaneous and SDF-1-induced migration in vitro (P <.05). Moreover, CD34+ cells from healthy G-CSF-mobilized donors exhibited significantly higher spontaneous and SDF-1-induced (P <.01) migration than CD34+ cells from patients mobilized with chemotherapy and G-CSF. The lower migratory capacity in vitro of patient-derived CD34+ cells was not due to lower expression of CXCR-4 but probably reflected decreased motogenic behavior of the cells. These results indicate that the migratory capacity of the cells is important for hematopoietic recovery. The data suggest that the engraftment potential of autologous stem cells is more or less impaired by treatment before or during the mobilization procedure and might possibly be restored by in vitro manipulation of the cells. In addition, an exponential relation between CXCR-4 expression and number of CD34+ cells that mobilized to the peripheral blood was found (P <.001), suggesting that CXCR-4 expression plays a role in the mobilization of CD34+ cells.

Adolescent↗
PubMed2001

Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia.

OBJECTIVE: To determine first-trimester fetal sex by isolating free fetal DNA from maternal plasma. METHODS: The index case was a pregnant woman who previously delivered a girl with congenital adrenal hyperplasia. The SRY gene as a marker for the fetal Y chromosome was detected in maternal serum and plasma by quantitative polymerase chain reaction analysis. Simultaneously, we performed the same test in 25 and 19 women in the first and second trimester, respectively, and compared plasma results with fetal gender as assessed by prenatal karyotyping or as seen at ultrasound or birth. RESULTS: In 44 of 45 patients at gestational ages ranging from 8 3/7 to 17 3/7 weeks, we correctly predicted fetal sex using quantitative polymerase chain reaction analysis of the SRY gene in maternal plasma. In one case, the test result was inconclusive. Overall, fetal sex was correctly predicted in 97.8% of cases (95% confidence interval 88.2%, 99.9%). CONCLUSION: Amplification of free fetal DNA in maternal plasma is a valid technique for predicting fetal sex in early pregnancy. In case of pregnancies at risk for congenital adrenal hyperplasia, the technique allows restriction of dexamethasone treatment to female fetuses resulting in a substantial decrease of unnecessary treatment and invasive diagnostic tests.

Adrenal Hyperplasia, Congenital↗