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Background: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation., Study Design and Methods: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files., Results: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per microL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per microL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files., Conclusion: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.

Background: Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry., Materials and Methods: DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression., Results: The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation., Conclusion: Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.

Objective: Allogenic transplantation of umbilical cord blood (UCB) from HLA-identical siblings is a therapeutic concept of increasing importance. Prenatal HLA typing results can provide important information as to whether the UCB should be collected. Therefore, we tested the suitability of two methods based on polymerase chain reaction (PCR) for low-resolution HLA genotyping of uncultured amniocytes contaminated with maternal blood., Study Design: Amniotic fluid samples (~10 mL, n = 4) were collected and divided into 5 equal sized portions. Subsequently, maternal blood was added to produce a series of varying degrees of artificial contamination ranging from 0% to 5% (vol/vol). Corresponding maternal blood and cord blood samples were collected and served as reference material. After DNA preparation, all samples were subjected to both PCR-SSP (sequence-specific primers) and PCR-SSO (sequence-specific oligonucleotides) typing procedures, designed for low-resolution typing of the highly polymorphic HLA-B locus., Results: Both PCR tests were able to accurately predict the fetal HLA-B type when pure amniotic fluid was used in all cases. The PCR-SSP method could still determine the fetal HLA type at 0.1% contamination but not at levels above this; in contrast, PCR-SSO failed if any degree of contamination was present., Conclusion: Fetal cells from amniotic fluid represent a reasonable source for prenatal testing of the fetal HLA genotype with either the PCR-SSP or the PCR-SSO method. Low levels of contamination with maternal blood in the amniotic fluid are tolerated by the more robust PCR-SSP method. In contrast, the PCR-SSO procedure is more sensitive to any degree of contamination, but it is the method of choice if the amount of DNA is limited because of the very low volume of specimens of amniotic fluid available.

Background: Dendritic cells (DCs) for immunotherapy of malignant melanoma can be generated from partially enriched monocytes prepared from PBMNCs. The feasibility of a single steady-state leukapheresis procedure to enrich monocytes for a complete vaccination series with up to 10 vaccinations was investigated., Study Design and Methods: Thirty-eight patients (27 males and 11 females) with metastatic melanoma were enrolled in the study. All leukapheresis procedures were performed by a continuous flow method (Spectra, Cobe BCT) with a standard MNC program., Results: An average of 11.7 L (range, 8-14 L) of whole blood was processed within 197.3 +/- 23.7 minutes, and a mean of 13.5 +/- 5.7 x 109 WBCs in a final volume of 191.0 +/- 24.2 mL was collected. The MNC purity in the apheresis component was 81.5 +/- 15.1 percent, from which 29.8 +/- 14.7 percent were monocytes. Thus, 11.0 +/- 5.0 x 109 MNCs and 3.2 +/- 2.0 x 109 monocytes were collected per procedure. Linear regression analysis revealed a high correlation between the absolute number of monocytes in peripheral blood before the apheresis procedure and the number of monocytes in the collected component (r=0.74, p < 0.0001). For the generation of DCs, 1.6 +/- 0.8 x 109 MNCs were plated into culture dishes; 3.2 +/- 1.8 percent of the cultured cells matured to DCs, which resulted in 56.5 +/- 49.4 x 106 DCs (range, 6.3-178) per patient for the complete vaccination series., Conclusion: A target dose of monocytes for the complete vaccination series could be obtained by a single convenient, safe, steady-state leukapheresis procedure in each patient without the need for G-CSF mobilization. The absolute number of monocytes in peripheral blood before the apheresis procedure is the best predictive variable for the yield of monocytes in the apheresis component.