Utility investigation of automated techniques in hematopoietic progenitor cell count and viability assessment in the Good Manufacturing Practice (GMP) settingg

Aim: To compare our parameters as regards: i) cell count via two different automated cell count techniques, and ii) viability via automated trypan blue exclusion and 7-aminoactinomycin D (7-AAD) staining. Method: We used the trypan blue exclusion technique and an automated cell counter and for viability testing, and the trypan blue exclusion technique and the 7-AAD evaluation by flow cytometry. The trypan blue exclusion and the radio frequency techniques were used for automated cell counting. Flow cytometric analysis was performed by evaluating the yielded cellular products for 7-AAD uptake during the cell count of CD34+ cells. Results: The mean values for cell count were estimated as 3.44±1.22x106/ml (range, 2.48-5.71x106/ml) and 4.14±1.94x106/ml (range, 1.77-7.43x106/ml) for the trypan blue exclusion and radio frequency techniques, respectively. Additionally, the mean values for viability analyses via the automated trypan blue exclusion and 7-AAD were 93.38±6.09% (range, 79.00-98.00%) and 99.49±0.60% (range, 98.40-100.00%), respectively. Conclusions: Our study has responded to two fundamental questions: whether the results of both of the automated techniques for cell count correspond with each other, and whether the results of the automated viability assessment conform those of the 7-AAD technique during the manufacturing processes of cellular therapy products intended for clinical use. Even though we have the opportunity to use the hemocytometer in our laboratory setting, the automated trypan blue exclusion technique gives cell count results in concordance within the range of the expectations of our Quality Management System (QMS)

Role of Killer Immunoglobulin-Like Receptor and Ligand Matching in Donor Selection

Despite all efforts to improve HLA typing and immunosuppression, it is still impossible to prevent severe graft versus host disease (GVHD) which can be fatal. GVHD is not always associated with graft versus malignancy and can prevent stem cell transplantation from reaching its goals. Overall T-cell alloreactivity is not the sole mechanism modulating the immune defense. Innate immune system has its own antigens, ligands, and mediators. The bridge between HLA and natural killer (NK) cell-mediated reactions is becoming better understood in the context of stem cell transplantation. Killer immunoglobulin-like receptors (KIRs) constitute a wide range of alleles/antigens segregated independently from the HLA alleles and classified into two major haplotypes which imprints the person's ability to suppress or to amplify T-cell alloreactivity. This paper will summarize the impact of both activating and inhibitory KIRs and their ligands on stem cell transplantation outcome. The ultimate goal is to develop algorithms based on KIR profiles to select donors with maximum antileukemic and minimum antihost effects

Flow Cytometric Aldehyde Dehydrogenase (ALDH) Assay Enables a Fast and Accurate Human Umblical Cord Blood Hematopoietic Stem Cell Assessment.

OBJECTIVE: Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method. MATERIALS AND METHODS: In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units. RESULTS: Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant. CONCLUSION: In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques

Donor-recipient killer immunoglobulin like receptor (KIR) genotype matching has a protective effect on chronic graft versus host disease and relapse incidence following HLA-identical sibling hematopoietic stem cell transplantation

Impact of donor-recipient killer immunoglobulin-like receptor (KIR) gene-gene matching on transplant outcomes is still inconclusive. Recent data suggest that killer cell immunoglobulin-like receptor (KIR) regulated natural killer cell (NK cell) activity may contribute to graft versus leukemia (GvL) effects and graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This case-control study aims to evaluate the effects of both aKIR and iKIR donor-recipient genotype matching on the outcomes of T cell replete HLA-identical sibling allo-HSCTs in a homogenous young patient population with myeloid leukemias. Five transplant outcomes including relapse rate (RR), disease-free survival (DFS), overall survival (OS), cumulative incidences of acute GvHD (aGvHD), and chronic GvHD (cGvHD) are investigated. Out of 96 HLA-identical sibling donor-recipient pairs, 34 were matched for activating KIR (aKIR), 38 for inhibitory KIR (iKIR), and 20 for both aKIR and iKIR. Fourty-four pairs were mismatched for both iKIR and aKIR. In univariate analysis, aKIR-matching resulted with a decrease in relapse rate (RR) (hazard ratio [HR]: 0.4; p = 0.04) and an increase in disease-free survival (DFS) (HR: 0.5; p = 0.03). In addition, cGvHD ocurred less frequently in the aKIR-matched (odds ratio [OR]: 0.4; p = 0.04) or iKIR-matched (OR: 0.3; p = 0.009) cohorts. Matching for both aKIR and iKIR was also associated with a decrease in cGvHD incidence (OR: 0.3; p = 0.02). iKIR-matching had no effects on RR, OS, or DFS. Analysis of donor haplotype effects showed haplotype-BB to have a tendency towards reduced relapse rate (HR: 0.4; p = 0.08) and better OS (HR: 0.4; p = 0.04); haplotype-Bx to increase the incidence of cGvHD (OR: 4.1; p = 0.03). In multivariate analysis, DFS advantage remained significant for aKIR-matching (HR: 0.5; p = 0.04); cGvHD incidence was reduced in the presence of iKIR-match (OR: 0.3; p = 0.02) and increased in the presence of haplotype-AB and -BB donors (OR: 7.9; p = 0.02; OR: 5.1; p = 0.03, respectively). In an attempt to investigate the pathogenesis underlying KIR-matching, we searched for residual NK/T cells on day 0 peripheral blood samples of six additional recipients and noted the presence of CD3(+) (7.0-91.4 x 10(6)/L) and CD56(+)57(+) (0.8-12.7 x 10(6)/L) cells. In conclusion, conditioning regimen surviving recipient NK/T cells potentially influenced by KIR-matching may contribute to GvL/GvH reactions

Flowcytometric evaluation of cell cycle regulators (cyclins and cyclin dependent kinase inhibitors) expressed on bone marrow cells of patients with chronic myelogenous leukemia and multiple myeloma

OBJECTIVE: Etiopathology of malignancy can be demonstrated by the comparison of the quantified changes in the different phases of the cycle about cyclins and cyclin dependent kinase inhibitors (CDKI) in healthy and malignant proliferated cells. The aim of this study is to analyze flow cytometric expression of cell cycle regulating elements in the malignant diseases with low and high proliferative signature.\ud METHODS: The levels of cyclin D, E, A, B and CDKI's p16, p21 were studied by flowcytometry in patients with chronic myeloid leukemia (CML) (n=16), multiple myeloma (MM) (n=13) and control subjects (n=15). \ud RESULTS: The distributions of the cell cycle S phase were 10, 63%, 6, 72% and 3, 59%; for CML, MM and control subjects, respectively. Among all the cyclins expressed during the S phase, cyclin D expression was the lowest, in CML patients. While the distribution of cyclins and CDKI’s was similar between MM and control groups in G2/M phase; cyclins expressions were parallel in all three phases in MM and chronic myeloid leukemia groups.\ud CONCLUSION: CML and MM are diseases presenting with variable degrees of proliferation. The increase of cyclins in cell cycle phases in patient group was not associated with the augmentation of the expression of CDKI’s. This finding may contribute the mechanisms effective in the etiopathogenesis of hematological malignancy