Australasian CD34+ quality assurance program and rationale for the clinical utility of the single-platform method for CD34+ cell enumeration

BACKGROUND: Enumeration of CD34(+) cells should be accurate and comparable between institutions, particularly when making clinical decisions, evaluating data, and in clinical trials. An Australasian CD34(+) quality assurance program (QAP) has been established to compare CD34(+) cell results and method (Part 1). Unexpected variation in WBCCs led to Part 2 of this report. METHODS: Part 1: Methods reagents and results were evaluated for 12 QAP samples analyzed by 36-43 centers. Part 2: The effects of different anticoagulants on WBCC of 12 peripheral blood samples (PBs) were compared using three cell counters. To test the validity of applying the conclusions to clinical samples, the WBCCs of leukapheresed products and BM harvest were also compared. RESULTS: Part 1: In some samples, WBCCs determined by certain cell-counter groups were significantly different. Results for percentage of CD34(+) and CD34(+)/microL suggest that standardization on the lyse-no-wash and single platform (SP) method reduces variation of results between institutions. Part 2: Using different counters, PB WBCC in ACD-A showed greater variation than the same PB in EDTA. For PB in different anticoagulants, the extent of difference in WBCC for the same PB is dependent on the counter used. DISCUSSION: This CD34 QAP has identified ACD-A as an additional factor that contributes to the disparate WBCCs, which may further compromise the accuracy of CD34(+) cell counts obtained by the dual platform (DP) method, especially for leukapheresed products. In order to achieve greater accuracy within individual institutions, as well as permitting more reliable inter-institutional comparisons, our data supports the adoption of the SP as the standard method for CD34(+) cell enumeration.     

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units

Background aimsIn 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards.MethodsTwelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures.ResultsExcellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells.ConclusionsThe current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.     

Aldehyde dehydrogenase as an alternative to enumeration of total and viable CD34(+) cells in autologous hematopoietic progenitor cell transplantation

We validated the correlation of aldehyde dehydrogenase ALDH(br) cells with total and viable CD34(+) cells in fresh and thawed hematopoietic progenitor cell (HPC) products, and looked for a correlation with time to white blood cell (WBC) and platelet engraftment after autologous transplantation, using simple linear regression analyzes. We found a significant correlation between pre-freeze ALDH(br) cell numbers and pre-freeze total CD34(+) (P < 0.001), viable CD34(+) (P < 0.001) and post-thaw viable CD34(+) (P < 0.001) cell numbers. We suggest that ALDH(br) may be substituted for CD34(+) cell numbers when evaluating HPC. As post-thaw viability testing apparently adds no significant information, we suggest that it may not be necessary. Finally, neither marker correlated with time to engraftment in our patients, supporting previous data suggesting the existence of a threshold dose for timely engraftment around 2.5 × 10(6) cells/kg.     

Frataxin-mediated iron delivery to ferrochelatase in the final step of heme biosynthesis

Human ferrochelatase, a mitochondrial membrane-associated protein, catalyzes the terminal step of heme biosynthesis by insertion of ferrous iron into protoporphyrin IX. The recently solved x-ray structure of human ferrochelatase identifies a potential binding site for an iron donor protein on the matrix side of the homodimer. Herein we demonstrate Hs holofrataxin to be a high affinity iron binding partner for Hs ferrochelatase that is capable of both delivering iron to ferrochelatase and mediating the terminal step in mitochondrial heme biosynthesis. A general regulatory mechanism for mitochondrial iron metabolism is described that defines frataxin involvement in both heme and iron-sulfur cluster biosyntheses. In essence, the distinct binding affinities of holofrataxin to the target proteins, ferrochelatase (heme synthesis) and ISU (iron-sulfur cluster synthesis), allows discrimination between the two major iron-dependent pathways and facilitates targeted heme biosynthesis following down-regulation of frataxin.     

胃癌及相应癌旁组织中VEGF、CD34、CD44v6的检测及临床意义

目的:探讨血管内皮生长因子(Vascular endothelial growth factor,VEGF)、CD34和CD44v6在胃癌及相应癌旁组织中的表达及其与临床病理意义.方法:应用免疫组化技术检测60例胃癌以及相应癌旁组织中VEGF、CD34和CD44v6的表达.结果:胃癌组织VEGF阳性率为71.67%(43/60)明显低于癌旁组织88.34%(53/60),两组间有显著性差异(P=0.022);VEGF阳性表达与胃癌病理分级、浸润深度、淋巴结转移和临床分期密切相关(P＜0.05).癌旁组织中微血管密度(MVD)明显高于癌组织MVD(P=0.000),有淋巴结转移癌组织中MVD高于无淋巴结转移者(P=0.043),并随浸润深度、临床分期MVD升高(P=0.046,P=0.000).癌旁组织中CD44v6阳性率为48.34%(29/60)低于癌组织的61.67%(37/60),两者无明显差别.CD44v6的阳性率随胃癌浸润的加深而升高,与淋巴结转移和TNM分期呈正相关.VEGF、CD34及CD44v6三指标间两两相关(P＜0.05).结论:VEGF、CD34和CD44v6三者联合检测有助于判断胃癌的浸润、转移及预后情况.     

VEGF、CD34、VEGF—C和VEGFR-3在胃癌中的表达及临床意义

目的：探讨胃癌组织中VEGF、CD34、VEGF-C和VEGFR-3的表达情况及临床意义。方法：采用免疫组化方法测定81例胃癌组织VEGF、CD34、VEGF—C和VEGFR-3表达情况,并结合患者的临床病理资料进行分析。结果：81例胃癌组织中MVD平均值为（42.95±14．79）个／视野,范围为13．00-68．33个／视野,VEGF、VEGF—C、VEGFR-3阳性表达率分别为74．1％、64．2％、67．9％。VEGF的表达与肿瘤的TNM分期、浸润深度、淋巴结转移有关,CD34的表达与肿瘤的分化程度、TNM分期、浸润深度、淋巴结转移有关,VEGF—C的表达与肿瘤的分化程度、浸润深度、淋巴结转移有关,VEGFR-3的表达与肿瘤的浸润深度、淋巴结转移有关。结论：VEGF、CD34、VEGF—C和VEGFR-3的表达与胃癌的浸润转移密切相关。     

VEGF、CD34、VEGF-C和VEGFR-3在胃癌中的表达及临床意义

目的：探讨胃癌组织中VEGF、CD34、VEGF-C和VEGFR-3的表达情况及临床意义。方法：采用免疫组化方法测定81例胃癌组织VEGF、CD34、VEGF—C和VEGFR-3表达情况,并结合患者的临床病理资料进行分析。结果：81例胃癌组织中MVD平均值为（42.95&#177;14．79）个／视野,范围为13．00-68．33个／视野,VEGF、VEGF—C、VEGFR-3阳性表达率分别为74．1％、64．2％、67．9％。VEGF的表达与肿瘤的TNM分期、浸润深度、淋巴结转移有关,CD34的表达与肿瘤的分化程度、TNM分期、浸润深度、淋巴结转移有关,VEGF—C的表达与肿瘤的分化程度、浸润深度、淋巴结转移有关,VEGFR-3的表达与肿瘤的浸润深度、淋巴结转移有关。结论：VEGF、CD34、VEGF—C和VEGFR-3的表达与胃癌的浸润转移密切相关。     

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.     

Maximum likelihood estimation of agreement in the constant predictive probability model, and its relation to Cohen's kappa

The alpha agreement parameter is defined as the proportion of a population of items that are classified identically "for cause" by two classifiers, the remaining items being classified at random. The parameters of the corresponding constant predictive probability model are shown to be estimable by the method of maximum likelihood, and a simulation study indicates applicability of the asymptotic results to finite samples. The new estimator tends to be larger than Cohen's kappa, except in the case of uniform margins. An application is made to the validity of cancer risk items included in a cancer registry.     

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38- umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38- cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38- cells.     

Expression of lumican related to CD34 and VEGF in the articular disc of the human temporomandibular joint

Lumican belongs to the small leucine-rich repeat proteoglycan (SLRP) gene family and has been reported to exist in the cornea, intervertebral disc and tendon. Lumican plays a significant role in the assembly and regulation of collagen fibres. The human temporomandibular joint (TMJ) disc is made up of fibrocartilage with an extracellular matrix (ECM) composed of collagen and proteoglycans. The existence and behaviour of lumican have not been studied in the human TMJ disc. Therefore, we used immunohistochemical methods to detect lumican, CD34 and vascular endothelial growth factor (VEGF) and histochemical staining with toluidine blue in 13 human TMJ specimens (10 surgically removed and 3 obtained from autopsy). In both normal and deformed discs we observed staining with toluidine blue. We found that the area of metachromasia inside the deformed disc was uneven and expression of lumican was strong in the areas negative for metachromasia. Staining of VEGF and CD34 inside the deformed disc was seen. We confirmed the expression of lumican in the human TMJ disc and showed that a large number of fibroblast-like cells existed in the area of strong lumican expression. These new findings about the behaviour of lumican suggest that it may play a key role in the generation of a new collagen network by fibroblast-like cells.Key words: TMJ disc, lumican, CD34, VEGF, immunohistochemistry, metachromasia.