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Objectives: Trichomonas vaginalis ( TV) infection is the most common non-viral STI globally and can result in adverse pregnancy outcomes and exacerbated HIV acquisition/transmission. Nucleic acid amplification tests (NAATs) are the most sensitive diagnostic tests, with high specificity, but TV NAATs are rarely used in Brazil. We investigated the TV prevalence and compared the performance of the US Food and Drug Association-cleared Aptima TV assay with microscopy (wet mount and Gram-stained) and culture for TV detection in women in Pelotas, Brazil in an observational study., Methods: From August 2015 to December 2016, 499 consecutive asymptomatic and symptomatic sexually active women attending a Gynaecology and Obstetrics Outpatient Clinic were enrolled. Vaginal fluid and swab specimens were collected and wet mount microscopy, Gram-stained microscopy, culture and the Aptima TV assay performed., Results: The median age of enrolled women was 36.5 years (range: 15-77). The majority were white, had a steady sexual partner and low levels of education. The TV detection rate was 4.2%, 2.4%, 1.2% and 0% using the Aptima TV assay, culture, wet mount microscopy and Gram-stained microscopy, respectively. The sensitivity of culture and wet mount microscopy was only 57.1% (95% CI 36.5 to 75.5) and 28.6% (95% CI 13.8 to 50.0), respectively., Conclusions: A 4.2% positivity rate of T. vaginalis was found among women in Pelotas, Brazil and the routine diagnostic test (wet mount microscopy) and culture had low sensitivities. More sensitive diagnostic tests (NAATs) and enhanced testing of symptomatic and asymptomatic at-risk women are crucial to mitigate the transmission of TV infection, TV-associated sequelae and enhanced HIV acquisition and transmission., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The number of aldehyde dehydrogenase-bright (ALDH(br) ) cells has been suggested as a viable marker of hematopoietic stem cell function. We evaluated the suitability of ALDH(br) cell analysis in the quality assessment of postthaw cord blood (CB) units., Study Design and Methods: A total of 245 CB units were obtained for estimating the numbers of total nucleated cells (TNCs), CD34+ cells, ALDH(br) cells, ALDH(br) cells among CD34+ cells (CD34+ALDH(br) cells), CD34+ cells among ALDH(br) cells (ALDH(br) CD34+ cells), colony-forming unit (CFU)-granulocyte-macrophages (GMs), and CFU-granulocyte-erythrocyte-macrophage-megakaryocytes (GEMMs). Simple linear regression analysis was performed to assess the correlation between the number of TNCs and CD34+ cells before and after crypreservation and CD34+ALDH(br) cells, ALDH(br) cells, and ALDH(br) CD34+ cells after cryopreservation and the number of CFU-GEMMS and CFU-GMs., Results: The number of CFU-GMs was found to be significantly correlated with the number of CD34+ cells before and after cryopreservation (r = 0.418 and r = 0.359, respectively), CD34+ALDH(br) cells, ALDH(br) cells, and ALDH(br) CD34+ cells (r = 0.426, r = 0.455, and r = 0.469, respectively). The number of CFU-GEMMs was found to be significantly correlated with the number of TNCs and CD34+ cells before and after cryopreservation (TNCs, r = 0.251 and r = 0.250, respectively; CD34+ cells, r = 0.391 and r = 0.347, respectively), CD34+ALDH(br) cells, ALDH(br) cells, and ALDH(br) CD34+ cells (r = 0.297, r = 0.297, and r = 0.252, respectively)., Conclusion: The high correlation found between ALDH activity and CFU-GM number supports the suitability of ALDH analysis in the quality assessment of postthaw CB units., (© 2014 AABB.)

Background Aims: Wide acceptance of the colony-forming unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay with an eye towards identifying practices that could be standardized to improve overall reproducibility., Methods: A survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed., Results: There were 105 respondents to the survey, of whom 68% performed CFU assays. Most survey recipients specified that an automated rather than a manual cell count was performed on pre-diluted aliquots of stem cell products. Viability testing methods employed various stains, and when multiple sites used the same viability stain, the methods differed. Cell phenotype used to prepare working cell suspensions for inoculating the CFU assay differed among sites. Most respondents scored CFU assays at 14-16 days of incubation, but culture plates were read with various microscopes. Of 57 respondents, 42% had not performed a validation study or established assay linearity. Only 63% of laboratories had criteria for determining if a plate was overgrown with colonies., Conclusions: Survey results revealed inconsistent inter-laboratory practices for performing the CFU assay. The relatively low number of centers with validated CFU assays raises concerns about assay accuracy and emphasizes a need to establish central standards. The survey results shed light on numerous steps of the methodology that could be targeted for standardization across laboratories., (Copyright © 2013 International Society for Cellular Therapy. All rights reserved.)

Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133(+) cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained., (2009 FRAME.)

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.

Aims: The aim of the present report was to evaluate current methods of in vitro analysis of three-dimensional (3D) scaffolds seeded with human bone marrow stromal cells (hBMSCs) from six bone marrow aspirates for tissue engineered growth of bone., Methods: A series of experiments was conducted to compare methods of cell expansion and to validate analysis of proliferation and differentiation of hBMSCs in long term cultures of up to 40 days in 3D scaffolds of calcium carbonate (CaCO(3)) and mineralized collagen. Proliferation within the seeded scaffolds was monitored using cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), neutral red (NR) and DNA fluorescence assays and compared with empty controls. Differentiation was assessed by means of ELISA for osteocalcin (OC) and real time PCR for OC and collagen I (Coll I)., Results: The results showed that the scaffold differed in seeding efficacy (CaCO(3): 53.3%, min. Coll.: 83.3%). The precise identification of the number of cells in biomaterials by MTT, NR and DNA assays was problematic, as MTT and NR assay overestimated the number of cells, whereas DNA assay grossly underestimated the number of cells on the scaffolds. Monitoring of changes over time may be biased by unspecific material-dependent background activity that has to be taken into account. Identification of osteogenic differentiation is not reliable by identifying osteogenic markers such as OC in the supernatant but has to be done on the transcriptional level., Conclusions: It is concluded that monitoring of in vitro procedures for the construction of biohybrid scaffolds requires more emphasis in order to make the cell based approach a reliable treatment option in tissue engineering.

For more than a decade the 'neurosphere assay' has been used to define and measure neural stem cell (NSC) behavior, with similar assays now used in other organ systems and in cancer. We asked whether neurospheres are clonal structures whose diameter, number and composition accurately reflect the proliferation, self-renewal and multipotency of a single founding NSC. Using time-lapse video microscopy, coculture experiments with genetically labeled cells, and analysis of the volume of spheres, we observed that neurospheres are highly motile structures prone to fuse even under ostensibly 'clonal' culture conditions. Chimeric neurospheres were prevalent independent of ages, species and neural structures. Thus, the intrinsic dynamic of neurospheres, as conventionally assayed, introduces confounders. More accurate conditions (for example, plating a single cell per miniwell) will be crucial for assessing clonality, number and fate of stem cells. These cautions probably have implications for the use of 'cytospheres' as an assay in other organ systems and with other cell types, both normal and neoplastic.

The reliability of the assay of endogenous erythroid colony (EEC) formation in serum-free, cytokine-free collagen-based media was investigated in a multicentric study including 140 patients with polyglobuly (80 polycythemia vera (PV), 54 secondary erythrocytosis (SE), six idiopathic erythrocytosis (IE)) and 10 healthy donors. In each center, EEC assays were performed in parallel with progenitor cells from bone marrow (BM) and peripheral blood (PB); two commercialized media and 'low' and 'high' cell plating densities were tested. Negativity of EEC assays was considered certain only when sufficient BFU-E growth was obtained in control cultures with cytokines. In the two media, EEC formation was specific - never observed in cultures of healthy donors or SE patients - and comparable. BM EEC assays were positive (presence of eythroid colonies) for 75% ('low' plating) to 100% ('high' plating) of PV patients; PB EEC assays were positive for 83.3% ('low' plating) to 93.7% ('high' plating) of PV patients (differences not significant). Depending on the medium, 86.2-93.7% of patients with a positive BM EEC assay had a positive PB EEC assay. Hence, a standardized collagen-based EEC assay can be performed with either BM or PB progenitors; the EEC assay described here is positive for at least 75% of PV patients when a single EEC assay is performed, and for at least 94% of PV patients when both BM and PB EEC assays are performed.

Autologous peripheral blood stem cell (PBSC) transplantation has a low treatment-related morbidity and mortality when using appropriate criteria for patient selection and graft quality evaluation. It will be important to use simple and standardised procedures for evaluation of progenitor cell numbers when considering autografting in patients with malignant or non-malignant disorders and increased risk of prolonged posttransplant cytopenia. We determined the number of clonogenic cells in PBSC autografts after 7 days of in vitro culture, and these results were compared with both the total number of colonies and the numbers of colony subsets in conventional 14 days colony assays (colony-forming unit granulocyte-erythrocyte-macrophage-megakaryocyte, CFU-GEMM; CFU-E, CFU-GM; CFU-megakaryocyte). The total colony number after 7 days of culture correlated significantly with (i) the CD34+ cell number; (ii) the total colony number as well as the numbers of erythroid, nonerythroid and mixed colonies in a conventional assay using 14 days of culture; (iii) the number of megakaryocyte colonies. The total colony number after 7 days of in vitro culture is a simple in vitro parameter that seems to reflect the proliferative capacity of various progenitor subsets in PBSC autografts. This simple analysis may be used in combination with other in vitro techniques (e.g. estimation of stem cell viability and CD34+ cell subset analysis) for pretransplant evaluation of autografts. However, the possible clinical use of this parameter has to be examined in prospective clinical studies.

We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

The colony-forming unit (CFU) assay is exposed to a lot of variation, part of which is introduced by several enrichment strategies that are routinely performed before assessment of clonogenic capacity in mobilized peripheral blood (PB), bone marrow (BM), or cord blood (CB). We investigated the possibility to perform a single-step CFU assay by direct plating of PB, BM, or CB into CFU culture medium to obtain more reproducible results than after a standard Ficoll or lysis procedure. Direct plating implies the presence of red blood cells (RBC), white blood cells (WBC), and plasma in the CFU assay, which could possibly influence the outcome of the assay. Of all components, only the RBC was found to negatively influence CFU-GM growth if a concentration of > 0.02 x 10(9)/ml was present in the CFU culture medium. Subsequently, depending on the RBC concentration PB, BM, and CB samples were prediluted in triplicate or quadruplicate and plated into CFU medium. Lysis and/or Ficoll procedures were also performed in triplicate or quadruplicate on the same samples, and the mean colony number and coefficient of variation (CV) of the three techniques were compared. Significantly smaller CV values were found using the direct plating technique (all assays, mean 7.5%, range 1.6-15.6%) than after Ficoll separation (mean 18.0%, range 2.2-62.5%). Intermediate results were obtained with the lysis method (mean CV 11.6%, range 3.3-29%). In most samples, and especially in those with a very low number of clonogenic cells per milliliter, more colonies were detected with the direct plating method than with either the lysis or Ficoll method. In conclusion, the single-step direct plating method significantly enhances reproducibility of the CFU assay for PB, BM, and CB samples in comparison with standard techniques by circumvention of loss of colony formation and by decreasing variability. Furthermore, the direct plating technique is a timesaving assay.

Various chemical mixtures exist in soil contaminated with petrochemical wastes, yet no comprehensive assessment of their impact on terrestrial ecosystems has been conducted. The purpose of this study was to evaluate hematotoxicity risks to wild populations of cotton rats (Sigmodon hispidus) residing in habitats previously contaminated by petroleum industrial wastes. Resident cotton rats were monitored on nine contaminated sites and nine ecologically matched reference sites in Oklahoma. The possible toxicological interactions of petrochemical wastes on bone marrow was investigated by using the assay of colony formation of granulocyte-macrophage progenitor cells. There was a consistent significant 21 to 39% decrease in the number of colony-forming units of granulocyte-macrophage (CFU-GM) in cotton rats from petrochemical-contaminated sites compared to matched reference sites, with no marked changes in hematological or histopathological parameters. These results suggest that bone-marrow progenitor cell culture is a sensitive indicator for the assessment of ecotoxicity risks associated with petrochemical wastes that are generated by the oil refining industry. Long-term exposure to hazardous wastes associated with the petroleum industry may represent a subtle risk to the hematopoietic system in humans.

Bone marrow is extremely sensitive to toxicants, and in vitro culture of bone-marrow progenitor cells has been shown to be a sensitive indicator of bone-marrow injury in laboratory rodents. The ability of a bone-marrow progenitor cell assay to detect myelotoxicity in a wild rodent model (cotton rat, Sigmodon hispidus) that inhabits many contaminated ecosystems in the southern United States was examined. Responsiveness of progenitor cells to recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and cotton rat lung-conditioned medium (LCM) was determined to optimize culture conditions for cotton rats. Myelotoxicity was induced in cotton rats by treating animals with either cyclophosphamide (8 or 80 mg/kg) or dexamethasone (500 microg/kg) over a 5-d period. Administration of a high dose of cyclophosphamide caused nearly total suppression of colony formation of granulocyte-macrophage progenitor cells (CFU-GM). Marked histological changes in both the bone marrow and spleen were also observed in cotton rats treated with a high dose of cyclophosphamide. Although histological lesions were not apparent, the number of CFU-GM in the bone marrow of low-dose cyclophosphamide- and dexamethasone-treated cotton rats was significantly suppressed compared to controls. The number of CFU-GM was consistently higher using LCM than recombinant murine GM-CSF. This reproducible, quantitative, in vitro bone-marrow progenitor cell culture system was a sensitive indicator of myelotoxicity in wild cotton rats and should be useful for monitoring chronic exposures to low levels of environmental toxicants in wild rodent populations.

Cord blood has proved itself, if correctly stored with rational criteria, an excellent source of stem cells for related and unrelated transplants. It has been recently proven that the factor which predicts the best the speed of engraftment in cord blood transplants in the dose of progenitor cells injected per kg of body weight of the recipient. This result has been obtained thanks to a careful standardization of the neonatal progenitor cell assay. This manuscript describes such a standardization realized as a joined effort by the Istituto Superiore di Sanità, Rome, and the pivotal cord blood bank founded as a feasibility study by the National Institutes of Health, Bethesda at the New York Blood Center.

Background: The stroma-based long-term culture is the assay of choice when a functional detection of primitive hematopoietic cells in vitro is sought. However, different stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in published frequencies of LTCIC alternative CAFC., Methods: In order to identify the most suitable stromal source in terms of supportive capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma in secondary long-term culture set-ups., Results: Seeking an approximation to the supportive capacity of human BM stroma we found the FBMD-1 cell line supplemented with G-CSF and IL-3 superior to FBMD-1 cells alone, and to M2-10B4 and Sl/Sl cells. Moreover, in co-cultures of CD34(+) cells and the FBMD-1 line, we found week 5 CAFC content highly reproducible (50.5 +/- 6.66 - 54.6 +/- 7.07/10(4) plated cells, p value > 0.95) and the assay was suitable for inter-individual comparison in a clinical setting. In fact, the week 5 CAFC results were even more reproducible than those of the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU assays). On the other hand, when extending the culture period to 8 weeks, the cobblestone area formation was best maintained by human BM stroma and the high reproducibility in CAFC enumeration in cultures supported by the FBMD-1 was lost., Discussion: Among the stromal cell sources tested, the FBMD-1 line was found to be superior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.

Introduction: The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay., Materials and Methods: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo)., Results: In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients., Conclusion: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.

To investigate the hypothesis that commercial kits for CFU-GM (colony forming unit granulocyte-macrophage) assay will reduce the interlaboratory variation noted by many workers, we carried out a quality assurance exercise in 2 parts. There were 8 participants in the first study and each performed CFU-GM assays using their in-house method and a commercial kit (Stem Cell CFU Kit, Gibco) in parallel. In the second exercise there were 10 participants and each performed CFU-GM with in-house methods and with a different commercial medium (Methocult GF H4534, Stem Cell Technologies). Twelve samples of cryopreserved peripheral blood progenitor cells (PBPC) were analysed by each participant in each part of the study. A very wide range of results was found for the different in-house methods, but standardizing the clonogenic assay with the commercial kits did not reduce the variation seen. To improve the reproducibility of CFU-GM assays between laboratories, scrupulous attention should be paid to all the steps involved in the assays, as little progress will be made by using commercial medium in isolation from efforts to reduce other sources of variation.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay, an essential test in evaluation of the quality of autologous grafts of hematopoietic stem cells, has yet to be standardized. With this aim in view, we carried out a multicenter study of five commercially available culture kits for CFU-GM evaluation. Four kits were methylcellulose-based (H4431, H4434, H4435, StemBio1d) and one was collagen-based (EasyClone-Multi). Using fresh and frozen samples of PBSC grafts, we compared CFU-GM and burst-forming unit-erythrocytes (BFU-E) growth using the EasyClone kit to each of the methylcellulose kits. BFU-E and CFU-GM clonogenicity of both fresh and frozen PBSC was clearly inferior with the H4431 kit, which provides conditioned medium only. CFU-GM numbers obtained with fresh and frozen PBSC samples were significantly higher with the EasyClone kit than with the H4434 and StemBio kits. BFU-E numbers were also higher with the EasyClone kit, but only when colonies were scored after May-Grünwald-Giemsa (MGG) staining. Finally, although the H4435 kit provides higher doses of recombinant cytokines than the EasyClone kit, CFU-GM and BFU-E numbers obtained for fresh or frozen PBSC with both kits were similar. In addition, CFU-GM and BFU-E numbers correlated well with CD34+ cell numbers for all five kits for both fresh and frozen PBSC. In summary, our study shows that the EasyClone-Multi and H4435 kits provide the best CFU-GM growth. The collagen-based EasyClone kit has the additional advantage of allowing gel staining and storage, which facilitates colony identification and, more importantly, makes gel exchange possible for standardization of the CFU-GM assay.

The results for colony forming unit granulocyte-macrophage (CFU-GM) assays vary substantially between centres. It is possible that colony counting is largely responsible for this discrepancy. In order to examine this exclusively from the many factors that make up the CFU-GM assay, we performed a colony counting exercise involving 11 laboratories. Two-way analysis of variance showed a highly significant difference (P = 0.0001) in the counts obtained from the centres. One centre was found to score consistently high and two others scored consistently low numbers of colonies. This suggests that identification of colonies is a major source of variation between centres.

A serum-free method for the culture of megakaryocyte progenitor cells (burst-forming units megakaryocyte, BFU-Mk) from human bone marrow is described, using recombinant cytokines (interleukin[IL]-3, IL-6, IL-11 and stem cell factor [SCF]) to provide megakaryocyte colony-stimulating activity. IL-3 alone, but not IL-6, IL-11 or SCF alone, promoted BFU-Mk formation. The combination of IL-3 with IL-11 or with IL-6+SCF was necessary for optimal maturation of the megakaryocytes. No significant difference in BFU-Mk numbers was found between the combinations of IL-3+IL-11 and IL-3+IL-6+SCF. The development of BFU-Mk was found to be maximal at 12-14 d. Using these conditions, normal ranges for human bone marrow BFU-Mk were derived which may provide the basis for studying megakaryocyte colony growth in pathological conditions.

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to > 100 Mk, detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions, the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However, optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors, of which a combination of IL-3, IL-6, GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large "pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+, approximately half of which are CD41+. Thus, CFC-Mk are more similar to primitive clonogenic erythroid progenitors than to their granulopoietic counterparts in their expression of CD34, CD45RA and CD71. Taken together, these findings support the concept that some erythroid and Mk progenitors may share a common developmental pathway. The availability of sensitive and reproducible procedures for isolating and detecting human Mk progenitors should facilitate future investigations of their biology and role in a variety of haematological conditions.

Many new developments in tissue engineering rely on the culture of primary tissues which is composed of parenchymal and mesenchymal (stromal) cell populations. Because stroma regulates parenchymal function, the parenchymal:stromal cell (P:S) ratio will likely influence culture behavior. To investigate parenchymal-stromal cell interactions, the P:S ratio was systematically varied in a human bone marrow (BM) culture system, measuring the output of mature cells, immature progenitors (colony forming units-granulocyte/macrophage [CFU-GM]), and primitive stem cells (long-term culture-initiating cells [LTC-IC]). When parenchymal CD34-enriched cells were grown without stroma, cell and CFU-GM output increased linearly as inoculum density was increased, resulting in constant cell and CFU-GM expansion ratios. On irradiated preformed stroma (IPFS), culture output was significantly higher and less dependent on CD34-enriched cell inoculum density, resulting in greater expansion ratios at lower inoculum densities. The number of IPFS cells required to support CD34-enriched cells was independent of the CD34-enriched cell number, suggesting that IPFS did not provide discrete niches, but instead acted through soluble signals. Experiments using conditioned medium (CM) from IPFS confirmed the presence of soluble signals, but CM did not completely substitute for direct contact between CD34-enriched cells and IPFS. Because of known differences between IPFS and stroma growing within BM mononuclear cell (MNC) cultures, experiments were next performed using mixtures of CD34-enriched and CD34-depleted fractions of MNC. When inoculated with a fixed CD34+lin- cell number, culture output was optimal near the P:S ratio of the unmanipulated MNC sample and declined as CD34- cell number was increased or decreased. In cultures inoculated with a fixed total cell number, CFU-GM output increased as CD34+lin- cell number was increased, whereas LTC-IC output reached a plateau. These data suggest that a limited number of LTC-IC supportive niches were present in MNC stroma, whereas IPFS lacks these niches and acts predominantly through a less potent soluble mechanism. These studies underscore the importance of parenchymal-stromal cell interactions in the ex vivo reconstitution of tissue function and offer insight into the nature of these interactions in the human BM culture system.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay is used commonly to assess adequacy of progenitor number in bone marrow transplantation. The assay is poorly standardized, resulting in variability of results between and within laboratories. We assessed three variables that contribute to the lack of standardization. The colony-stimulating activity of human placental-conditioned medium (HPCM) was compared with combinations of recombinant hematopoietic growth factors (HGF) in 5 normal bone marrow donors. A protocol for batch testing of fetal calf serum (FCS) is described. In addition, a rigid training program has been introduced to minimize interstaff and intrastaff variability in the counting of colonies. We show that a five-factor combination of interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and stem cell factor (SCF) produces a mean increase of 85% in colony number. Some combinations of three HGF produce similar growth to HPCM, and all four HGF combinations are equivalent or superior to HPCM. Batch testing of FCS shows variability between batches. We show significant interstaff and intrastaff variability between a new and experienced staff member that improves following a period of training. In summary, the use of recombinant HGF in association with a rigorous program of batch testing of FCS and staff training results in a CFU-GM assay that can be standardized between laboratories.

We evaluated the efficacy of in vitro clonogenic assays for acute myeloid leukemia (AML) (CFU-Leuk) and granulocyte-macrophage progenitor cells derived from normal bone marrow (BM) (CFU-GM) to predict hematopoietic engraftment, median survival time (MST) and leukemia-free survival (LFS) in LBN rats that received injections of untreated or drug-treated AML and/or normal BM cells. Injection of untreated AML cells resulted in a log-linear relationship between AML cell dose and time of death from leukemia; LBN rats given 10(6) cells died with AML (MST, 24 days; range, 19-28) after injection. A minimum of 0.5-1.0 X 10(6) untreated normal BM cells was needed to insure satisfactory hematopoietic reconstitution in at least 50% of lethally irradiated LBN rats. After ex vivo incubation with graded concentrations of 4-hydroperoxycyclophosphamide (4HC) or bleomycin (BLEO), LBN AML or normal BM cells were cultured for CFU-Leuk or CFU-GM and injected into untreated or lethally irradiated syngeneic recipients. Over a variety of drug concentrations (4HC, 3-30 micrograms/ml; BLEO, 100-10,000 mU/ml) and cell doses (10(6)-10(7)/animal) examined, the log-kill estimates derived from in vitro CFU-Leuk assays correlated with the observed MST or LFS. Recovery of greater than 1% CFU-GM from 4HC- or BLEO-treated suspensions of normal BM was associated with satisfactory engraftment in lethally irradiated LBN rats. Clonogenic assays also predicted for engraftment and LFS in animals that received mixtures of AML and normal BM cells (1:10) treated with 4HC and/or BLEO. We conclude that CFU-Leuk and CFU-GM assays are useful screening techniques to develop and evaluate strategies for ex vivo purging with chemotherapeutic agents in this preclinical model of autologous marrow transplantation for AML.

A linear model for the errors of the 'spleen colony' assay for haemopoietic stem cells has been derived. The components emerging from the model are interpreted and practical recommendations given for interpreting measurements made with this assay. The model permits correction for the effect of overlapping colonies and gives average errors for single measurements of the number of CFU-s. More reliable and more precise information can be obtained using this model. The spleen colony technique detects a population of immature precursor cells designated as CFU-s (Till & McCulloch, 1961). The relative error of measurement is often large when compared with the changes in the phenomena studied. Consequently a better knowledge of the errors of this technique is highly desirable. This paper should be regarded as an extension of the previous analysis of Till (1972). The theory for the errors of the spleen colony technique was applied to 905 determinations of the CFU-s numbers performed on random-bred mice. Data from random-bred mice rather than those from inbred mice have been used because the error components can be expected to be larger and, consequently, more easily detectable. The model of errors has also been validated using data published by Till (1972) and has subsequently been applied to data from several inbred mice strains (Znojil & Necas, 1988).

Through our retrospective analysis of HTCA and clinical effects as well as fundamental studies, the following results were obtained: In this retrospective study, HTCA for lung cancer showed a predictive accuracy of 71%, a true positive rate of 50% and a true negative rate of 77%. To obtain good predictive accuracy, HTCA should be modified to provide conditions comparable to those in vivo with regard to drug concentration and drug exposure time. More precise analysis of the pharmacokinetics of anticancer agents might yield methodological improvement. A decrease in the chemosensitivity spectrum in vitro was observed after chemotherapy. This might be related to evidence that patients with prior chemotherapy exhibited a poor response rate to chemotherapy. There were no active anticancer agents against specimens with aplating efficiency of more than 0.04%. More extensive prospective trials will be necessary to determine the clinical value of HTCA. HTCA could be a superior assay for detecting the anti-tumor activity of new agents and a useful method for in vitro phase II study.

We report here the development of a rapid and quantitative method for measuring in vitro T cell transformation by human T cell leukemia viruses type I (HTLV-I) and type II (HTLV-II). This method is based on our finding that cocultivation of lethally irradiated HTLV-producing cells with peripheral blood lymphocytes (PBLs) preactivated for 24 hours with phytohemagglutinin and interleukin-2 (IL-2) induces colony formation in methylcellulose-containing medium. Colonies of about 200 cells can be clearly distinguished from background aggregates within four to six days after cocultivation. These colonies gradually increase in size and reach 300 to 1,000 cells within 14 days after cocultivation. Cells of these colonies were infected, as evidenced by expression of viral p19 antigen and the presence of HTLV proviral sequences. These cells proved to be transformed in terms of IL-2-independent continuous growth in liquid medium. Colony formation was found to depend in a linear fashion upon the percentage of the infected cells present in the irradiated cell population and is sufficiently sensitive to detect as few as 1% of virus-producing cells.

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation or transformation assays and is a necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.

An advantage of established tumor cell lines compared to fresh human tumor specimens used in sensitivity assessments is the possibility of repeated experiments. Ultimately a database of sensitivity profiles on a panel of cell lines can be made and the sensitivity to new drugs compared with historical data. A prerequisite of this strategy is a minimal interexperimental variation. The sensitivity of eight human small cell lung cancer cell lines to adriamycin, daunomycin, aclacinomycin A, and mitoxantrone was tested in the clonogenic assay. A covariation in the sensitivity to the drugs emphasized the importance of simultaneous drug testing on the same batch of cells. On one cell line (NCI-N592) the interexperimental variation was further evaluated and a significant correlation was found between preexposure culture conditions, size of S-phase, and sensitivity to adriamycin, daunomycin, and mitoxantrone. Rigorous standardization of the growth conditions prior to clonogenic assay reduced the variation in the sensitivity to adriamycin from a factor of five to only 10-15%. It is concluded that simultaneous experiments on the same batch of cells in drug comparisons should be used if possible. Specification and standardization of culture conditions are necessary in the comparison of drugs tested in different experiments. Inclusion of the same reference drug in all experiments may further increase the validity of comparisons in different experiments.