A Single-Step Colony-Forming Unit Assay for Unseparated Mobilized Peripheral Blood, Cord Blood, and Bone Marrow

Abstract

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.

Red blood cell depletion with a semiautomated system or hydroxyethyl starch sedimentation for routine cord blood banking: a comparative study

“…We investigated the causes of this difference, and we found a negative influence of RBC content in CB sample before cryopreservation on CFU counts. Other authors, also with a direct plating method, have found the negative influence of RBC content on CFU–GM growth if a concentration of greater than 0.02 × 10 9 per mL was present in the CFU medium 24 . We have not determined the concentration of RBCs in medium, and we have no explanation for these data.…”

“…Other authors, also with a direct plating method, have found the negative influence of RBC content on CFU-GM growth if a concentration of greater than 0.02 ¥ 10 9 per mL was present in the CFU medium. 24 We have not determined the concentration of RBCs in medium, and we have no explanation for these data. A possible influence of HES in protecting HPCs during the cryopreservation process could explain these results.…”

Stem Cell-Like Human Endothelial Progenitors Show Enhanced Colony-Forming Capacity After Brief Sevoflurane Exposure: Preconditioning of Angiogenic Cells by Volatile Anesthetics

“…2B). 19 Human umbilical cord blood (55-95 mL) was collected from placentas immediately after cesarean delivery, and MNCs were isolated, as described earlier (Fig. 2B).…”

Development of a novel assay to evaluate the functional potential of umbilical cord blood progenitors

“…Thus, when comparing a UCB product with high ATP and low CFC numbers to a UCB unit with low ATP and high CFC numbers, one might interpret this to mean that progenitors are more immature in the former than in the latter UCB unit. Although there are reports showing some level of success in minimizing the magnitude of variability associated with the CFC assay, [20][21][22][23] it has been difficult to standardize this method among laboratories for the purpose of measuring the functional potential of progenitors contained within UCB. This is probably because there are a number of possible sources of error contributing to assay variability, which include the use of different cytokines by different laboratories, sample preparation, the plating of cells into methylcellulose, and the subjective interpretation of the colony number and type as the final readout for the CFC assay.…”