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The Number of Nucleated Cells Reflects the Hematopoietic Content of Umbilical Cord Blood for Transplantation

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The Number of Nucleated Cells Reflects the Hematopoietic Content of Umbilical Cord Blood for Transplantation Bone Marrow Transplantation, (1999) 24, 965–970 1999 Stockton Press All rights reserved 0268–3369/99 $15.00 http://www.stockton-press.co.uk/bmt The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation FTH Lim1,2, JM v Beckhoven3, A Brand3, JC Kluin-Nelemans1, JMH Hermans4, R Willemze1, HHH Kanhai2 and JHF Falkenburg1 Departments of 1Hematology, 2Obstetrics and 4Medical Statistics from the Leiden University Medical Center; and 3Red Cross Blood Bank, Leidsenhage, The Netherlands Summary: irradiated recipients. Since the colony-forming unit granulocyte–monocyte (CFU-GM) defined in a semi-solid A single umbilical cord blood (UCB) collection may con- culture assay, has been associated with hematopoietic tain sufficient hematopoietic stem cells to achieve engraftment of human bone marrow cells, this assay has engraftment and repopulation of the hematopoietic sys- been used as a quality control method for hematopoietic tem of children and adults after myeloablative therapy. stem cell grafts.12,13 The hematopoietic potential of a UCB unit is often The human hematopoietic stem cells reside in the frac- defined by the number of CD34+ cells or the number of tion of cells expressing the CD34 antigen.14,15 CD34+ cells colony-forming units as measured in semisolid hemato- can be identified by flow cytometry, but the various tech- poietic progenitor cell (HPC) cultures. However, these niques for CD34 enumeration that have been advocated assays are relatively difficult to standardize between may result in different estimated concentrations.16–18 Both UCB banks. The number of nucleated cells infused per the CD34+ cell content, and the numbers of hematopoietic kilogram body weight of the recipient is also reported progenitor cells (HPC) as determined in semi-solid medium to be a significant factor in the speed of recovery of culture assays are frequently used to predict the hematopoi- neutrophils and platelets after transplantation. To ana- etic potential of a stem cell transplant, and to estimate the lyze which parameters could be used to evaluate the viability of the stem cells after processing or cryopreserv- hematopoietic potential of a UCB graft, we evaluated ation. However, both the functional enumeration of HPC almost 300 UCB units that were collected for banking and the CD34+cells enumeration are difficult to standardize. for unrelated transplantation. A strong correlation was + The number of nucleated cells infused per kilogram of found between the frequencies of CD34 cells and the the recipient has been reported to be a significant factor HPC as measured in semi-solid medium cultures. From in the speed of recovery of neutrophils and platelets after the various leukocyte subpopulations, the concentration transplantation. In reports by Wagner et al19 on UCB trans- and total numbers of nucleated cells correlated best 20 + plants in 44 siblings, and Kurtzberg et al on 25 unrelated with both the HPC content and the number of CD34 UCB transplantations, a trend was found for the time to cells. Differentiation of these nucleated cells into subsets myeloid or platelet engraftment to decrease with the dose of leukocytes offered no advantage for better prediction + of clonogenic precursors or CD34 cells infused, but the of HPC or CD34+ cells. These results indicate that the correlations were not statistically significant. Gluckman et nucleated cell count probably reflects the hematopoietic al21 reported a significant correlation between the number potential of a UCB graft, and may for that reason corre- of nucleated cells per kg body weight infused and neutro- late with the speed of engraftment after transplantation. phil recovery. Recently Wagner et al22 showed a correlation Keywords: umbilical cord blood; transplantation; hema- + topoietic cells between the number of CD34 cells and the speed of recov- ery in 143 patients transplanted with unrelated UCB transplants. In this study, we evaluated the correlation between the Human umbilical cord blood (UCB) can be used as a source two methods of enumeration of HPC, and analyzed whether of hematopoietic stem cells for transplantation purposes.1 the nucleated cell counts or numbers of leukocyte subpopu- A single cord blood collection may contain enough hemato- lations correlated with the CD34+ cells or HPC content of poietic stem cells to achieve engraftment and repopulation UCB units. From the Cord Blood Bank of the Red Cross of the hematopoietic system of both children and adults.2–11 Blood Bank, Leidsenhage, which is part of the national Functionally, hematopoietic stem cells can be defined as umbilical cord blood bank EuroCord Nederland, we evalu- cells that are capable of lymphomyeloid reconstitution of ated data from the first 300 UCB units. We found that the number of nucleated cells correlated well with HPC and CD34 content of a UCB unit. Therefore we conclude that Correspondence: Dr JHF Falkenburg, Department of Hematology, Build- as long as the enumeration of CD34+ cells and cultured ing 1, C2-R, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands HPC from an UCB transplant are not standardized, the Received 24 November 1998; accepted 16 June 1999 number of nucleated cells can be used as an alternative to Hematopoietic content of umbilical cord blood FTH Lim et al 966 indicate the hematopoietic potential of UCB units for the gated CD14−/CD66e− population, the CD34+ cells could transplantation. be identified in a CD34 vs SSC dot plot (Figure 1). The sample that was stained with the mixture containing mouse- IgG1, was used as a negative control. In the calculation of + Materials and methods CD34 cells, the percentage of isotype control binding cells was subtracted from the percentage of CD34+ cells. Con- + Collection of umbilical cord blood centrations of CD34 cells and leukocyte subpopulations were calculated by multiplying the percentage with the After informed consent was obtained from the mother number of nucleated cells per ml UCB counted by the Sys- before delivery, UCB was collected from the umbilical cord mex. White blood cells (WBC) were defined as lympho- after the expulsion of the placenta. After puncture of the cytes, monocytes and granulocytes; mononuclear cells umbilical vein, the UCB was collected into a 250 ml collec- (MNC) were defined as lymphocytes and monocytes. tion bag containing 15 ml of Hanks’ balanced salt solution (BioWhittaker, Verviers, Belgium) with 75 IU/ml of pre- servative-free heparin as anticoagulant. Two samples of the Hematopoietic progenitor cell cultures UCB were sent to two different laboratories. The cell counts and phenotype were independently analyzed in one For the culture of HPC, UCB samples were diluted 10 times laboratory, while the HPC culture frequencies were defined in Iscove’s modified Dulbecco’s medium (IMDM) by the other laboratory. (BioWhittaker). Cells were seeded in three 35-mm tissue culture dishes with different concentrations of cord blood ␮ ␮ ␮ Cell counts and FACS analysis cells. Quantities of 10 l, 20 lor40 l of the diluted UCB were added to 1.5 ml of a semisolid culture-mixture Nucleated cells were counted using a Sysmex K1000 (Toa of IMDM, containing 30% fresh-frozen AB-heparin Medical Electronics, Kobe, Japan), which was validated in plasma, 0.5% deionized bovine serum albumin (BSA), quality rounds. human transferrin (0.47 g/l) (Behringwerke, Marburg, × −5 Phenotype analysis of umbilical cord blood was perfor- Germany) saturated with FeCl3-H2O, 5 10 m beta-mer- med within 36 h after harvest, using a Becton Dickinson captoethanol (Sigma Chemicals, St Louis, MO, USA), FACScan flow cytometer. For the determination of CD45 1.1% methylcellulose (Methocel 4000 cps; Fluka, Freiburg, and CD34 expressing cells, a method for whole blood coun- Germany), and the following human hematopoietic growth ting was used which was based on a protocol for CD34+ factors: recombinant granulocyte colony-stimulating factor cell enumeration with flow cytometry, described by the (G-CSF) 10 ng/ml, kindly provided by Amgen (Thousand national workshop SIHON.16 Oaks, CA, USA); granulocyte–macrophage colony-stimul- Three samples of 100 ␮l blood were labeled with combi- ating factor (GM-CSF) 10 ng/ml, Sandoz (Basel, nations of directly fluorescein isothiocyanate (FITC)- or Switzerland); stem cell factor (SCF) 50 ng/ml, kindly pro- phycoerythin (PE)-labeled monoclonal antibodies: CD45 vided by Amgen; interleukin-3 (IL-3) 25 ng/ml, a gift from FITC + CD14 PE for leukocyte subsets, CD14 Sandoz; and human recombinant erythropoietin (Epo) 2 FITC/CD66e FITC + CD34 PE for CD34+ cell assessment, U/ml, a kind gift from Organon Technica (Turnhout, and CD14 FITC/CD66e FITC + mouse-IgG1 PE for isotype Belgium). After 14 days of culture in 35 mm tissue culture ° control. The CD45 FITC/CD34 PE combination, CD14 dishes (37 C, fully humidified atmosphere, 5% CO2), col- FITC, mouse-IgG1 PE and CD34 PE were from Becton onies were counted. Colonies were defined as aggregates Dickinson (San Jose, CA, USA); CD66e FITC, was of more than 50 cells. Erythroid burst-forming unit (BFU- obtained from the Central Laboratory of the Blood Trans- E) colonies were defined as bursts of colonies consisting fusion Services (Amsterdam, The Netherlands). Cellquest of hemoglobinized cells. Granulocyte–macrophage colony- software was used to determine percentages lymphocytes, forming unit (CFU-GM) colonies contained granulocytes or monocytes, granulocytes and CD34+ cells. Briefly, LDS macrophages or both. Granulocyte–erythroid–macrophage– 751 (Exciton, Daton, OH, USA)23 was used to set a live megakaryocyte CFU (CFU-GEMM) colonies were defined gate, discriminating between damaged and intact nucleated as aggregates containing at least both erythroid and myeloid cells (LDS 751+ cells). Within this live gate, 50000 events cells. HPC was defined as the sum of BFU-E, CFU-GM and were analyzed, and a distinction between subpopulations of CFU-GEMM. From the three samples plated in different CD45+ leukocytes was made; CD45− nucleated cells were concentrations, the concentrations of HPC/ml UCB were considered nucleated red blood cells.
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