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 Lim 1,2, JM v Beckhoven 3, A Brand 3, JC Kluin-Nelemans 1, JMH Hermans 4, R Willemze 1, HHH Kanhai 2 and JHF Falkenburg 1 Departments of 1 Hematology, 2 Obstetrics and 4 Medical Statistics from the Leiden University Medical Center; and 3 Red Cross Blood Bank, Leidsenhage, The Netherlands Summary: A single umbilical cord blood (UCB) collection may contain sufficient hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of children and adults after myeloablative therapy. The hematopoietic potential of a UCB unit is often defined by the number of CD34 + cells or the number of colony-forming units as measured in semisolid hematopoietic progenitor cell (HPC) cultures. However, these assays are relatively difficult to standardize between UCB banks. The number of nucleated cells infused per kilogram body weight of the recipient is also reported to be a significant factor in the speed of recovery of neutrophils and platelets after transplantation. To analyze which parameters could be used to evaluate the hematopoietic potential of a UCB graft, we evaluated almost 300 UCB units that were collected for banking for unrelated transplantation. A strong correlation was found between the frequencies of CD34 + cells and the HPC as measured in semi-solid medium cultures. From the various leukocyte subpopulations, the concentration and total numbers of nucleated cells correlated best with both the HPC content and the number of CD34 + cells. Differentiation of these nucleated cells into subsets of leukocytes offered no advantage for better prediction of HPC or CD34 + cells. These results indicate that the nucleated cell count probably reflects the hematopoietic potential of a UCB graft, and may for that reason correlate with the speed of engraftment after transplantation. Keywords: umbilical cord blood; transplantation; hematopoietic cells Human umbilical cord blood (UCB) can be used as a source of hematopoietic stem cells for transplantation purposes. 1 A single cord blood collection may contain enough hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of both children and adults. 2 11 Functionally, hematopoietic stem cells can be defined as cells that are capable of lymphomyeloid reconstitution of Correspondence: Dr JHF Falkenburg, Department of Hematology, Building 1, C2-R, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands Received 24 November 1998; accepted 16 June 1999 irradiated recipients. Since the colony-forming unit granulocyte monocyte (CFU-GM) defined in a semi-solid culture assay, has been associated with hematopoietic engraftment of human bone marrow cells, this assay has been used as a quality control method for hematopoietic stem cell grafts. 12,13 The human hematopoietic stem cells reside in the fraction of cells expressing the CD34 antigen. 14,15 CD34 + cells can be identified by flow cytometry, but the various techniques for CD34 enumeration that have been advocated may result in different estimated concentrations. 16 18 Both the CD34 + cell content, and the numbers of hematopoietic progenitor cells (HPC) as determined in semi-solid medium culture assays are frequently used to predict the hematopoietic potential of a stem cell transplant, and to estimate the viability of the stem cells after processing or cryopreservation. However, both the functional enumeration of HPC and the CD34 + cells enumeration are difficult to standardize. The number of nucleated cells infused per kilogram of the recipient has been reported to be a significant factor in the speed of recovery of neutrophils and platelets after transplantation. In reports by Wagner et al 19 on UCB transplants in 44 siblings, and Kurtzberg et al 20 on 25 unrelated UCB transplantations, a trend was found for the time to myeloid or platelet engraftment to decrease with the dose of clonogenic precursors or CD34 + cells infused, but the correlations were not statistically significant. Gluckman et al 21 reported a significant correlation between the number of nucleated cells per kg body weight infused and neutrophil recovery. Recently Wagner et al 22 showed a correlation between the number of CD34 + cells and the speed of recovery in 143 patients transplanted with unrelated UCB transplants. In this study, we evaluated the correlation between the two methods of enumeration of HPC, and analyzed whether the nucleated cell counts or numbers of leukocyte subpopulations correlated with the CD34 + cells or HPC content of UCB units. From the Cord Blood Bank of the Red Cross Blood Bank, Leidsenhage, which is part of the national umbilical cord blood bank EuroCord Nederland, we evaluated 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 as long as the enumeration of CD34 + cells and cultured HPC from an UCB transplant are not standardized, the number of nucleated cells can be used as an alternative to

966 Hematopoietic content of umbilical cord blood indicate the hematopoietic potential of UCB units for transplantation. Materials and methods Collection of umbilical cord blood After informed consent was obtained from the mother before delivery, UCB was collected from the umbilical cord after the expulsion of the placenta. After puncture of the umbilical vein, the UCB was collected into a 250 ml collection bag containing 15 ml of Hanks balanced salt solution (BioWhittaker, Verviers, Belgium) with 75 IU/ml of preservative-free heparin as anticoagulant. Two samples of the UCB were sent to two different laboratories. The cell counts and phenotype were independently analyzed in one laboratory, while the HPC culture frequencies were defined by the other laboratory. Cell counts and FACS analysis Nucleated cells were counted using a Sysmex K1000 (Toa Medical Electronics, Kobe, Japan), which was validated in quality rounds. Phenotype analysis of umbilical cord blood was performed within 36 h after harvest, using a Becton Dickinson FACScan flow cytometer. For the determination of CD45 and CD34 expressing cells, a method for whole blood counting was used which was based on a protocol for CD34 + cell enumeration with flow cytometry, described by the national workshop SIHON. 16 Three samples of 100 l blood were labeled with combinations of directly fluorescein isothiocyanate (FITC)- or phycoerythin (PE)-labeled monoclonal antibodies: CD45 FITC + CD14 PE for leukocyte subsets, CD14 FITC/CD66e FITC + CD34 PE for CD34 + cell assessment, and CD14 FITC/CD66e FITC + mouse-igg1 PE for isotype control. The CD45 FITC/CD34 PE combination, CD14 FITC, mouse-igg1 PE and CD34 PE were from Becton Dickinson (San Jose, CA, USA); CD66e FITC, was obtained from the Central Laboratory of the Blood Transfusion Services (Amsterdam, The Netherlands). Cellquest software was used to determine percentages lymphocytes, monocytes, granulocytes and CD34 + cells. Briefly, LDS 751 (Exciton, Daton, OH, USA) 23 was used to set a live gate, discriminating between damaged and intact nucleated cells (LDS 751 + cells). Within this live gate, 50000 events were analyzed, and a distinction between subpopulations of CD45 + leukocytes was made; CD45 nucleated cells were considered nucleated red blood cells. First, monocytes were defined as CD45 + and CD14 +, and granulocytes and lymphocytes as CD45 + and CD14. These CD45 + /CD14 cells were gated and then plotted in a forward sideward light scatter pattern, which showed two subpopulations, granulocytes with a high sideward light scatter (SSC) pattern, and lymphocytes with a low SSC. In the samples that were stained with CD14 FITC/CD66e FITC + CD34 PE, an FL histogram gate was set on the population that was negative for CD14 and CD66e, to exclude mature myeloid (CD66e + ) and monocytic cells (CD14 + ) from further analysis. From the gated CD14 /CD66e population, the CD34 + cells could 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 CD34 + cells, the percentage of isotype control binding cells was subtracted from the percentage of CD34 + cells. Concentrations of CD34 + cells and leukocyte subpopulations were calculated by multiplying the percentage with the number of nucleated cells per ml UCB counted by the Sysmex. White blood cells (WBC) were defined as lymphocytes, monocytes and granulocytes; mononuclear cells (MNC) were defined as lymphocytes and monocytes. Hematopoietic progenitor cell cultures For the culture of HPC, UCB samples were diluted 10 times in Iscove s modified Dulbecco s medium (IMDM) (BioWhittaker). Cells were seeded in three 35-mm tissue culture dishes with different concentrations of cord blood cells. Quantities of 10 l, 20 l or40 l of the diluted UCB were added to 1.5 ml of a semisolid culture-mixture of IMDM, containing 30% fresh-frozen AB-heparin plasma, 0.5% deionized bovine serum albumin (BSA), human transferrin (0.47 g/l) (Behringwerke, Marburg, Germany) saturated with FeCl 3 -H 2 O, 5 10 5 m beta-mercaptoethanol (Sigma Chemicals, St Louis, MO, USA), 1.1% methylcellulose (Methocel 4000 cps; Fluka, Freiburg, Germany), and the following human hematopoietic growth factors: recombinant granulocyte colony-stimulating factor (G-CSF) 10 ng/ml, kindly provided by Amgen (Thousand Oaks, CA, USA); granulocyte macrophage colony-stimulating factor (GM-CSF) 10 ng/ml, Sandoz (Basel, Switzerland); stem cell factor (SCF) 50 ng/ml, kindly provided by Amgen; interleukin-3 (IL-3) 25 ng/ml, a gift from Sandoz; and human recombinant erythropoietin (Epo) 2 U/ml, a kind gift from Organon Technica (Turnhout, Belgium). After 14 days of culture in 35 mm tissue culture dishes (37 C, fully humidified atmosphere, 5% CO 2 ), colonies were counted. Colonies were defined as aggregates of more than 50 cells. Erythroid burst-forming unit (BFU- E) colonies were defined as bursts of colonies consisting of hemoglobinized cells. Granulocyte macrophage colonyforming unit (CFU-GM) colonies contained granulocytes or macrophages or both. Granulocyte erythroid macrophage megakaryocyte CFU (CFU-GEMM) colonies were defined as aggregates containing at least both erythroid and myeloid cells. HPC was defined as the sum of BFU-E, CFU-GM and CFU-GEMM. From the three samples plated in different concentrations, the concentrations of HPC/ml UCB were calculated. The mean of the three results was used to calculate the concentration of HPC for each UCB unit. Statistics Correlations were evaluated with Spearman correlation coefficients. Comparison of two independent correlation coefficients was done with Fisher s Z-test. Comparison of test results of two groups was done with the Mann Whitney test.

Hematopoietic content of umbilical cord blood 967 Figure 1 Flow cytometric enumeration of CD34 + cells in UCB. (a) Forward-sideward scatter pattern. (b) Labeling with CD45 FITC and CD14 PE for leukocyte subsets. (c) Labeling with CD14 FITC/CD66e FITC and CD34 PE, FL histogram gate on the population that is negative for CD14 (monocytic cells) and CD66e (mature myeloid cells). (d) Identification of CD34 + cells in the CD14 /CD66e population. Results Only UCB collections that exceeded 50 ml were accepted as UCB units for transplantation and entered in this evaluation. Table 1 shows the volumes, cell counts, CD34 + cell frequencies and HPC content of the 295 UCB units that were evaluated, expressed as concentration as well as total numbers per unit harvested. Figure 2 illustrates the significant correlation between the number of CD34 + cells and the number of HPC in the UCB units. The correlation of r = 0.68 appeared to be influenced by subjective interpretation of scoring of colonies in the HPC culture by different technicians. Therefore, we compared test results from the two technicians who performed most of the HPC culture assays, 104 and 105 UCB units, respectively. The HPC cultures from the remaining 75 UCB units were scored by eight other technicians. The results indicate that there were significant interpersonal differences in scoring HPC expressed in relation to CD34 counts. The correlation coefficient for the first technician (r = 0.84) differed significantly from the correlation coefficient for the second technician (r = 0.63) (P 0.001). The CD34 content of the units analyzed by both technicians were similar, 3.30 10 6 and 3.36 10 6 CD34 + cells, respectively. How- Table 1 Volumes and cell counts of 295 UCB units Median Range Mean s.d. Volume (ml) 87 50 227 92.5 27.8 Nucleated cells concentration ( 10 6 /ml) 12.1 5.1 33.1 12.6 4.2 total numbers a ( 10 9 ) 1.60 0.2 5.1 1.20 0.62 White blood cells concentration ( 10 6 /ml) 11.1 4.8 32.0 11.6 3.8 total numbers ( 10 9 ) 0.95 0.2 4.9 1.09 0.55 Mononuclear cells concentration ( 10 6 /ml) 5.2 0.9 20.8 5.5 2.0 total numbers ( 10 9 ) 0.45 0.07 3.2 0.51 0.29 CD34 + cells concentration ( 10 3 /ml) 37.0 0 264.5 45.4 36.0 total numbers ( 10 6 ) 3.32 0 36.8 4.40 4.12 HPC concentration ( 10 3 /ml) 15.4 0.7 87.0 20.3 16.3 total numbers ( 10 6 ) 1.37 0.07 14.8 1.94 1.91 BFU-E concentration ( 10 3 /ml) 5.7 0.1 50.5 7.7 7.6 total numbers ( 10 6 ) 0.49 0.01 7.0 0.75 0.86 CFU-GM concentration ( 10 3 /ml) 6.4 0.1 43.0 8.8 7.2 total numbers ( 10 6 ) 0.56 0.01 6.4 0.84 0.83 CFU-GEMM concentration ( 10 3 /ml) 2.5 0 23.6 3.8 3.8 total numbers ( 10 6 ) 0.24 0 3.0 0.36 0.42 a Total numbers per unit harvested.

Hematopoietic content of umbilical cord blood 968 Figure 2 Correlation between the total numbers of CD34 + cells and HPC. = technician 1, = technician 2, = other technicians. Correlation for all technicians: n = 295, r = 0.68, P 0.01; for technician 1: n = 104, r = 0.84, P 0.01; for technician 2: n = 105, r = 0.63, P 0.01. ever technician 1 scored significantly lower numbers of HPC (median number of 1.33 10 6 HPC) than technician 2 (median of 1.66 10 6 HPC) (P = 0.014). This illustrates that the HPC culture assay is partly dependent on a subjective component. In Table 2, the correlations of the frequencies and total numbers of nucleated cells, leukocyte subpopulations with CD34 + cells and cultured HPC in UCB were evaluated. The best correlation was found between the total numbers of CD34 + cells and the total numbers of nucleated cells, as illustrated in Figure 3. Discrimination of the leukocytes into subpopulations including mononuclear cells, monocytes, lymphocytes or granulocytes did not improve the correlation. Table 2 Correlations between hematopoietic progenitor cells and leukocyte subpopulations n = 295 As concentration As total numbers Subsets CD34 + HPC CD34 + HPC cells cells CD34 + 0.62 0.68 Nucleated cells 0.61 0.50 0.71 0.60 White blood cells 0.55 0.44 0.68 0.57 Mononuclear cells 0.46 0.31 0.64 0.48 Lymphocytes 0.39 0.25 0.60 0.44 Monocytes 0.45 0.31 0.60 0.47 Granulocytes 0.40 0.35 0.55 0.49 Figure 3 Correlation between the total numbers of CD34 + cells and nucleated cells (NC) in individual UCB units. n = 295, r = 0.71, P 0.01. Discussion The use of UCB as a source of hematopoietic stem cells for transplantation, is limited by the volume of cord blood that can be harvested. Therefore the estimation of the hematopoietic potential of a UCB unit to be used for transplantation is critical for deciding which graft should be used for transplantation if more than one suitable unit is available. 24,25 In this study, we evaluated whether a correlation could be found between leukocyte subset cell counts and the two most commonly used methods of enumeration of progenitor cells, namely flow cytometric counts of CD34 + cells and culture of HPC. These parameters are considered to indicate the hematopoietic cell content, although none of these parameters indicate long-term repopulation. The cobblestone area forming cell assay (CAFC) 26 and the long-term culture initiating cell assay (LTC-IC) 27 may be methods to predict more accurately the frequency of stem cells but they are time and skilled man-power consuming, and therefore less suitable for standard tests for routine use in cord blood banking. As shown in Table 2, the correlation between the frequencies of CD34 + cells and HPC in semi-solid medium cultures was 0.68. In scoring of HPC in semi-solid medium cultures, however, subjective factors in the enumeration of progenitor cells were most obvious, as illustrated by the significant interpersonal differences found. The correlation coefficient of 0.84 between the HPC scores of one technician and the enumeration of CD34 + cells by flow cytometry may indicate that it is technically possible to achieve standardization of the HPC enumeration, but that on a rou-

tine basis the assay may be too dependent on long lasting experience of the technicians. Correlations have been found between the numbers of nucleated cells as well as CD34 positive cells with clinical outcome of allogeneic stem cell transplantation. 14,19 22 Therefore, we analyzed whether leukocyte subsets also reflected the hematopoietic content of UCB grafts. Since UCB grafts can contain a significant number of nucleated red cells, both the total numbers of nucleated cells, the total numbers of CD45 + white blood cells, lymphocytes, monocytes and granulocytes were compared with frequencies of CD34 + cells and HPC in each unit. The best correlation was found between the total numbers of nucleated cells and the number of CD34 + cells or HPC. Although it is known that hematopoietic precursors reside in the mononuclear fraction of leukocytes, any differentiation of leukocytes decreased the correlation with CD34 + cells or HPC. Variation of hematopoietic precursors in UCB does not directly influence the number of nucleated cells in UCB by their small absolute numbers, but certain factors like the course of parturition, may influence both the absolute numbers of progenitor cells as well as the absolute numbers of nucleated cells. 28 In conclusion, flow cytometric analysis of CD34 + cells in UCB transplants appeared to be more suitable to enumerate the frequency of hematopoietic precursors in UCB units than the HPC culture assay, due to interpersonal differences in scoring HPC. Moreover, flow cytometry is less timeconsuming, and therefore cheaper than HPC culture. The total number of nucleated cells correlated with the number of CD34 + cells, although there was still considerable variation. Because it is still difficult to achieve a standard of CD34 enumeration performed by all providers of UCB transplants, the total number of nucleated cells of UCB units at the time of harvest may be used to compare the hematopoietic potential of the units from different UCB banks. The number of CD34 + cells may help as an additional parameter to choose when there are more suitable UCB units available. Although HPC cultures appeared not to be very suitable for selecting UCB units, comparison of HPC cultures before and after cryopreservation plays an important role in quality control of cryopreservation and storage of UCB units in umbilical cord blood banks. Determination of the HPC frequencies in a small aliquot from the unit left aside during freezing and storage may indicate the viability of the unit selected for use at the time of the transplantation. Acknowledgements This study was supported by grants from the JA Cohen Institute for Radiopathology and Radiation Protection, and the Dutch Cancer Society. We are grateful to M Aalders and Y Breuger for obtaining informed consent from patients at the Department of Obstetrics, to A vd Veen and M Blijleven for harvesting, processing and preservation of the UCB units, to A vd Marel, J Overdevest, G d Groot-Swings and G Brouwer-Mandema for flow cytometric analysis and to R Bongaerts and E Neerincx for the HPC culture assays. Hematopoietic content of umbilical cord blood References 1 Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86: 3828 3832. 2 Gluckman E, Broxmeyer HA, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical cord blood from an HLA-identical sibling. New Engl J Med 1989; 321: 1147 1178. 3 Rubinsten P. 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