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January 2001, Volume 27, Number 2, Pages 133-138
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Cord Blood Stem Cells
Cord blood collection before and after placental delivery: levels of nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clots
A Wong1, P M P Yuen1, K Li1, A L M Yu2 and W C Tsoi3

1Department of Paediatrics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong

2Centre for Clinical Trials and Epidemiological Research, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong

3Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong

Correspondence to: Dr P MP Yuen, Department of Paediatrics, The Chinese University of Hong Kong, 6th Fl, The Prince of Wales Hospital, Shatin, NT, Hong Kong

Abstract

The number of nucleated cells infused into the recipient of a cord blood (CB) transplant has emerged as the most important factor affecting the probability and speed of engraftment. At present, there is no international consensus on the procedure of CB collection in the maternity ward. In order to maximise the yield of viable cells in a CB unit, we aimed to investigate the efficiency of CB collection, with respect to the time of delivery of the placenta. We analysed stem and progenitor cells in terms of CD34+ cell content and colony-forming activities, lymphocyte subpopulations and the presence of macroscopic clots in 93 paired CB samples, collected before and after the delivery of the placenta. Our results demonstrated that the median concentrations of nucleated cells and total colony-forming unit (CFU) were significantly lower in CB collected after placenta delivery by 9.5% (P < 0.001) and 11.6% (P = 0.015), respectively, when compared to their counterparts collected before placental delivery. A reduction of granulocytes (P < 0.001), monocytes (P < 0.001) and CD19+ B lymphocytes (P = 0.031) was observed, with no significant change in the proportion of T cell subsets (CD4+, CD8+ cells) or activated T cells (CD25+, CD45RO+ cells) in samples collected after placenta delivery. The incidence of macroscopic clots was also higher in these samples (31% vs 1%, P < 0.001). The reduction of stem and progenitor cells correlated significantly with that of major cell populations, indicating a general cell loss, possibly due to clotting activities developed with time. Our study has documented strong evidence for recommending the collection of CB before the delivery of the placenta. Bone Marrow Transplantation (2001) 27, 133-138.

Keywords

cord blood collection; placental delivery; stem and progenitor cells

Cord blood (CB) was first successfully used to reconstitute a young patient with Fanconi's anaemia in 1989 and the haematopoietic reconstitution has been sustained for over 10 years.1 Since then, CB transplants have been performed in over 700 patients with malignancies, haematological disorders and inborn errors of metabolism.2 Many CB banks have been set up worldwide. However, there are limitations in the application of CB for transplant. The yield of stem and progenitor cells in a collection has been a prime concern especially to patients with large body weights. Recently, the statistics of accumulated clinical data confirmed that the number of nucleated cells/kg and therefore the number of stem and progenitor cells infused into the recipient was the most important factor influencing the probability and speed of engraftment.3,4,5 It is thus critical that all efforts be made to ensure a large cell dose in the CB unit sufficient for a rapid and sustained engraftment.

In order to maximise the cell quantity in a single unit of CB, various studies have been carried out for their collection, processing and cryopreservation. Broxmeyer et al6 described a two-phase method by which CB was firstly collected from the severed cord during placental delivery and then additional CB was obtained from multiple needle aspirations from the umbilical vein. A modified method for overcoming the collapse of the umbilical vein was later described.7 Bertolini et al8 reported an extensive evaluation of the CB collection procedure which included the effect of vaginal vs Caesarian section delivery, recoveries of colony-forming units (CFU) and high proliferative potential colony-forming cells after density gradient centrifugation or gelatin sedimentation of both fresh and cryopreserved CB samples. The rates of bacterial contamination in open and closed collection systems were also compared.8 Elchalal et al9 studied the collection of CB by three different methods comparing single and two-step collections using open and closed systems.

Despite the several studies on the procedure of CB collection, some practical issues remain unresolved. So far, there has been little information on the yield of stem and progenitor cells as affected by the time of delivery of the placenta. Ballin et al10 reported the presence of clotting activities in CB collected before placental delivery. It is conceivable that any delay in the timing of collection would lead to increased clotting activities and thus reduce the quantity of stem and progenitor cells. At present, the procedure of CB collection has not been internationally standardised and is subject to the common practice of each individual institution. In some CB banks,11 collections were routinely performed after the placenta was delivered, whereas in others CB was collected while the placenta was still in utero.12,13 In some centres, a combination of the two protocols were used.14,15

In this study, we addressed the issue by measuring the levels of nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clots in 93 paired CB samples, collected before and after placental delivery. The data provide evidence applicable to the recommendation and standardisation of the CB collection procedure for clinical transplants.

Materials and methods

Sample collection

Umbilical CB samples (n = 93) were collected in pairs from the umbilical vein of normal full-term vaginal deliveries before and after the delivery of the placenta under the auspices of the Department of Obstetrics and Gynecology, Prince of Wales Hospital, The Chinese University of Hong Kong. The umbilical cord was clamped within 1 min after delivery of the infant. The cord was wiped free of any maternal blood and cleaned with alcohol followed by betadine. After clamping, a sample of CB (10-15 ml) was collected from the umbilical vein in a syringe within 3 min (interquartile range, 2-5 min) of the delivery and was transferred into a plastic universal container (Sterilin, Stone, UK) in the presence of 100 IU/ml preservative-free heparin (David Bull Laboratories, Victoria, Australia). A similar volume of CB was collected after delivery of the placenta. Both samples were analysed by flow cytometry, progenitor cell assay and examined for the presence of macroscopic clots. Informed parental consent was obtained for the CB collection and this study was approved by the Ethics Committee for Clinical Research of The Chinese University of Hong Kong.

Progenitor cell assay

Haematopoietic progenitor cells were assayed by CFU culture for 2 weeks in methylcellulose. Mononuclear cells (MNC) after Ficoll-Paque density separation (1.077 g/ml, Pharmacia, Piscataway, USA) and at a concentration of 105 cells/ml were seeded in 1% methylcellulose (Sigma Chemical, St Louis, MO, USA) in Iscove's modified Dulbecco's medium (IMDM; Gibco BRL, UK) and supplemented with 10% heat inactivated fetal calf serum (Gibco), 1% bovine serum albumin (Sigma), 100 mum beta-mercaptoethanol, 100 U/ml penicillin, 50 mg/ml streptomycin, 100 ng/ml of granulocyte-macrophage colony-stimulating factor (Sandoz, Basle, Switzerland), 20 ng/ml stem cell factor (Genzyme, Boston, MA, USA), 1 IU/ml erythropoietin (Eprex, Zug, Switzerland). CFU cultures were performed in triplicate, maintained in a fully humidified atmosphere at 37°C with 5% CO2 for 14 days. For the scoring of colony-forming units granulocyte-macrophage (CFU-GM), colony-forming units erythroid (CFU-E) and colony-forming units granulocyte, erythrocyte, macrophage and megakarocyte (CFU-GEMM), colonies of over 50 cells were counted.

Flow cytometry analysis

One hundred microlitres of whole blood were stained with directly conjugated monoclonal antibodies against various cell surface markers. The following conjugated monoclonal antibodies were purchased from Becton Dickinson (BD) (San Jose, CA, USA), CD34-fluorescein isothiocyanate (FITC), CD34-phycoerythrin (PE), HLA-DR- peridinin chlorophyll protein (PerCP) , CD33-PE, CD14-PE, CD45RO-PE, CD3-FITC,CD3-PerCP, CD19-PE, CD16-PE and CD56-PE. CD8-PE and CD4-FITC were obtained from Dako (Copenhagen, Denmark) and CD45-FITC was purchased from Immunotech (Marseille, France). All samples were incubated for 20 min in the dark at room temperature, lysed (FACS lysing solution, BD) for 10 min, washed and then acquired with a FACScan flow cytometer using the Lysis II software (BD). For each sample, the three different populations were identified by the CD45/CD14 and/or CD45/SSC plots. For the enumeration of CD34+ cells, the ISHAGE protocol16 was used and a minimum of 78000 events were collected. A minimum of 10000 events was collected for the analysis of lymphocyte subsets. Total white cell counts were determined by a haematocytometer and corrected for the presence of nucleated red cells. Nucleated red blood cells were determined in duplicate and counted twice, using the wedge slide technique after staining with Wright's eosin-methylene blue (Merck, Marmstadt, Germany).

Macroscopic clots

The frequency of macroscopic/visible clots in CB before Ficoll density separation was graded according to the degree of clotting: no visible clots, visible clots present and heavily clotted. The incidence of the failure of withdrawing CB from the umbilical vein due to heavy clotting was also recorded.

Statistical analysis

Cell contents of CB collected before and after placental delivery were compared using the Wilcoxon's signed rank test. All results were expressed as median (interquartile range). Correlations of the changes of various cell populations and time delays in placental delivery were calculated using the Spearman correlation coefficient (r). A P value of P < 0.05 was considered as statistically significant.

Results

Leukocyte counts and subpopulations

The total nucleated cell counts per ml of CB were higher in samples collected before placental delivery when compared to their counterparts after placental delivery (13.6 ´ 106/ml (9.8-19.0) vs 13.0 ´ 106/ml (8.91-17.1), P < 0.001). In terms of differential counts, higher concentrations of monocytes (P < 0.001) and granulocytes (P < 0.001) were observed in samples collected before delivery of the placenta (Figure 1). There was no difference in the proportions of nucleated red cells in the two groups (before samples 4.63% (2.44-5.88) and after samples 4.63% (2.19-5.79, P = 0.24)).

Haematopoietic progenitor cells

The concentration of CD34+ cells per ml CB was not different between CB samples collected before and after placental delivery (3.14 ´ 104/ml (1.84-5.22) vs 3.05 ´ 104/ml (2.13-4.98), P = 0.089) (Figure 2). Similar trends were observed in the subpopulations of CD34+ cells, including the CD33+cells and the HLA-DR+ cells. The total number of CFU was significantly higher in CB collected before placental delivery (4.43 ´ 103/ml (2.59-7.85) vs 3.33 ´ 103/ml (2.21-6.79), P = 0.015), as shown in Figure 3. A significantly higher number of CFU-GM was also observed in CB collected before placental delivery (3.25 ´ 103/ml (1.88-5.11) vs 2.71 ´ 103/ml (1.58-1.46), P = 0.025). For the numbers of CFU-E, BFU-E and CFU-GEMM, there was no significant difference between CB samples collected before and after placental delivery.

Phenotypic analysis of lymphocyte subsets

The levels of lymphocyte subsets are summarized in Table 1. There was a slight decrease in the number of CD19+ B lymphocytes (P = 0.031) and an increase in CD3-CD16+ CD56+ natural killer (NK) cells (P = 0.031) in samples after placental delivery. There were no significant differences on the absolute levels of T cells and subsets, including CD3+CD4+, CD3+CD8+, CD3+CD25+ and CD3+CD45RO+ cells.

Macroscopic clots

The incidence of macroscopic clots was low in the samples collected before delivery of the placenta when compared with those collected after placental delivery (1% (visible clot) vs 31% (15% visible clot, 16% heavily clotted), P < 0.001). We encountered a 6% failure in withdrawing any blood from umbilical cords obtained after placental delivery but none in those before placental delivery.

Correlation analysis

Correlations between the changes of specific cell types were calculated by the Spearman correlation coefficient in order to determine whether similar trends of changes occurred in the different cell populations. The change in the concentration of CD34+ cells correlated positively with that of total white cells (r = 0.561, P < 0.001) (Figure 4), granulocytes (r = 0.673, P < 0.001), lymphocytes (r = 0.572, P < 0.001), monocytes (r = 0.481, P < 0.001) and CFU (r = 0.392, P = 0.03).

The median time delay in the delivery of the placenta was 7 min (range, 5-10). The time intervals between the collection of the paired samples did not correlate with the change of levels of any of the parameters measured.

Discussion

Broxmeyer reported that the lowest number of CB nucleated cells transplanted and successfully engrafted was 1 ´ 107 per kg body weight of the recipient.17 A recent study by Gluckman et al18 revealed that the number of CB nucleated cells required for engraftment was about 1 log less than for a standard allogeneic bone marrow transplant and 10 times less than for a standard peripheral blood stem cell transplant.18 In their series of CB transplants, adult patients who received less than 2 ´ 107nucleated cells/kg had only a 69% probability of reaching 500/ml neutrophils and 49% reaching 20000/ml platelets on day 60. Overall the data indicated that transplants with a low number of nucleated cells were associated with both an increased risk of non-engraftment and a delay of engraftment. Our present study demonstrated that the median concentrations of nucleated cells and total CFU were significantly decreased by 9.5% and 11.6%, respectively, in samples collected after placental delivery when compared to their counterparts collected before placental delivery. Although the level of CD34+ cells was not significantly different between the paired samples, there was also a trend to fewer CD34+ cells in samples collected after placental delivery. In our study, we collected samples from the same umbilical cord for paired comparisons, thus we did not have data concerning the volume of CB that can be obtained independently using the two collection methods. This issue was recently addressed by Surbek et al19 who demonstrated a significantly larger volume of CB collected before placental delivery. The reduction in CB volume after placental delivery, together with the decrease in cell concentrations could lead to substantial cell losses in a CB unit. As a consequence, the engraftment kinetics and clinical outcome might be adversely affected, particularly in CB transplants given to recipients with large body weights.

As for lymphocyte subsets, there was a slight decrease in the level of B cells and increase of NK cells collected after placental delivery. The subsets of T cells, in terms of the T helper and suppressor populations and the activation status (CD25+ and CD45RO+ cells) were similar in the paired samples. Consequently, the method of collection would likely have no influence on the severity of graft-versus-host disease or graft-versus-leukemia effects after transplantation. Lim et al20 reported that fetal and/or maternal stress during delivery could affect the level of leukocyte subsets in CB. It is unlikely that our collection of CB after placental delivery fell into this category as all the deliveries were classified as normal, full-term and spontaneous.

Our data on the general loss of cells in samples after placental delivery indicated that clotting activities might have occurred. Indeed, the frequency of visible clots was significantly higher in samples collected after placental delivery. Our observation of visible clots was less frequent than those reported by Anderson et al,21 who used a Fenwall PDF-20 paediatric transfusion filter (Fenwall, Deerfield, IL, USA) for trapping the clots and noted them in 7% of CB collected before placental delivery. Without a standard procedure for its assessment, the influence of clotting activity on the collection of CB might have been underestimated. Our data showed that the changes of CD34+ cells and CFU correlated with those of other white cell subsets, indicating that clotting activities might be a general phenomenon and not cell specific. However, we cannot rule out the possibility that a certain cell type might be less susceptible to clotting such as the NK cells which were slightly higher in the delayed samples. In addition, the presence of clots may also adversely affect the efficiency of subsequent processing procedures such as volume and red cell reduction, cryopreservation and thawing as practised by most cord blood banks and transplant institutions.

To date, there has been no standard guideline as to the timing of CB collection in the maternity ward. We documented the presence of more nucleated and progenitor cells in CB collected before the delivery of the placenta. The decrease in these cells after placental delivery might be a result of the increase of clotting activities with time. In conclusion, our study provided strong evidence that the collection of CB before the delivery of the placenta should be the preferred procedure, bearing in mind there is a finite amount of stem and progenitor cells available in these circumstances. We fully realise that the collection of CB while the placenta is in utero might constitute an invasion of privacy of the mother at the time of delivery and the possibility of diverting the medical staff from their primary responsibility for the care of the mother and her newborn infant. It is therefore important that informed consent be obtained from the mother and the reason for the collection of CB before delivery of the placenta be fully explained to her. To ensure the safety and minimal interference to both the mother and her newborn infant, only CB from normal full-term vaginal deliveries should be obtained, and ideally this should be carried out by a well-trained member of the medical staff assigned specifically for that purpose. The strategy of maximizing stem and progenitor cells by collecting them prior to placental delivery is particularly relevant to CB transplants targeted for recipients within the family.

Acknowledgements

We wish to thank the nurses at the Department of Obstetrical and Gynecology, Prince of Wales Hospital, Hong Kong for helping with the collection of CB. This work was financially supported by the Children Cancer Fund and the Industrial Support Fund, Government of Hong Kong Special Administrative Region. The antibodies were partially sponsored by Becton Dickinson Ltd.

References

1 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: 1174-1178, MEDLINE

2 Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 1997; 90: 4665-4678, MEDLINE

3 Lim F, Beckhoven J, Brand A et al. The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation. Bone Marrow Transplant 1999; 24: 965-970, MEDLINE

4 Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors (see comments). New Engl J Med 1998; 339: 1565-1577, MEDLINE

5 Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. New Engl J Med 1997; 337: 373-381, MEDLINE

6 Broxmeyer HE, Kurtzberg J, Gluckman E et al. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells 1991; 17: 313-329, MEDLINE

7 Turner CW, Luzins J, Hutcheson C. A modified harvest technique for cord blood hematopoietic stem cells. Bone Marrow Transplant 1992; 10: 89-91, MEDLINE

8 Bertolini F, Lazzari L, Lauri E et al. Comparative study of different procedures for the collection and banking of umbilical cord blood. J Hematother 1995; 4: 29-36, MEDLINE

9 Elchalal U, Fasouliotis SJ, Shtockheim D et al. Postpartum umbilical cord blood collection for transplantation: a comparison of three methods. Am J Obstet Gynecol 2000; 182: 227-232, MEDLINE

10 Ballin A, Arbel E, Kenet G et al. Autologous umbilical cord blood transfusion (see comments). Arch Dis Child Fetal Neonatal Ed 1995; 73: F181-F183, MEDLINE

11 Navarrete C, Warwick R, Armitage S et al. The London Cord Blood Bank. Bone Marrow Transplant 1998; 22: (Suppl. 1) S6-S7, MEDLINE

12 Dal Cortivo L, Marolleau JP, Gluckman E et al. The Paris Cord Blood Bank. Bone Marrow Transplant 1998; 22: (Suppl. 1) S11, MEDLINE

13 Querol S, Gabarro M, Amat L et al. The placental blood program of the Barcelona Cord Blood Bank. Bone Marrow Transplant 1998; 22: (Suppl. 1) S3-S5, MEDLINE

14 Pojda Z, Machaj E, Debski R et al. Organization of the cord blood bank in Warsaw, Poland: current status and future prospects. Bone Marrow Transplant 1998; 22: (Suppl. 1) S13, MEDLINE

15 Jacobs HC, Falkenburg JH. Umbilical cord blood banking in The Netherlands. Bone Marrow Transplant 1998; 22: (Suppl. 1) S8-S10, MEDLINE

16 Sutherland DR, Anderson L, Keeney M et al. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother 1996; 5: 213-226, MEDLINE

17 Broxmeyer HE. Questions to be answered regarding umbilical cord blood hematopoietic stem and progenitor cells and their use in transplantation (see comments). Transfusion 1995; 35: 694-702, MEDLINE

18 Gluckman E, Rocha V, Chastang C. Cord blood banking and transplant in Europe. Eurocord. Vox Sang 1998; 74: (Suppl. 2) 95-101, MEDLINE

19 Surbek DV, Schonfeld B, Tichelli A et al. Optimizing cord blood mononuclear cell yield: a randomized comparison of collection before vs after placenta delivery (letter). Bone Marrow Transplant 1998; 22: 311-312, MEDLINE

20 Lim FT, van Winsen L, Willemze R et al. Influence of delivery on numbers of leukocytes, leukocyte subpopulations, and hematopoietic progenitor cells in human umbilical cord blood. Blood Cells 1994; 20: 547-558, MEDLINE

21 Anderson S, Fangman J, Wager G, Uden D. Retrieval of placental blood from the umbilical vein to determine volume, sterility, and presence of clot formation. Am J Dis Child 1992; 146: 36-39, MEDLINE

Figures

Figure 1 Contents of leukocytes and subpopulations in cord blood collected before and after placental delivery. Results were expressed as median, interquartile range (box) and range. There were significantly lower white cell (P < 0.001), monocyte (P < 0.001) and granulocyte (P < 0.001) counts in cord blood collected after placental delivery (shaded box).

Figure 2 Levels of stem and progenitor cells and subsets in cord blood collected before and after placental delivery. There were no significant differences between the two groups of paired samples.

Figure 3 Levels of colony-forming units and subsets in cord blood collected before and after placental delivery. There were significantly less total CFU (P = 0.015) and CFU-GM (P = 0.025) per ml cord blood collected after placental delivery.

Figure 4 The correlation between the changes in the concentrations of CD34+ cells and total white cell counts. Change = after minus before values.

Tables

Table 1  Lymphocyte subsets collected in CB before and after placental delivery

Received 27 June 2000; accepted 23 October 2000
January 2001, Volume 27, Number 2, Pages 133-138
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