Effect of the umbilical cord blood acid-base status and gas values on the yield of mononuclear cells and CD34+ cells

Authors


Professor Ikuo Kashiwakura, Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki 036-8564, Japan. Email: ikashi@cc.hirosaki-u.ac.jp

Abstract

Aim:  To investigate the influence of umbilical cord blood (CB) acid-base status and gas values on the yield of mononuclear cells and CD34+ cells, pH, pCO2, pO2, HCO3- and base excess were measured in arterial CB samples obtained from normal full-term deliveries. The relationship of these values with the yield of mononuclear cells and CD34+ cells detected in venous CB was analyzed.

Material and Methods:  A total of 145 CB units were collected from full-term vaginal deliveries at a single hospital. Immediately after delivery, a segment of the umbilical cord was double clamped, and arterial CB was analyzed to determine the acid-base status and gases. Venous CB was collected in a sterile collection bag and processed for cell separation within 24 h of collection. The relationship between umbilical arterial acid-base status, each gas value, and the yield of mononuclear cells and CD34+ cells was analyzed.

Results:  Statistically significant correlations were observed between the net weight of CB and the total mononuclear and CD34+ cell counts. In addition, there was a negative correlation between the mononuclear cell counts and pH, but a positive correlation between the mononuclear cell counts and pCO2. However, no significant differences were observed between the primipara and multipara groups in terms of the net weight of CB, total mononuclear cell counts and total CD34+ cell counts.

Conclusion:  The findings of the present study show that the mononuclear cell counts are correlated with arterial CB pH and pCO2, suggesting the involvement of fetal hypoxia on the yield of mononuclear cells.

Introduction

Umbilical cord blood (CB) is the fetal peripheral blood that plays a role in the exchange of nutrients and gases with fetal capillary blood within the connective tissue of the villous core. As CB contains high amounts of hematopoietic stem/progenitor cells, such as CD34+ cells,1 it has been increasingly used as an alternative source to obtain these cells for treating patients with diseases such as hematopoietic malignancies.2,3 Furthermore, except for hematopoietic stem/progenitor cells, it has already been shown that CB contains multipotent stem cells that can differentiate into blood vessel, muscle, and neuronal cells.4–6 Previous reports have shown that the clinical outcomes of CB transplantation are generally influenced by the number of mononuclear cells per CB unit transplanted. However, a reduction in the number of cells per CB unit often causes serious issues during transplantation in adult primates, even though the hematopoietic stem cells contained in CB have a higher proliferative potential than that of bone marrow cells. Therefore, it is important to obtain and use CB units that contain a sufficient number of mononuclear cells.3,7–13 The importance of CB has therefore greatly increased as a source of cells for performing both clinical and basic scientific research. However, it is difficult to predict the number of nucleated/CD34+ cells per CB unit in advance because of the extremely wide variations in individual samples.

Perinatal factors, such as birth weight, placental weight, gestational age, neonatal sex, the mode of birth, and the collection and length of the umbilical cord, generally influence the cellular content of CB and the volume that can be collected.14–18 In addition, arterial CB acid-base status and gases provide useful information on the condition of the newborn before and at the time of birth. Uterine labor contractions cause fetal hypoxemia due to the intermittent obstruction of the uteroplacental circulation and blocked supply of oxygenated maternal blood to the placenta. Severe hypoxic stress leads to cellular and/or tissue damage, which can then result in neonatal mortality and severe morbidity.19 Therefore, the analysis of the gases from the umbilical artery is believed to be the best representation of the fetal acid-base status immediately before birth. However, little is known about the influence of CB acid-base status and gas values on the yield of mononuclear and CD34+ cells. In the present study, to investigate these issues, pH, pCO2, pO2, HCO3-, and base excess levels were analyzed in umbilical artery derived from normal, full-term deliveries. The relationship of these values to the yield of mononuclear and CD34+ cells detected in umbilical vein was then analyzed. This is a retrospective analyses and the result was introduced from the data based on the limited specimens.

Material and Methods

Collection of CB units

Between January 2009 and December 2010, CB units were collected at a single hospital (Hirosaki National Hospital, Hirosaki, Japan) after obtaining informed consent from every mother and approval from the Committee of Medical Ethics of Hirosaki National Hospital (Hirosaki, Japan) and the Committee of Medical Ethics of Hirosaki University Graduate School of Medicine (Hirosaki, Japan). The criteria for inclusion were low-risk pregnancies, singleton gestations, vaginal deliveries, and the newborns would be born without resuscitation or immediate rescue procedures. Immediately after delivery, a segment of the umbilical cord was double clamped, and blood was drawn from the umbilical artery into preheparinized plastic syringes for the determination of pH, base excess, and arterial CB gas values. At the same time, according to the guidelines of the Tokyo Cord Blood Bank, CB units were collected before placental delivery (in utero collection). CB was collected in a sterile collection bag that contained 28 mL citrate-phosphate dextrose anticoagulant (CBC-20; Nipro, Osaka, Japan) until the flow ceased. A total of 202 CB units were available for cell separation within 24 h of CB collection. Relevant perinatal data (such as maternal age, gestational age, duration of labor, birth weight, and Apgar score) were obtained from the hospital. Gestational ages <37 and ≥42 weeks were excluded from this study. Unknown or missing data regarding these concerning obstetric factors were not taken into account.

Separation of mononuclear cells and purification of CD34+ cells

Mononuclear cells were separated by the use of Ficoll-Paque (GE Health care Japan, Tokyo, Japan). Twenty milliliters of CB was diluted two-fold with phosphate-buffered saline (PBS) (−) plus 5 mM ethylenediamine-tetraacetic acid (EDTA) (Wako Pure Chemicals, Tokyo, Japan) and layered onto 15 mL Ficoll-Paque (1.077 g/mL, Amersham Pharmacia Biotech AB, Uppsala, Sweden). The samples were at centrifuged 300 g for 30 min at room temperature. The buffy coat was harvested and diluted with EDTA-PBS solution. To remove the platelets, cells were washed twice with EDTA-PBS and centrifuged at 100 g for 10 min at 4°C. The cells were then resuspended in EDTA-PBS at 4°C and counted using Türk solution. Following the manufacturer's instructions, magnetic cell sorting (Miltenyi Biotec, Germany) was used for the positive selection of CD34+ cells. At the end of the procedure, the amount of CD34+ cells recovered from the mononuclear cells was approximately 0.1%–0.6% and the purity, as determined by flow cytometry, was 80–95%. Cell viability was verified using the trypan blue exclusion method. CD34+ cells were not purified when the CB unit contained an extremely low number of mononuclear cells (<8 × 107 cells) because of poor recovery.

Umbilical artery blood analysis

Umbilical artery was analyzed to determine pH, pCO2, pO2, HCO3-, and base excess values using a portable blood analyzer (i-STAT300F, Abbott Point of Care, IL, USA). This analysis was usually performed within 30 min of delivery, and in no case longer than 60 min after delivery.

Statistical analysis

Statistical analysis was performed using SPSS software, version 16.0 (SPSS Japan, Tokyo, Japan), and Origin (Origin Lab, Northampton, MA, USA) for Windows. Descriptive statistics are shown as arithmetic median (range). Multiple linear regression analysis was performed to determine the association between mutually adjusted obstetric characteristics (independent variables) and cell yields (dependent variables). After normality tests, data were also analyzed by univariate analysis using either Mann–Whitney U-test or Spearman's rank correlation coefficient and stepwise regression analysis, depending on the distribution pattern of the data. A value of P < 0.05 was considered significant.

Results

Perinatal data of the study population

A total of 202 CB units were collected from healthy newborns, 7 units <37 weeks in gestational age, 1 unit ≥42 weeks, 21 units with unknown or missing perinatal or gas data, and 28 units that were unavailable for cell separation were excluded from this study. In total 145 CB units were used in this study. In total, 145 CB units were used in this study. Perinatal data of the study population are summarized in Table 1. The median maternal age was 30 years; 61.4% of the mothers were primipara and 38.6% were multipara. The median gestational duration was 39 weeks, and 45.5% of the newborn infants were male and 54.5% were female. The median birth weight was 3098 g. The median Apgar scores at 1 and 5 min were 9 and 9, respectively.

Table 1.  Characteristics of cord blood (CB) and obstetrics factors
Factor n (%)Median (range)
  • *

    P < 0.001 as determined by Mann–Whitney U-test. ROM, rupture of membranes.

Maternal age (years) 30 (18–43)
Parity  
 Primipara89 (61.4) 
 Multipara56 (38.6) 
Total duration of labor (min) 479 (55–3011)
 Primipara 590* (141–3011)
 Multipara 344 (55–1166)
First stage of labor (min) 440 (25–2985)
 Primipara 525* (60–2985)
 Multipara 320 (25–1140)
Second stage of labor (min) 23 (1–490)
 Primipara 36* (6–490)
 Multipara 12 (1–203)
Time from ROM to birth (min) 30 (2–5189)
Meconium staining23 (15.69) 
Nuchal cord57 (39.3) 
Gestational age (weeks) 39 (37–41)
Birth weight (g) 3098 (2136–4100)
Neonatal sex  
 Male66 (45.5) 
 Female79 (54.5) 
1-min Apgar score 9 (6–10)
5-min Apgar score 9 (8–10)
Placental weight (g) 540 (385–865)

Characteristics of CB

The characteristics of the units, the number of mononuclear/CD34+ cells, CB acid-base status and gas values, are summarized in Table 2. The median net weight of the CB units was 43.1 g. The median total mononuclear and CD34+ cell counts were 1.28 × 108 cells and 1.28 × 106 cells, respectively. Each labor was placed into one of two groups, primipara or multipara, and each characteristic was analyzed based on this group placement. As a result, although all of the median values were higher in the primipara group, no significant differences were observed between these groups.

Table 2.  Summary of cord blood (CB) volumes and total mononuclear and CD34+ cell counts
FactorTotal laborsPrimiparaMultipara
(n = 145)(n = 89)(n = 56)
  1. Each value shows the median value for each birth. Values shown in parentheses indicate the range of each birth.

Net weight of CB (g)43.143.840.9
(10.1–118.7)(10.1–118.7)(12.9–98.7)
Total mononuclear cells (×108)1.281.151.38
(0.03–6.10)(0.03–6.10)(0.03–4.50)
Total CD34+ cells (×106)1.281.321.09
(0.06–9.76)(0.06–9.78)(0.11–4.50)

Assessment of CB acid-base status and gas values

As the CB acid-base status and gases provide useful information on the condition of the newborn before and at the time of birth, assessments of the CB acid-base status and gas values were performed (Table 3). The median umbilical arterial pH was 7.32, and median pCO2 and pO2 were 45.2 and 16.0 mmHg, respectively. The base excess value was −3.0 mM/L. In comparison, the umbilical arterial pH, HCO3-, and base excess were significantly lower in the primipara group compared with the multipara group. However, no significant differences were found between the two groups in terms of pCO2 and pO2.

Table 3.  Blood acid-base/gas assessment in the umbilical artery
FactorTotal laborsPrimiparaMultipara
(n = 145)(n = 89)(n = 56)
  1. **P < 0.001, *P < 0.01 as determined by Mann–Whitney U-test. Each value shows the median value for each birth. Values shown in parentheses indicate the range of each birth.

pH7.327.30**7.34
(7.18–7.51)(7.18 −7.42)(7.21–7.51)
pCO245.245.543.5
(24.2–79.8)(30.5 –79.8)(24.2–62.8)
pO216.016.017.0
(4–43)(4–41)(7–43)
HCO3-22.922.4*23.5
(17.5–32.6)(18.2–27.6)(17.5–32.6)
Base excess−3.0−4.0**−2.0
(−10–7)(−10–7)(−7–3)

Relationship between mononuclear/CD34+ counts, CB acid-base status and gases

A univariate analysis was performed to determine the association between net CB weight and the total mononuclear and CD34+ cell counts (Fig. 1). As a result, statistically significant correlations were observed between the net CB weight and either the total mononuclear cell count or the total CD34+ cell count. These results were consistent with previously published reports18,19 and confirmed that the CB samples used in the present study were relevant subjects for this investigation. Next, the total mononuclear and CD34+ cell counts were analyzed to determine their relationships with CB acid-base status and gas values (Fig. 2). The mononuclear cell count was negatively correlated with pH and positively correlated with pCO2. However, no correlations were found between these values and the CD34+ cell count. In addition, base excess correlated with neither mononuclear nor CD34+ cell counts. A stepwise regression analysis was used to identify independent variables that are associated with elevated mononuclear counts. The net weight of CB, placental weight, birth weight, umbilical artery pH and base excess were identified as the variables that independently predict elevated mononuclear counts (R2 = 0.48, anovaP < 0.001).

Figure 1.

Correlation between the net weight of cord blood (CB) collected and cell counts of CB units in all deliveries. Significantly positive correlations were observed as follows: (a) net weight of CB collected and total mononuclear cell count (n = 145); and (b) net weight of CB collected and total CD34+ cell count (n = 105).

Figure 2.

Correlations between umbilical arterial pH, gas values, and cell counts of cord blood (CB) units in all deliveries. A significantly negative correlation was observed in (a) the umbilical arterial pH and the total mononuclear cell count (n = 145). A significantly positive correlation was observed between (b) the umbilical arterial pCO2 and the total mononuclear cell count (n = 145). No significant correlation was observed between (c) the umbilical arterial base excess value and total mononuclear cell count (n = 145). No significant correlations were observed in relation to the CD34+ cell count (n = 105) (d,e,f).

Discussion

As CB is the fetal peripheral blood, reductions in the blood flow from the mother to the fetus due to umbilical cord abnormalities in the uterus or compression of the umbilical cord can cause fetal hypoxia, leading to non-reassuring fetal status. Fetal hypoxic stress causes acute redistributions in fetal blood flow away from the periphery and viscera to the vital organs, such as the brain, heart, and adrenal glands.20 However, prolonged hypoxia can lead to fetal acidosis and rhexis, resulting in the redistribution of fetal blood flow. As CB acid–base status and gas analyses are recommended in all high-risk deliveries,21 these analyses in umbilical artery provide useful support if an intrapartum hypoxic-ischemic event is suspected.22 Umbilical arterial pH is routinely measured in many maternity wards after placental delivery to evaluate the acid-base status of the infant. A value of less than 7.0 is considered morbid hypoxia, which causes nervous after-effects.23–25 In the present study, the median umbilical arterial pH of all the deliveries examined was 7.32, thus indicating healthy newborns without severe fetal acidemia (Table 3). A negative correlation was observed between the total mononuclear cell count and umbilical arterial pH, while a positive correlation was observed between the total mononuclear cell count and pCO2 (Fig. 2), thus suggesting the possibility that fetal hypoxic conditions may influence the number of mononuclear cells. Juutistenaho et al. has shown that umbilical arterial pH is associated with mononuclear, CD34+, and total hematopoietic progenitor cell counts.26 However, no correlation was found between the umbilical arterial pH and the total CD34+ cell count in the present study. The previous study used an automated centrifugation-based cell separation system utilizing hydroxyethyl starch for mononuclear cell separation from the CB, and the counting of mononuclear cells and CD34+ cells was performed with a hematology analyzer and a flow cytometer, respectively, suggesting that these cell processing techniques may have been responsible for the differences in the results.

Uterine labor contractions can lead to the intermittent obstruction of the uteroplacental circulation and block the supply of oxygenated maternal blood to the placenta. Therefore, prolonged labor frequently causes fetal hypoxia due to uterine contractions, thus increasing the risk of dysfunction in the fetus. Lower umbilical arterial pH or perinatal adverse outcomes is associated with prolonged duration of the second stage of labor.27–30 Although large individual differences exist in the duration of labor, generally the duration of labor of a primipara is twice as long as that of a multipara.31,32 In the present study, the median duration of labor of the primipara group was 590 min, approximately twice as long as the median of 344 min for the multipara group (Table 3), indicating general delivery conditions. The present results show lower umbilical arterial pH, HCO3-, and base excess levels in the primipara group compared with the multipara group, indicating a statistically significant difference between the parity and umbilical arterial acid-base status (Table 3). Lim et al. reported that longer durations of labor and stress during delivery increase the number of mononuclear cells, granulocytes, CD34+ cells, and hematopoietic progenitor cells, possibly by mobilizing various cell populations of endogenous cytokines.33 In addition, Aufderhaar et al. reported that large blood volume, long duration of labor, and lower arterial and venous pH values positively correlate with higher levels of mononuclear cells.19 However, no significant differences were observed between the primipara and multipara groups in terms of the net CB weight, total mononuclear cell count, or total CD34+ cell count (Table 2). To resolve these issues, a more precise comparison between selective caesarean section cases, which have no stresses caused by uterine contraction before delivery, and normal vaginal delivery, will be required in the future.

In conclusion, the findings of the present study show that the number of mononuclear cells is correlated with umbilical arterial pH and pCO2, suggesting that the fetal hypoxic status affects the yield of mononuclear cells. Juutistenaho et al. concluded that stress-related perinatal factors, particularly umbilical arterial pH, are associated with the number of hematopoietic progenitor cells present in CB sample and may improve unit selection.26 Currently, it is difficult to predict the number of mononuclear cells per CB sample prior to cell processing due to the extremely wide amount of variation that exists in individual samples. Therefore, the detailed mechanisms that influence the relationship between CB acid-base status, gas values, and the yields of mononuclear and hematopoietic stem/progenitor cells need to be elucidated.

Acknowledgments

This study was supported by a grant for Hirosaki University Institutional Research (2008–2010). The study was performed by Ikuo Kashiwakura's research group, who designed the study and performed a series of pilot experiments, and had overall responsibility. Ayako Tarakida, Tomoko Ogasawara, Mami Manabe and Sei-ichi Katagiri collected CB units from normal vaginal deliveries at the Obstetrics and Gynecology Department of Hirosaki National Hospital. They also took the all perinatal data of the study population and measured all the perinatal data. Satoko Ebina and Atsuko Omori analyzed the data obtained in this study. Satoko Ebina and Ikuo Kashiwakura wrote the manuscript following discussions with the other authors. All authors contributed equally to the preparation of the manuscript.

Disclosure

All authors have no conflict of interest.

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