Efficiency assessment of cord blood banking and compatibility with delayed cord clamping

Abstract

Background

There is debate whether delayed umbilical cord clamping following delivery, the current gold standard, affects the proportion of cord blood units (CBU) suitable for public cord blood banking. This study was designed to assess the impact of delayed cord clamping on the number of CBU suitable for therapeutic uses.

Materials and methods

To minimize variability, data from the four most active collection centers within the Programa Concordia (Spain) were included. Data on CBU collected in utero from mothers following normal vaginal deliveries from July 2018 to December 2021 were analyzed. The weight of the collection bags (as a surrogate of volume) and total nucleated cell (TNC) count were analyzed according to three defined clamping times: 30 s, 60 s and ≥120 s. The CBU were stratified as suitable for stem cell transplantation (≥110 g and ≥1,500x10⁶ TNC/unit) or other clinical applications (≥100 g but TNC count below the threshold).

Results

There were 131 (18%), 548 (76%), and 40 (5%) CBU collected at 30 s, 60 s and ≥120 s, respectively. The median weight of the CBU decreased gradually with time, with a significant difference between units collected when the cord was clamped at 30 s or 60 s (p=0.036), so significantly fewer CBU met the minimal weight criterion (100 g) at 60 s than at 30 s (p=0.002). However, this was not reflected by the TNC available, resulting in non-statistical differences in CBU eligible for banking between these times. The major predictor of collection success was the neonate’s birth-weight.

Discussion

Despite decreases in the volume of cord blood collected when cord clamping at 30 s or 60 s, TNC count is maintained resulting in similar numbers of CBU eligible for banking. The different clamping delays investigated in this study are, therefore, compatible with public cord blood banking needs.

INTRODUCTION

The human placenta, essentially a fetal organ closely attached to the maternal uterine wall, contains a large reservoir of fetal blood. Once the fetus is delivered, placental blood flows into the neonate’s circulation until the umbilical cord is clamped and separated; in babies born at term, approximately 80 mL of blood is transferred by 1 minute after the birth, reaching approximately 100 mL at 3 minutes^1–3. In the concept of cord blood banking (CBB), residual blood remaining in the placental tissue after clamping can be collected into sterile bags by puncturing the umbilical vein, and then processed and banked for subsequent clinical use. It is recommended that cord blood units (CBU) of high quality, in terms of volume and total nucleated cell count (TNC), are used in order to achieve a good outcome following transplantation^4,5.

The transfer of placental blood into the neonate’s circulation has been shown to be beneficial for both term and preterm babies^6,7 with no added risk to maternal health. Considering the short-term and long-term benefits for infants, in 2014, the World Health Organization published a guideline on delayed cord clamping containing strong recommendations to delay cord clamping until at least 1 minute after birth⁸. The American College of Obstetricians and Gynecologists also released a committee opinion in 2017 recommending delaying cord clamping until at least 30 s to 60 s after birth⁹.

With the implementation of the practice of delayed cord clamping, researchers were keen to assess the impact of delayed clamping on the quality of CBU collected for banking purposes^10–12. Among them, Frändberg et al. demonstrated that delaying clamping by 60 s did not have a major effect on CBU collection efficiency¹⁰. According to the study carried out by Ciubotariu et al., a cord clamping delay of up to 60 s had a small negative impact on CBU with high TNC. When the clamping delay was more than 60 s, both the volume and TNC of CBU were significantly decreased and the chance of obtaining clinically useful CBU for stem cell transplantation was drastically reduced¹¹.

In the setting of high TNC thresholds for CBB, the discard rate of CBU may reach as high as 80% of those collected^8,13. Under the CBB 2.0 concept¹⁴ (use of CBU beyond transplantation), new pathways are opening up to utilize cord blood-derived products for transfusional as well as non-transfusional applications to help more patients in need. Within this concept, much research and many clinical trials are ongoing to determine the feasibility of utilizing products derived from cord blood and placental tissue for cell therapy support, regenerative medicine and specialized transfusion medicine¹⁴. On this background, we at the Banc de Sang i Teixits (BST) have developed a specialized hemotherapy program to produce different blood components for clinical applications from CBU unsuitable for transplantation purposes, but meeting the acceptance criteria for manufacturing¹⁵. This program will make the fullest use of donated products with the goal of achieving a substantial clinical conversion rate. Given that the quality of collected CBU depends mainly on the volume and TNC of the units, it is important to ascertain whether delayed clamping is compatible with public CBB. Therefore, this retrospective study was planned to assess the impact of different cord clamping delays on key quality variables of CBU, such as volume and TNC, collected within the Programa Concordia BST.

MATERIALS AND METHODS

Cord blood collection procedure

The Programa Concordia BST is one of the public cord blood banks authorized to collect CBU in six regions of Spain and Andorra. All collection centers send CBU daily to a single processing center located at the BST premises in Barcelona, Spain. The whole program is accredited by FACT-Netcord and CAT Cord Blood. CBU are collected in utero by trained staff, mainly obstetricians and midwives, following internal procedures. The time the cord is clamped is measured and recorded by a nursing officer or midwife attending the delivery. Collected CBU are transported daily to the central processing BST center for testing, processing, banking and distribution. At reception, all units are registered in the cord blood bank database and the weight of the collection bag is determined as a surrogate for volume. In the units weighing ≥110 g and fulfilling the registration criteria, TNC and CD34+ cell counts are performed. CBU with an initial TNC ≥1,500×10⁶ per unit are processed in a controlled environment and cryopreserved for transplantation. CBU weighing ≥100 g but that do not meet the cell threshold are transferred for further processing intended for other clinical applications under development. Units weighing <100 g are sent to the research biobank for clinical research purposes without testing.

The cell count was performed using a hematology analyzer (XN550, Sysmex, Kobe, Japan; XE2000, Sysmex UK, Milton Keynes, UK) and included evaluation of nucleated cell concentration, percentage of mononuclear cells, hematocrit and platelet concentration. Phenotyping was done by flow cytometry (Navios, Beckman Coulter, Brea, CA, USA; BD FACS Canto II, Oxford, UK) assessing CD34+ cell expression according to the single-platform International Society for Hematotherapy and Graft Engineering (ISHAGE) protocol¹⁶.

Data collection and statistical analysis

Ethical approval for this retrospective study was given by Vall d’Hebron University Hospital research ethics committee for medicines under reference number PR(BS)58/2023. The research was conducted ethically, with all study procedures being performed in accordance with the requirements of the World Medical Association’s Declaration of Helsinki.

Out of all maternity hospitals involved in cord blood collection, relevant, anonymized data were extracted from the four most active collection centers to minimize variations. The four centers selected were Hospital Universitari Germans Trias i Pujol, Badalona (A), Hospital Sant Joan de Deu de Esplugues, Barcelona (B), Hospital de Vic, Catalunya Central (C) and Hospital Universitari Arnau de Vilanova, Lleida (D). Data regarding CBU collected from mothers following normal vaginal deliveries from July 2018 to December 2021 were included.

After retrieving all relevant data, three groups were defined with respect to the cord clamping time following delivery; namely (i) at 30 s, (ii) at 60 s and (iii) at 120 s or more. The main variables analyzed were the bag’s initial weight (as a surrogate of volume) and TNC count. A statistical analysis was performed, comparing the median values of variables between groups, using the Kruskal-Wallis test. Furthermore, we analyzed the impact of cord clamping time on the proportions of CBU suitable for transplantation (CBU weighing ≥110 g and with ≥1,500×10⁶ TNC) and for other clinical applications (such as cell therapy or specialized hemotherapy) (any CBU weighing ≥100 g and with <1,500×10⁶ TNC). Percentages were compared between delayed clamping categories using Pearson’s chi-squared test.

To identify other possible factors affecting CBU eligibility for banking purposes, descriptive data were compared between clinically suitable and unsuitable categories using the Wilcoxon rank test. Variables considered were CBU collection center, maternal age, mode of delivery, birth weight and gender of the neonate. p values <0.05 were considered statistically significant for all the statistical tests used.

RESULTS

Number of cord blood units collected during the study period

Overall, 719 CBU were collected in the four selected collection centers during the study period. The contribution of each hospital is shown in Table I. The numbers and proportions of CBU collected following cord clamping at 30 s, 60 s and ≥120 s were 131 (18.19%), 548 (76.25%) and 40 (5.56%), respectively. The initial weight of 233 (32.40%) units was within the acceptance range for processing and these CBU, therefore, proceeded to undergo TNC evaluation.

Table I.

Number and percentage of cord blood units collected at each maternity hospital

Maternity hospital		No. of CBU	%
A)	Badalona - Hospital Universitari Germans Trias i Pujol	166	23.1
B)	Barcelona VH - Hospital Sant Joan de Deu de Esplugues	184	25.6
C)	Catalunya Central - Hospital de Vic	162	22.5
D)	Lleida - Hospital Universitari Arnau de Vilanova	207	28.8
Total		719	100

CBU: cord blood units.

Impact of cord clamping time on the weight of collection bags

Table II and Figure 1A summarize the analysis of weight with respect to cord clamping time. The median weight of CBU gradually decreased as the cord clamping delay increased. The median weight difference between bags collected at 30 s and 60 s was statistically significant (p=0.036).

Table II.

Analysis of weight and total nucleated cell count of cord blood units according to clamping delay

1. Analysis of CBU weight (g) according to clamping delay
Clamping delay	Sample size No. 719 (100%)	Mean	Q1	Median	Q3	IQR
30 s	131 (18.2%)	107.32	87	104	127.50	40.50
60 s	548 (76.2%)	102.24	84	97	116.25	32.25
≥120 s	40 (5.6%)	100.00	81	91.5	111.50	30.50
2. Analysis of TNC (×10 6 ) according to clamping delay
Clamping delay	Sample size No. 158 (100%)	Mean	Q1	Median	Q3	IQR
30 s	34 (21.5%)	1,686.28	1,213.11	1,431.14	1,965.11	751.99
60 s	118 (74.6%)	1,604.76	1,154.29	1,555.01	1,872.89	718.59
≥120 s	6 (3.8%)	1,780.59	1,375.47	1,537.66	1,933.83	558.35

CBU: cord blood units; Q1: 1^st quartile; Q3: 3^rd quartile; IQR: interquartile range; TNC: total nucleated cells.

Figure 1.

Graphical representation of median weight and median total nucleated cell count of cord blood units with cord clamping time

(A) Graphical representation of the median weight of cord blood units (CBU) with cord clamping time. (B) Graphical representation of median total nucleated cells (TNC) of CBU with cord clamping time. Box-and-whisker plots represent median values, and first and third quartile values with the range defined by the lines (outliers are shown as dots).

Impact of cord clamping time on total nucleated count of cord blood units

Of 233 CBU tested, TNC was available for analysis for only 158 (22%) units. As shown in Table II, the highest median TNC was seen in the group of units collected after clamping at 60 s (1,555×10⁶), followed by the group collected after clamping at ≥120 s (1,537.66×10⁶) and at 30 s (1,431.14×10⁶). No statistically significant differences were observed in TNC collected between clamping time categories (p=0.94) (Figure 1B).

Impact of cord clamping time on proportion of cord blood units suitable for clinical applications

For transplantation

Table III shows the proportion of CBU that would meet the criteria for banking established at the BST (minimum weight of 110 g and initial TNC 1,500×10⁶) per unit), with respect to clamping times. Out of the total 719 units collected, 75 (10.4%) units that weighed ≥110 g were excluded from analysis due to unavailability of TNC. Of the rest, 564 (78.4%) CBU were ineligible for banking because of the failure to reach the cut-off weight and/or TNC, while 80 (11.1%) CBU met both criteria and became eligible for banking. Percentage differences between clamping time categories were not statistically significant (p=0.785). Fifty percent of the CBU reaching the cut-off weight for banking (≥110 g) had the cut-off TNC level and became eligible for banking at the BST.

Table III.

Number and percentage of cord blood units eligible and ineligible for banking with respect to cord clamping time

Clamping delay	Ineligible CBU		Eligible CBU		CBU without data		Total
	No.	%	No.	%	No.	%	No.	%
30 s	103	78.6	14	10.7	14	10.7	131	100
60 s	430	78.5	63	11.5	55	10	548	100
≥120 s	31	77.5	3	7.5	6	15	40	100
Total	564	78.4	80	11.1	75	10.4	719	100

CBU: cord blood units.

For other clinical applications

To assess the suitability of CBU for other clinical applications, we calculated the number of CBU that met the weight threshold of 100 g but failed to reach the TNC threshold in the three cord clamping groups (Table IV). In the 30 s category, 47% of CBU were usable, whereas in the other two categories, only 30% reached the weight limit. The proportion of CBU that weighed ≥100 g was significantly higher in the group of CBU clamped at 30 s than in the group clamped at 60 s (p=0.002).

Table IV.

Proportion of cord blood units suitable for clinical applications other than stem cell transplantation

Clamping delay	CBU ≥100 g but failed to reach TNC threshold		CBU weighing <100 g		Total
	No.	%	No.	%	No.	%
30 s	48	46.60	55	53.40	103	100
60 s	129	30.00	301	70.00	430	100
≥120 s	9	29.02	22	70.98	31	100
Total	186	32.98	378	67.02	564	100

CBU: cord blood units; TNC: total nucleated cells.

Factors affecting the suitability of cord blood units for banking

Among the variables compared between CBU eligible and ineligible for banking (Table V), only the birth weight of the neonate showed a statistically significant difference (p<0.001).

Table V.

Comparison of descriptive data between cord blood units ineligible and eligible for banking

Descriptive data	Ineligible CBU (No.=564)	Eligible CBU (No.=80)	OR (95% CI)	p value
Collection center				0.090
A	121 (21.5%)	22 (27.5%)	Ref.
B	148 (26.2%)	19 (23.8%)	0.71 (0.36;1.37)
C	117 (20.7%)	23 (28.7%)	1.08 (0.57;2.06)
D	178 (31.6%)	16 (20%)	0.50 (0.25;0.98)
Median maternal age (yrs)	33 (29;36)	34 (31;37)	1.04 (1.00;1.09)	0.092
Maternal age categories				0.087
18–30 yrs	162 (28.7%)	16 (20%)	Ref.
30–35 yrs	202 (35.8%)	26 (32.5%)	0.71 (0.36;1.37)
>35 yrs	200 (35.5%)	38 (47.5%)	1.08 (0.57;2.06)
Type of delivery				0.425
Difficult delivery	57 (10.1%)	11 (13.8%)	Ref.
Normal delivery	507 (89.9%)	69 (86.2%)	0.70 (0.36;1.47)
Median birth weight (g)	3,351	3,565	1.00 (1.00;1.00)	<0.001
	(3,098; 3,600)	(3,352;3,810)
Birth weight				<0.001
<3,250 g	201 (40.2%)	13 (17.6%)	Ref.
3,250–3,750 g	214 (42.8%)	37 (50%)	2.65 (1.40;5.33)
>3,750 g	85 (17%)	24 (32.4%)	4.32 (2.12;9.18)
Gender of neonate				0.144
Female	271 (48%)	46 (57.5%)	Ref.
Male	293 (52%)	34 (42.5%)	0.68 (0.42;1.10)

CBU: cord blood units; OR: odds ratio estimation; CI: confidence interval; A: Hospital Universitari Germans Trias i Pujol, Badalona; B: Hospital Sant Joan de Deu de Esplugues, Barcelona; C: Hospital de Vic, Catalunya Central; D: Hospital Universitari Arnau de Vilanova, Lleida; Ref:- Reference; g: grams; yrs: years.

DISCUSSION

Public CBB aims to provide potential donors for any patient in need. Banks are providers of stem cell grafts for patients requiring allogeneic transplantation, but other applications are also being developed. Delayed cord clamping is the recommended practice after delivery, but there is some debate about whether this practice jeopardizes the potential quality of CBU collected for banking. To analyze this issue, we designed a retrospective study to compare key parameters that determine the clinical use of collected samples assessed according to the times of cord clamping following the current recommendations of the World Health Organization.

In the present study, the initial weight of CBU collected in-utero following normal vaginal delivery decreased as the cord clamping delay was longer, with a statistically significant difference between units collected at 30 s and 60 s. The longer the delay, the more the weight was affected. Our results are compatible with those of previous studies that analyzed the volume (or weight) of umbilical cord blood collected in utero with respect to cord clamping time. For example, Frändberg et al. demonstrated that the volume collected was significantly reduced when changing from a practice of clamping immediately to one of waiting until 60 s¹⁰. However, for ex-utero CBU collections, weight reduction was only significant when the clamping time exceeded 60 s¹¹. This difference might be because the volume of units collected in utero is significantly greater than that of units collected ex utero^17,18.

A comparison of TNC with different clamping times in some previous studies demonstrated significant reductions of TNC with increased clamping delays. The proportion of CBU with more than 1,500×10⁶ TNC was significantly reduced in all categories of delayed clamping time compared to immediate clamping in the research conducted by Allan et al.¹². However, the study by Ciubotariu et al. on CBU collected ex-utero demonstrated that the TNC, like the weight of units, was maintained in CBU collected with a clamping delay of up to 60 s¹¹. Similarly, Frändberg et al. reported that cell recovery was still maintained even after changing clamping practice from immediate to 60 s¹⁰. These observations are aligned with the findings of this study in which the TNC of CBU was not affected by clamping delays ranging from 30 s to 60 s. The findings of the present study are, however, different from those of previous similar works because the cell count was maintained even when the clamping delay exceeded 60 s. It is difficult to comment on this difference, as there were relatively few CBU in this category compared to in the others. Ultimately, this paradoxical behavior of volume and cell transfer results in no decrease in cell content, at least in units collected up to 120 s after birth. The volume of blood collected, birthweight, mode of delivery¹⁹, gestational age, infant’s race, parity and infant’s sex²⁰ have also been significantly associated with the cell count of CBU.

What is the impact of this finding for banking activities? Around 14% of CBU in the present study met the threshold weight and TNC levels for transplantation. Importantly, the percentage of CBU eligible for banking was not significantly changed by different cord clamping delays. This finding differs from that by Ciubotariu et al., who also analyzed the percentage of CBU that qualified for transplantation (successful recovery of CBU) with different cord clamping delays. In contrast to the present study, around 22% and 20% CBU in the 0–30 s and 30–60 s clamping groups, respectively, met the banking criteria (≥1,600×10⁶ TNC). When the clamping delay exceeded 60 s, this percentage decreased dramatically and only 2.5% CBU were eligible for banking¹¹. Some research has demonstrated banking of almost 40% of collected units, even after a clamping delay of 60 s, which is far higher than the current findings¹⁰. Our study further confirmed the association between banking eligibility of CBU and the birth weight of the neonate, as found in previous, similar research^19–21.

Altogether the data suggest that: (i) a clamping delay up to 60 s is fully compatible with the purpose of CBB of collecting suitable CBU for transplantation, and (ii) exploring new therapeutic avenues, such as those proposed by our bank within the concept of CBB 2.0¹⁴, will further improve the clinical conversion rate making the most of donated units. A recently published international study explored the utilization of CBU ineligible for transplantation through multicomponent cord blood fractionation. The implementation of the MultiCord12 protocol show-cased its effectiveness in establishing a standardized approach for the production of platelet concentrate, plasma and red blood cells derived from cord blood²². The platelet fraction can be used in regenerative medicine such as eye drops^23,24 or for cutaneous applications^15,24. The red blood cells in CBU are attractive for transfusion for preterm babies because they contain fetal hemoglobin^25,26. In this regard, our study showed that around 50% of CBU collected after clamping at 30 s were eligible for broader clinical applications. When clamping was delayed for 60 s or more, there was a 17% significant reduction of usable CBU. It is, in any case, interesting to note that with any tested clamping delay, over 45% of CBU were usable at the BST. Thus, in summary, the effect of the different delays in cord clamping tested in this study on the clinical conversion rate of donated CBU is minimum.

One of limitations of our study is that it did not include a control arm with “no delay in clamping”, so preventing conclusions being drawn on the true impact of the delays. Since this study was conducted retrospectively, we were unable to extend the analysis further. For example, as all samples were collected in utero following vaginal delivery, the potential effect of clamping delays on units collected ex utero or following Cesarean section, which was considered in other papers, cannot be commented upon. Furthermore, the number of CBU collected with a clamping delay of ≥120 s was much smaller than the numbers in the other clamping delay categories, which might compromise the significance of our findings. In addition, the absence of TNC for some of CBU with an acceptable weight for processing might have had a negative impact on the overall results.

CONCLUSIONS

Our data showed that TNC was maintained independently of the clamping time assessed and only a progressive decrease of volume collected was observed. Expanding the use of CBU beyond transplantation further increases the clinical conversion rates making collection programs and banks more efficient. Therefore, the practice of delaying cord clamping to various extents (ranging from 30 s to 120 s) is compatible with public CBB needs.