Four phases of checks for exclusion of umbilical cord blood donors

Abstract

Aim

The aim of this study was to analyse umbilical cord blood (UCB) collection over 1 year between October 2008 and September 2009, seeking ways to improve the number of suitable banked UCB units. Four phases of the process were investigated, from the consent form to the banking procedure, paying attention to the discarded UCB units.

Material and methods

We recruited couples at 35 weeks of gestation and took an accurate history, focusing on genetic, immunological and infectious diseases. We collected UCB from pregnant women who delivered vaginally or by Caesarean section between the 37–41⁺⁶ weeks of gestation. Some units were discarded on the basis of the patients' history, obstetric events or biological criteria. In utero collection was the preferred method of collection.

Results

During the study period, between October 2008 and September 2009, there were 1,477 deliveries in our unit. The number of couples interested in UCB donation was 595 (40.2%-595/1,477). We collected 393 UBC units. We excluded 122 patients at the phase of the history taking, counselling and informed consent (first phase check). Of the 393 units collected, 162 (41.3%) were banked whereas 231 (58.7%) were discarded because they did not fulfil biological criteria (third phase check). The volume of UCB units collected after Caesarean section was greater than the volume of units collected after vaginal delivery (95.4 mL versus 85.0 mL, respectively; p <0.01). The UCB units collected after vaginal delivery contained a higher number of total nucleated cells compared to the units collected after Caesarean section (970x10⁶ cells versus 874x10⁶ cells, respectively; p=0.037). None of the banked UCB units was discarded at the clinical check 6 months after delivery (fourth phase check).

Conclusions

Our study shows that strict observance of each of the checks and the collection strategy is important to guarantee the safety of the UCB units and to maximise the cost-benefit ratio. After the appropriate checks we banked UCB units from only 27.2% (162/595) of the couples who gave consent to the procedure and from only 11% (162/1,477) of all the deliveries in the 12 month study period, as 59.8% of couples were not properly informed about UCB donation.

Introduction

For more than a decade, umbilical cord blood (UCB) has been attracting attention as a source of haematopoietic stem cells in alternative to the bone marrow and peripheral blood^1–3. The number of UCB transplants in the world has been increasing, as 18 years of research on haematopoietic stem cells from placental blood have shown the benefits of these cells over bone marrow or peripheral blood stem cells. Indeed, the use of haematopoietic stem cells from placental blood has several advantages: (i) no risk for the mother or neonate during the collection procedure, (ii) the product is ready to be used when necessary, (iii) no refusals at the time of donation, (iv) low risk of viral contamination of the blood unit, (v) weak immunological properties due to the immaturity of the human leucocyte antigen (HLA) system, (vi) low incidence of Graft-versus-Host Disease, which is the main cause of transplant failure, and (vii) easier recruitment of ethnic minorities.

In many circumstances, therefore, UCB stem cells are the treatment of choice for haematopoietic diseases, considering also the importance of perfect compatibility between the donor and the recipient^4,5.

The aim of this study is to analyse the activity of our Centre (Centre for the Umbilical Cord Blood Collection, CRaSCO, Obstetrics and Gynaecology Unit, "Di Venere" Hospital, Bari, Italy), with regards to the collection of UCB during a period of 1 year, between October 2008 and September 2009, looking for strategies to increase the number of UCB units for banking and to improve the quality of such units.

To this end, four particular moments were investigated: (i) consent to the UCB donation, given at 35 weeks of gestation after proper counselling which should involve the couple, (ii) collection of the UCB in the labour ward or in the operating theatre, (iii) quality control of the UCB units by the Cord Blood Bank and (iv) the maternal-neonatal check, 6 months after delivery, just before the final banking (Figure 1).

Figure 1.

Four phases of checks at which UCB donations may be excluded.

Materials and methods

We studied the donation of UCB in our Unit from October 2008 to November 2009. We provided proper counselling and obtained informed consent to UCB donation from pregnant women at 35 weeks of gestation and their partners. We also carefully collected the couple's history, focusing on genetic, immunological and infectious diseases on both the maternal and paternal sides (anamnestic criteria). We collected UCB units each day of the week, from pregnant women at 37 weeks of gestational age, or beyond.

There are two main techniques for collecting UCB from the umbilical vein. With the in utero technique, the blood is collected in the delivery room while the placenta is still inside the uterus. When the ex utero method is used, the UCB is collected in a different room from that in which the birth occurred, after delivery of the placenta. In our experience the in utero technique is preferable, because it is faster and simpler. Only one person is necessary and there is a lower risk of blood contamination, as it is not necessary to move the placenta before the blood is collected. Thirty seconds after delivery, the umbilical cord is clamped and cleaned with betadine solution. The UCB is collected from the umbilical vein with a 12.5-gauge needle connected to a sterile 150 mL bag (Maco Pharma S.A. Laboratoires Pharmaceutiques, France), containing 21 mL of phosphate-citrate-dextrose anticoagulant. The umbilical cord is "milked" and the uterus gently massaged during the last period of the collection in order to increase the blood flow.

In certain circumstances, units were not collected or were discarded on the basis of the following maternal and foetal obstetric criteria: (i) rupture of membranes more than 12 hours before the delivery, (ii) maternal fever >38 °C, (iii) green amniotic fluid, (iv) too quick expulsion of the placenta, (v) dystocia, pre-eclampsia, placental abruption, (vi) low Apgar score at birth (<7 at 1 minute and <5 at 5 minutes), (vii) low birth weight (<2,600 g), and (viii) congenital malformations.

After collection the UCB was sent to the haematology department, with the anamnestic form and six samples of maternal blood. After a first laboratory check, all the material was sent within 24–48 hours to the UCB bank, located in the "Casa Sollievo della Sofferenza" Research Institute in San Giovanni Rotondo (Italy), where the units were processed for cryopreservation.

Cord blood units were characterized, processed and cryopreserved within 48 hours of collection. Our threshold for accepting cord blood for banking is a total nucleated cell (TNC) count of 8x10⁶. Before cryopreservation, volume reduction, blood cultures (to verify sterility), CD34⁺ cell counts, and plating for colony-forming unit (CFU) assays were performed.

After a few weeks the UCB bank reports the results of the laboratory studies of the UCB units sent.

The laboratory criteria for excluding UCB from banking (biological criteria) are: (i) low cellularity (<8x10⁸), (ii) weight of the UCB unit bag <80 g, (iii) presence of blood clots, (iv) damage to the UCB bag, and (v) infections of the blood collected during the delivery. The last check of the units before definitive banking is performed 6 months after delivery, when it is determined whether the mother or infant has any clinical problems. The statistical significance (defined as p<0.05) of any differences between two groups was determined by Pearson's correlation coefficient and Student's t test (SPSS software version 10.0).

Results

During the study period from October 2008 to September 2009, there were 1,477 deliveries in our Unit (Obstetrics and Gynaecology Unit, "Di Venere" Hospital, Bari, Italy ).

Five hundred and ninety-five couples (40.2%) were interested in and consented to the collection of umbilical cord blood, but 122 were excluded for anamnestic reasons.

A further 80 UCB units were excluded at the time of collection in the labour ward or operating theatre. We, therefore, sent 393 units to the UCB bank, collected during 26.6% of the 1,477 deliveries in our Unit in 1 year. Following laboratory investigations, the UCB bank of the "Casa Sollievo della Sofferenza" Research Institute excluded 231 units (58.7% of the 393 donations sent from our Unit). Thus, in the end, just 162 UCB units were banked (41.3% of the 393 units collected), meaning that of 1,477 deliveries over the period of 1 year, only 10.9% culminated with the banking of UCB (Figure 2).

Figure 2.

Data from the Umbilical Cord Blood Collection Centre, Obstetrics and Gynaecology Unit, "Di Venere" Hospital, from October 2008 to November 2009.

We excluded 122 patients on the basis of anamnestic criteria at the time of taking the history: in 73 cases (60%) because of drug use/abuse, in 39 cases (32%) because of an autoimmune disease, unsafe sexual behaviour, or a positive blood test for viral infections; in 8% of the cases we found other diseases. We discarded 80 units in the labour ward/operation theatre, at the time of the UCB collection, according to obstetric criteria: there were 22 cases (27.5%) of green amniotic fluid, 22 cases (27.5%) of early separation of the placenta, during the blood collection, 18 cases (22.5%) of fever, 10 cases (12.5%) of membrane rupture more than 12 hours prior to delivery and 8 cases (10%) of low birth weight babies (weight less than expected according to the gestational age of the delivery, and anyway less than 2600 g) or neonates with an Apgar score less than 5 at 1 minute and 5 minutes after birth.

Of the 393 units collected, 205 (52.2%) were discarded on the basis of biological criteria because of either low cellularity or low weight of the UCB unit (Table I).

Table I.

Data from the Umbilical Cord Blood Collection Centre, Obstetrics and Gynaecology Unit, “Di Venere” Hospital, from October 2008 to September 2009.

Month	Deliveries			Units collected		Excluded		Low cellularity
	VD	CS	Total	Total	%	Total	%	VD	CS	Total	%
October	53	75	128	29	22.6	15	51.7	3	12	15	51.7
November	47	68	115	37	32.0	19	51.3	5	10	15	40.0
December	54	68	122	36	29.7	15	41.6	7	8	15	41.6
January	51	67	118	16	29.4	5	31.2	2	3	5	31.2
February	43	66	109	24	21.1	19	79.1	10	8	18	75.0
March	57	79	136	44	32	22	50	7	9	16	36.3
April	35	73	108	39	36	27	69.2	8	15	23	58.9
May	48	73	121	43	35.5	25	58.2	10	10	20	46.5
June	49	74	123	33	26.8	26	78.7	10	13	23	69.7
July	43	84	127	31	24.4	22	70.9	6	14	20	64.5
August	56	74	130	32	24.6	20	62.5	8	11	19	59.3
September	56	84	140	29	21	16	55.0	6	10	16	55.0
Total	592	885	1,477	393	26.6	231	58.7	82	123	205	52.2

VD, vaginal delivery; CS, Caesarean section.

We observed that the volume of UCB collected correlated positively with cell recovery (r=0.74, p<0.01 with TNC and r=0.4, p<0.01 for CD34⁺ cells). Both these biological features were positively correlated with birth weight (r=0.26, p<0.01 for volume, and r=0.33, p<0.01 for TNC). An interesting (although modest) positive correlation was found between TNC content and gestational age (r=0.16, p=0.012).

A subsequent analysis was performed comparing biological features and type of delivery, considering that the preferred technique for collecting UCB was the in utero technique. It was found that the volume of the UCB units collected after Caesarean section was higher than that of units collected after vaginal delivery (95.4 mL versus 85.0 mL, respectively; p<0.01). UCB units collected after vaginal delivery contained more TNC than units collected after Caesarean delivery (9.70x10⁸ cells versus 8.74x10⁸ cells, respectively; p=0.037) (Table II).

Table II.

Biological characteristics of 393 UCB donations according to the type of delivery in the "Di Venere" Collection Centre.

	Vaginal deliveries	Caesarean sections	P
Volume (mL) (n=383)	85.0±26.3	95.4±23.7	0.01
TNCx106 (n=380)	970±497.0	874±352.0	0.037
CD34+x104 (n=169)	317.5±210.0	309.8±203.8	NS

Data are expressed as mean ± standard deviation. P values <0.05 are considered statistically significant. NS, not significant.

Six months after donation of the units, the fourth and last phase of the assessment of the suitability of the UCB was made. This clinical check showed that the health status of all 162 mother and infant pairs was normal and, therefore, no UCB units were discarded after this last check.

Discussion and conclusions

In our population the main cause of exclusion of the couple at the time of history taking and consent (first phase check) was "drug abuse", accounting for 60% of the 122 cases excluded at this phase. In the second phase check, in the labour ward/operating theatre, the most frequent cause of exclusion was "green amniotic fluid" and "too quick expulsion of the placenta, during the blood collection", accounting for 27.5% of the units discarded in this phase. No UCB units were discarded as a result of the fourth phase check.

Absolute requirements for the success of a transplant are the cellularity of the blood unit (haematopoietic precursor cells and CD34⁺ cells), and the HLA compatibility between the donor and recipient⁴. In our study many units had to be excluded either because of the low cellularity of the unit or because the volume of blood collected was insufficient (third phase check). To be precise, 205 blood units were excluded for these reasons, that is, 52.2% of the 393 units collected. (Table I).

The collection strategy is the first key step in collecting good quality UCB units, with a proper volume of blood, suitable for banking and donation. According to the literature, factors that minimise the problems of low cellularity and small volume of blood collected include: (i) placing the neonate on the mother's abdomen immediately after birth; (ii) the correct timing of clamping the cord⁷; (iii) the position of the placenta at the time of blood collection^10–14.

Several studies have shown that the total blood volume collected and the cellularity of UCB units could be influenced by several obstetric factors: placental weight (volume and cellularity improved when the placenta was >600 g), the neonate’s birth weight (>3390 g), gestational age (>39 weeks), foetal blood pH, duration of labour, the length of the umbilical cord (>55 cm), the experience of the person collecting the blood, placing the neonate on the mother’s abdomen just after birth and the correct timing of clamping the cord (>30 sec, <1 min)^6–10.

We observed that the volume of blood collected was positively correlated with cell recovery: a larger volume of collected blood was related to a higher number of TNC (r=0.74, p<0.01 with TNC and r=0.4, p<0.01 for CD34⁺ cells). Both these biological features also positively correlated with birth weight, such that heavier infants gave richer units, both in terms of volume and TNC number (r=0.26, p<0.01 for volume, and r=0.33, p<0.01 for TNC). An interesting (albeit modest) positive correlation was found between TNC content and gestational age, focusing our attention on longer lasting pregnancies (r=0.16, p=0.012).

As already described, there are two main techniques for collecting UCB: in utero and ex utero. Surbek and Solves compared both collection strategies after vaginal deliveries, and concluded that in utero collection was associated with a significantly higher volume and TNC number. Solves found that the proportion of UCB units excluded, before processing, was 33% for the ex utero technique and 25% for the in utero technique^11,12. With regards to the ex utero technique, the presence of haemorrhagic foci in the delivered placenta and of blood clots in the placental foetal vessels could explain the lower levels of haematopoietic progenitors in UCB collected using this technique. Solves and Tamburini compared the in utero and ex utero techniques after delivery by Caesarean section and found that there was no significant difference in cell content between UCB units collected with the two techniques^13,14. We evaluated whether the type of delivery (vaginal delivery versus Caesarean section) influenced the volume and the biological features, such as white blood cell count, TNC count and CD34⁺ cell fraction, of the collected UCB units.

We collected UCB while the placenta was in utero. We obtained units of larger volume after delivery by Caesarean section, but it was after vaginal deliveries that we obtained UCB units with an higher content of TNC, which is an important requirement for the success of a transplant.

Considering that 595 patients were referred to our centre for donation of UCB and that all of them delivered in our hospital (among a total of 1,477 deliveries from October 2008 to November 2009), we can conclude that the percentage of patients interested in UCB donation was 40.2%. Although such a percentage could appear satisfactory, it is important to be aware that a high number of units were discarded through the four phases of assessment. Indeed, at the end of the assessment process we banked UCB units from only 27.2% of the couples who gave consent, and 11% of the deliveries in the 12-month study period.

In conclusion, the history taking must be scrupulous, the collection method performed correctly and the obstetric exclusion criteria applied rigorously in order to increase the number and improve the quality of UCB units available for biological laboratory checks and for the subsequent, potential banking. The strict observance of the various checks and the collection strategy is important to guarantee the safety of the UCB units and ensure a good cost-benefit ratio.

The best way to improve the number of banked UCB units appears to be the choice of a proper collection strategy, which should take into account the collection technique (vaginal delivery and in utero collection are associated with higher quality UCB units) and the time of delivery (the quality of UCB units is positively correlated with birth weight and gestational age). Finally, in order to increase the number of units banked it is also important to make as many couples as possible aware of the possibility of UCB donation.