Title: Allogeneic umbilical cord red blood cell transfusion for children with

severe anaemia in a Kenyan hospital: an unmasked, single arm trial to assess

safety, harm and efficacy.

Oliver W. Hassall1, 2, Johnstone Thitiri1, Greg Fegan1, 3, Fauzat Hamid1, Salim

Mwarumba1, Douglas Denje4, Kongo Wambua5, Kishor Mandaliya4, 5, Prof.

Kathryn Maitland1, 6, Prof. Imelda Bates2

1 Centre for Geographic Medicine Research (Coast), Kenya Medical Research

Institute/ Wellcome Trust Research Programme, Kilifi 80108, Kenya2 Liverpool School of Tropical Medicine, Liverpool L1 5QA, United Kingdom3 Centre for Clinical Vaccinology & Tropical Medicine, University of Oxford,

Oxford OX3 7LJ, United Kingdom.4 Coast Provincial General Hospital, Mombasa 80100, Kenya5 Regional Blood Transfusion Centre, Mombasa 80100, Kenya6 Department of Paediatrics, Imperial College London, London SW7 2AZ,

United Kingdom

Corresponding author: Dr. Oliver Hassall (address as 2. above)

ohassall@gmail.com

+ 44 (0) 7774 354716

1

AbstractBackground

Severe anaemia requiring an urgent blood transfusion is common in

hospitalised children in sub-Saharan Africa but blood is frequently

unavailable. Where conventional blood supplies are inadequate, allogeneic

umbilical cord blood may be a feasible alternative. Transfusion of cord blood

is a novel concept in sub-Saharan AfricaT he aim of this trial was to assess

theso safety and efficacy of cord blood transfusion in children with severe

anaemia.need to be demonstrated.

Methods

Cord blood was donated at Coast Provincial General Hospital and screened

for transfusion-transmitted infections and bacterial contamination. Red cells

were produced by sedimentation during refrigerated storage. Children with

severe anaemia but without signs of critical illness were recruited at Kilifi

District Hospital and received a maximum of two group identical/compatible

cord blood units. Participants were closely monitored for adverse events and

followed up for one month. The primary outcome measure was the frequency

and nature of adverse reactions associated with the transfusion. Secondary

outcome measures were change in haemoglobin at 24 hours and one month

after transfusion compared to pre-transfusion levels. The study has been

completed. (Trial registration:ISRCTN66687527)

Findings

Fifty-five children received sedimented red cells from 74 cord blood donations.

Ten children experienced 10 serious adverse events and 43 children

experienced 94 non-serious adverse events. In none of these cases did an

independent expert panel consider cord blood transfusion to be probably or

certainly implicated (one-sided 97.5% confidence interval; 0 to 6.5%). The

median rise in haemoglobin was 2.6g/dL (IQR 2.1g/dL to 3.1g/dL) 24 hours

after transfusion, and 5.0g/dL (IQR 1.0g/dL to 6.8g/dL) 1 month after

transfusion.

Interpretation

2

The results haematological efficacy and absence of any adverse reactions

associated with umbilical cord red blood cell transfusion in children with

severe anaemia demonstrated by this single arm trialstudy, justify further

studiestrials comparing the safety and efficacy of cord blood transfusion and

conventional adult-donated blood transfusion. Such trials should include

operational analyses of the availability of cord blood and conventional blood.

Challenges associated with cost, infrastructure and scale up also need

exploring. Cord blood may be an important supplementary source of blood for

transfusion in children in sub-Saharan Africa. Funding Wellcome Trust

Training Fellowship (073604)

3

IntroductionBackground

Sub-Saharan Africa has the highest risk of death in the first month of life and

is one of the regions showing the least progress in reducing this high mortality

rate (1).1 Severe anaemia is a major public health problem in sub-Saharan

Africa and children aged less than 2 years are the most frequently affected.

The prevalence of severe anaemia in hospitalised children is reported to

range from 8-29% with case fatality rates of 8-18% (2).2 In children with

severe, uncompensated anaemia, blood transfusion can substantially reduce

mortality (3).3 Over 50% of deaths occur within 4 hours of admission and early

intervention and the ready supply of safe blood are key components of the

hospital treatment of severe anaemia in childhood (4, 5).4,5 The supply of

conventional blood for transfusion in sub-Saharan Africa is insufficient with

only an estimated 52% of demand being met and a shortfall of at least 2

million units a year (6-8).6–8

Where the blood supply is limited and young children receive a significant

number of blood transfusions, umbilical cord blood is a novel and potentially

important source of blood for transfusion (9-11).9–11 Not only might cord blood

provide increased numbers of small volume transfusions but also reduce the

pressure on stocks of conventional, adult-donated blood thereby improving

the supply of blood for emergency transfusions for other vulnerable groups. In

sub-Saharan Africa, lack of blood for transfusion is implicated in 25% of

maternal deaths due to haemorrhage (12).12

In order to test the feasibility of cord blood transfusion, we have established a

cord blood donation programme on the labour ward at Coast General

Provincial Hospital in Mombasa, Kenya. Previously we have demonstrated the

acceptability to mothers of cord blood donation and transfusion; the feasibility

of a two-stage informed consent process for cord blood donation; and the

quality of variable volumes of whole cord blood stored in a fixed volume of

anticoagulant-preservative solution (13, 14).13,14 We have also shown that, for

cord blood collected by our study team, rates of both bacterial contamination

and seroreactivity for HIV, HBV, HCV and syphilis compare favourably to that

4

of conventional adult blood donated to the Regional Blood Transfusion Centre

in Mombasa (15).15 Here we report the findings of, to our knowledge, Here we

report the findings of the first clinical trial of allogeneic cord blood transfusion

in children with severe anaemia.

Objectives

The primary objective of the study was to assess the frequency and nature of

adverse reactions associated with umbilical cord red blood cell (UC-RBC)

transfusion. The secondary objective was to assess the haematological

efficacy of UC-RBC transfusion.

MethodsTrial design

This was an unmmasked, single arm trial designed to produce preliminary

data on safety, harm and haematological efficacy of umbilical cord blood

transfusion in children with severe anaemia. The protocol was reviewed and

approved by the Kenya National Ethics Committee and the Research Ethics

Committee of the Liverpool School of Tropical Medicine. The trial is registered

as an International Standardised Randomised Controlled Trial, number with

the ISRCTN (66687527).

Participants and study setting

Participants were recruited from children aged less than 12 years admitted for

paediatric in-patient care at Kilifi District Hospital (KDH), Kenya from 26th June

2007 to 20th May 2008. Eligibility criteria were designed to identify those

children for whom a transfusion would provide clinical benefit based on WHO

clinical guidelines (16) but exclude those who were critically ill.16

Research staff from the KEMRI-Wellcome Trust research programme provide

24 hour clinical cover at KDH and at admission all children have a structured

clinical assessment, including anthropometry and a standard set of; and

laboratory investigations, including a haemoglobin concentration (Hb)

estimation (Beckman Coulter, France), a blood film examination for malaria

and a blood culture. Haemoglobin electrophoresis to detect haemoglobin S

5

was done retrospectively for study children aged greater than 3 months of

age. Full investigation of the aetiology of severe anaemia was not part of the

study protocol.

Children were eligible for inclusion in the study if they had severe anaemia

(Hb 10g/dL in children aged 3 months or less; Hb 4g/dL in children aged

greater than 3 months) and the attending clinician requested a blood

transfusion. Children with any of the following clinical features of critical illness

were excluded: coma (Blantyre Coma Scale 2), prostration, shock, deep

(acidotic) breathing, and hyperbilirubinaemia requiring exchange transfusion.

In addition children were not eligible for the study if they had had a previous

UC-RBC transfusion as part of this trial or were already enrolled in another

intervention trial. A cChildren wasere only enrolled in the study if sufficient

cord blood was available and w.ritten consent was given by their caregiver.

The intervention

The intervention under investigation was the transfusion of umbilical cord red

blood cells (UC-RBC). Cord blood was collected from placentas donated at

Coast Provincial General Hospital in Mombasa and screened (for HIV,

hepatitis B and C and syphilis) as described previously (15).15

Screened cord blood units were transported at 2-6C 50km by road to Kilifi,

sedimented by storing vertically in racks at 2-6C and quarantined until

screened for bacterial contamination. This was done by incubation of a 4ml

sample in 40ml of brain heart infusion at 37C in the manner described

previously (15, 17).15,17 Incubation was for 48 hours and screening was by

microscopic examination of a Gram stained smear.

Volume, haemoglobin concentration and blood group of cord blood units were

entered on an electronic database, which was used to ascertain whether

sufficient cord blood was available as soon as a blood transfusion was

requested for an eligible child. This was defined as at least 2.2g/kg of

haemoglobin from a maximum of two group identical and/or blood group

6

compatible cord blood units. Thus cord blood units were selected based on

estimated haemoglobin content rather than volume. In addition, no child was

transfused more than 3.5ml/kg of CPDA-1.

The hospital clinical laboratory used standard methods of blood grouping and

crossmatching. In children without severe acute malnutrition (SAM) (defined

as weight-for-height Z-score (WHZ) < -3), UC-RBC were transfused over 4

hours with no co-administration of furosemide and a maximum permitted

volume of 20ml/kg. In children with SAM, UC-RBC were transfused over 3

hours with a maximum permitted volume of 10ml/kg; and 1mg/kg of

furosemide administered intravenously at the start of the transfusion as per

clinical guidelines (16).16

Outcomes

The outcome measure to achieve the primary objective was the frequency

and nature of adverse reactions occurring during, or within at least one month

of, UC-RBC transfusion. Serious adverse reactions (SAR) were defined as

any serious adverse event (SAE)1 that was judged probably or certainly

related to the transfusion. Adverse reactions were defined as any adverse

event (AE)2 judged probably or certainly related to the transfusion. The

detection of adverse reactions was a two-stage process comprising the

rigorous surveillance of adverse events (monitoring of harm) and an

independent, expert judgement about their relationship to UC-RBC

transfusion (assessment of imputability).

Monitoring of harm (Figure 1)

Monitoring of harm was by both passive and active surveillance. Children

recruited to the study were admitted to a paediatric high dependency unit until

24 hours after the start of the transfusion. During the transfusion and for two

hours afterwards children had continuous physiological monitoring.

Temperature, pulse rate, respiration rate, oxygen saturation, and blood

1 Any untoward medical occurrence that is fatal, life-threatening, disabling, prolongs

hospitalisation, or results in hospitalization.18 (18)2 Any untoward medical occurrence.18 (18)

7

pressure were recorded before the start of the transfusion, 15 minutes after

the start of a transfusion and every 30 minutes thereafter for the duration of

the transfusion and for 2 hours subsequently. Two hours after the start of the

cord blood transfusion, a blood sample was obtained for the estimation of

serum potassium (Ilyte Ion Selective Electrode Analyser; Instrumentation

Laboratory, US) and calcium (Selectra E; Vitalab, The Netherlands).

A clinician reviewed every child and performed a study-specific structured

clinical assessment designed to capture adverse events 2 hours after and 24

hours after the end of a transfusion, and at hospital discharge. For the rest of

the child’s admission, monitoring of harm was by review of the daily clinical

record kept by the attending clinicians.

At hospital discharge, carers of children recruited to the study were given the

cost of their fare home and the return fare back to the hospital and invited to

bring the child to the hospital one month after the cord blood transfusion. They

were encouraged to come back to the hospital before then if they had any

concerns about their child. In addition, details of their homestead location

were taken. Children who returned to the hospital had a structured clinical

assessment. Those who did not attend were followed up at home by a

fieldworker, who confirmed whether the child was alive and well by direct

observation of the child and/or discussion with an adult family member.

Carers were also encouraged to bring these children to the hospital for a full

review.

Assessment of imputability of adverse events

The Principal Investigator (OH) and the Local Safety Monitor (LSM; an

experienced consultant paediatrician) reviewed all serious adverse events

and prepared a case summary, which was sent to the Safety Review

Committee (SRC). The SRC comprised 3 paediatricians with extensive

experience of the clinical care of children in sub-Saharan Africa and who were

independent of the study. The SRC and the LSM came to a consensus

decision regarding the probability that an SAE was caused by the transfusion

8

of UC-RBC and assigned it an imputability score based on an established 4-

point scale (19).19

All other (non-serious) adverse events were reviewed by a study clinician and

the Principal Investigator. They were described according to an established

adverse reaction nomenclature (COSTART: Coding Symbols for a Thesaurus

of Adverse Reaction Terms (20))20 and the probability of a causative

relationship with UC-RBC transfusion scored according to the same 4-point

scale. A summary of these adverse events was reviewed by the LSM and

SRC.

Outcome measures for the secondary objective

The outcome measure used to achieve the secondary objective was median

change in haemoglobin concentration compared with pre-transfusion levels

one day and one month after UC-RBC transfusion. A blood sample for

haemoglobin estimation (Beckman Coulter, France) was taken 24 hours after

the start of UC-RBC transfusion; unless a haemoglobin was requested for the

clinical management of the child before this time in which case this result was

used. A further blood sample for haemoglobin estimation was obtained from

those children who returned for follow-up at one month.

Sample size

It was estimated that 100 children fulfilling the eligibility criteria for the trial

would be admitted to KDH during a period of one year and that cord blood

would be available and consent to transfuse given for 40-80% of these. Thus,

during one year of study 40 to 80 children might be recruited to the trial. We

intended to run the trial for one year and these numbers were set as a

minimum and maximum sample size. The precision, as indicated by a

confidence interval, of the frequency of adverse reactions (the primary

outcome measure) at different event frequencies and sample sizes is shown

in Table 1 of the appendix.

Stopping rules

9

The trial was to be stopped in the event of a Suspected Unexpected Serious

Adverse Reaction (SUSAR), and not recommenced until a full review had

been undertaken by the SRC and their recommendations seen and approved

by the ethics committees. In addition, in the event of an SAE the SRC advised

whether they felt that the trial should continue with no change to the protocol,

continue with a change to the protocol, or be stopped.

Statistical methods

Binary data were expressed as a percentage with 95% confidence intervals

where appropriate. Where event frequencies were zero, a one-sided 97.5%

confidence interval with a lower limit of zero was calculated. Continuous data

were summarised by the median with range (minimum and maximum) and

interquartile ranges (IQR). Observed differences in continuous data were

compared for statistical significance using non-parametric statistics (Wilcoxon

rank-sum).

Role of the funding source

OH was supported by a Wellcome Trust Training Fellowship (073604). The

funder had in no role in design of the study; in the collection, analysis, and

interpretation of data; in the writing of the report; and in the decision to submit

the paper for publication.

ResultsParticipant flow and recruitment (Figure 12)

There were 413 transfusion episodes to children over the period of the study

and 87 children were considered eligible for the trial. In 24 cases, UC-RBC of

sufficient haemoglobin content and/or blood group were not available and

consent was declined for 6 children. Thus, 57 children were recruited to the

study but 2 were withdrawn before UC-RBC transfusion. In one case, the

laboratory made an error during compatibility testing and no further cord blood

was available. In the second case, clinical review soon after recruitment

demonstrated deep breathing (see exclusion criteria).

Demographic and clinical characteristics of participants (Table 12)

10

Fifty-five children received UC-RBC from 74 cord blood donations. Ages

ranged from 2 days to 5 years and 8 months (median, 12 months) with 24

children aged 3 months or less. Weights of children ranged from 1.1kg to

14.5kg (median, 5.3kg). WHZ-scores ranged from -4.4 to -0.9 (median, 1.9)

and 7 children had severe acute malnutrition (defined as a WHZ< -3). In those

children aged 3 months or less, pre-transfusion haemoglobin ranged from

5.5g/dL to 10g/dL (median, 8.7g/dL). In those children aged greater than 3

months, pre-transfusion haemoglobin ranged from 1.9g/dL to 4.0g/d/L

(median, 3.2g/dL). All children with SAM received 10ml/kg of UC-RBC; for

those children without SAM, the median volume transfused was 13ml/kg

(range, 10ml/kg to 20ml/kg).

Numbers analysed

In the event of an adverse event after a child had received UC-RBC from 2

units it is very unlikely that imputability could have been assigned to one of

the two units, therefore the denominator for the primary outcome measure

was the number of children transfused. Children who received subsequent

conventional blood transfusions during the follow up period were included in

the analysis of the primary outcome as these transfusions could themselves

be evidence of harm related to UC-RBC transfusion. However, these children

were not included in the analysis of haemoglobin change at 1 month as the

subsequent transfusions would have confounded the effect of UC-RBC

transfusion.

Outcomes (Table 3)

Of the 55 children who received UC-RBC transfusion, 10 experienced 10

serious adverse events (SAE) and 43 experienced 94 adverse events (AE)

(Table 2). In no case (0/55) was UC-RBC transfusion considered probably or

certainly implicated and thus the frequency of serious adverse reactions and

adverse reactions was 0% (One-sided 97.5% confidence interval; 0 to 6.5%).

There is, therefore, a 2.5% chance that the frequency of adverse reactions

associated with UC-RBC transfusion exceeds 6.5%.

11

The median change in haemoglobin on the day after UC-RBC transfusion

(median 24 hours; IQR 17 – 24 hours) was a rise of 2.6 g/dL (IQR 2.1 – 3.1)

(Table 3). In the 33 children who did not receive a further transfusion and for

whom a blood sample was obtained, the median change in haemoglobin at

one month (median 29 days; IQR 28 – 35) was a rise of 5.0g/dL (Table 3).

Four of the seven life-threatening SAE were new signs of critical illness (deep

breathing and/or prostration) observed after recruitment at the pre-transfusion

assessment and before the transfusion of UC-RBC. UC-RBC transfusion was

therefore excluded as a potential cause of these SAE. Of the remaining 6

SAE, one was a death, 3 were judged life-threatening and 2 resulted in

hospitalisation after discharge (Table 4). These are described in detail below

(see also Table X in the appendix):

Study number: WG014

SAE severity: Fatal (Time after start of UC-RBC transfusion: approx. 7 days)

Imputability level: Unlikely

A 13-month old girl admitted with fever and cough and treated for pneumonia.

Her admission Hb was 4.6g/dL but 5 days later this had dropped to 4g/dL and

she received 100ml of UC-RBC, which raised her Hb to 6.5g/dL. Five days

later, she was diagnosed with an acute lympoblastic leukaemia by

microscopic examination of a peripheral blood film taken before UC-RBC

transfusion but reported afterwards (21% lymphoblasts). She was referred to

the local regional hospital for further management but left against medical

advice and taken by bus to her mother’s marital home in Kisumu in western

Kenya- a journey of approximately 850km. At discharge she had been noted

to be ‘pale, sick-looking and febrile’. On follow-up in the community, the child’s

grandfather reported that she died the day after arriving in Kisumu,

approximately 1 week after UC-RBC transfusion.

Study number: WG034

SAE severity: Life-threatening (26 hours)

12

Imputability level: Possible

A male infant with a birth weight of 1080g, delivered by emergency caesarean

section for maternal ante-partum haemorrhage at approximately 28 weeks

gestation. His initial Hb was 14.9g/dL and over the first 3 weeks of life he was

treated for neonatal sepsis and jaundice. On day 23 of life, weighing 1120g,

he was noted to be pale and had an Hb of 8.8g/dL. He was transfused 20ml of

UC-RBC, which increased his Hb to 15.5g/dL. Twenty-six hours after UC-RBC

transfusion, he became unwell with diarrhoea, dehydration and a metabolic

acidosis and was managed as probable neonatal sepsis. He made a full

recovery after intervention with oxygen, broad-spectrum antibiotics and

intravenous fluids.

Study number: WG044

SAE severity: Life-threatening (7 days)

Imputability level: Unlikely

A pre-term infant boy born unexpectedly while his mother was walking to work

on the family’s shamba and admitted on day 1 of life with an estimated

gestation of 30 weeks and a weight of 1440g. His admission Hb was 9.4g/dL

and he was transfused UC-RBC on day 2. Seven days after UC-RBC

transfusion, he developed abdominal distension, respiratory distress and

jaundice. This was managed as probable neonatal sepsis and he made a full

recovery after intervention with broad-spectrum antibiotics, intravenous fluid

therapy and phototherapy.

Study number: WG049

SAE severity: Life-threatening (14 hours)

Imputability level: Possible

A female infant born at home and admitted on day of birth weighing 1200g,

with an estimated gestation of 30 weeks and an Hb of 11.1g/dL. On day 9,

weighing 1180g, she was noted to be pale, her Hb was 7.6g/dL and she was

transfused 18ml UC-RBC raising her Hb to 11.0g/dL. Fourteen hours after

UC-RBC transfusion she was febrile, tachypnoeic and having apnoeas. She

was presumed septic and managed with broad-spectrum antibiotics,

intravenous fluids, supplemental oxygen and aminophylline. A chest

13

radiograph demonstrated right upper lobe consolidation consistent with a

diagnosis of pneumonia. She made a full recovery.

Study number: WG054

SAE severity: Hospitalisation (28 days)

Imputability level: Unlikely

A 4-year old boy with known sickle cell disease admitted with respiratory

distress and an Hb of 4.4g/dL. A blood transfusion was requested but he was

ineligible for UC-RBC transfusion. No adult-donated blood was available until

a replacement donor was found the following day, after which he was

transfused 20ml/kg of whole blood resulting in a post-transfusion Hb of

8.3g/dL. Over the next 10 days he was treated for sepsis and his Hb fell to

3.6g/dL over this period. He was transfused 150ml of UC-RBC from two cord

blood donations, his Hb rose to 6.2g/dL and he was discharged well with oral

haematinics 3 days later. He returned mistakenly for study follow-up at 21

days after UC-RBC transfusion and at that time had an Hb of 6.6g/dL and was

well. At his 28-day study follow-up assessment, he was found to have an Hb

of 4.1g/dL, was admitted for a conventional blood transfusion and was

discharged the following day with an Hb of 6.1g/dL.

Study number: WG056

SAE severity: Hospitalisation (28 days)

Imputability level: Possible

A female infant born in hospital and admitted to paediatric ward at 19 hours of

age with jaundice, low birth weight (2300g) and suspected prematurity, and

treated for possible sepsis. At 5 days of age was noted to be pale, had an Hb

of 9.7g/dL, and was transfused 25ml of UC-RBC which raised her Hb to

11.5g/dL. She was discharged well 3 days later fully breastfeeding and with a

weight of 2120g. At 28-day follow-up, she was found to have an Hb of 6.2g/dL

and was offered admission but her mother declined. She re-presented one

week later and was admitted. Her Hb was 8.4g/dL and a blood transfusion

was requested. No blood was available until a relative was found to donate,

after which she received 20ml/kg whole blood and was discharged well on

oral haematinics with an Hb of 14.1g/dL.

14

The median change in haemoglobin on the day after UC-RBC transfusion

(median 24 hours; IQR 17 – 24 hours) was a rise of 2.6 g/dL (IQR 2.1 – 3.1)

(Table 5). In the 33 children who did not receive a further transfusion and for

whom a blood sample was obtained, the median change in haemoglobin at

one month (median 29 days; IQR 28 – 35) was a rise of 5.0g/dL (Table 5).

Ancillary analyses

In children aged less than 3 months, median change in haemoglobin at 1

month (median 29 days; IQR 28 – 36) was a rise of 0.5g/dL (IQR 0.2 – 1.2)

compared with a rise of 6.1g/dL (IQR 5.3 – 8.1) in children aged greater than

3 months (median time to follow-up, 30 days; IQR 28 – 35) (Table 5). This

difference is statistically significant (p<0.001).

In those children aged greater than 3 months, the 7 children with severe acute

malnutrition who received a maximum of 10mL/kg UC-RBC showed a median

haemoglobin rise 1 day after transfusion of 2.1g/dL (IQR 2.0 – 2.9). In 23

children aged greater than 3 months without severe acute malnutrition who

received a maximum of 15mL/kg the median rise in haemoglobin 1 day after

transfusion was 2.6g/dL (IQR 2.2 – 3.1). The difference is not statistically

significant (p=0.15, Wilcoxon rank sum). For the 5 children aged greater than

3 months with severe acute malnutrition for whom a result was available, the

median rise in haemoglobin 1 month after UC-RBC transfusion was 8.1g/dL

(IQR 7.8 – 8.2) compared to 5.9g/dL in the 15 children without severe acute

malnutrition for whom there was a haemoglobin result.

DiscussionIn tThis study 55 children with severe anaemia were explored the novel

concept of transfusedusing allogeneic umbilical cord sedimented red blood

cells from 74 umbilical cord blood donations transfusion in children with

severe anaemia in sub-Saharan Africa and assessed the safety, efficacy for

improving haemoglobin levels and any potential harm. Of 10 serious adverse

events and 94 adverse events, none were certainly or probably attributable to

cord blood transfusion. Median A increasesrise in haemoglobin after

15

transfusion levels were 2.6g/dL at was noted within 24 hours24 hours and

5.0g/dL at 29 days.1 month after cord blood transfusion and no adverse

reactions were attributable to the transfusion. These findings areis is

consistent with the very few data concerning allogeneic cord blood transfusion

that have been reported previously (9).9

Although we excluded children with signs of critical illness from the study at

the time of recruitment, many children experienced adverse events which

were unrelated to the cord blood transfusion. In four children, signs of critical

illness not present at recruitment and which would have excluded them from

the study were detected at the clinical assessment undertaken just prior to

UC-RBC transfusion. To withdraw these children from the study at this stage

and to secure and crossmatch adult-donated blood would have introduced an

unacceptable delay in the management of critically ill children. This highlights

the challenge of conducting studies focusing on safety and harm in

hospitalised children in sub-Saharan Africa. Rand the need for a robust

monitoring frameworks are required to identifytease out potential associations

between the effects of the intervention and other confounding factors. A

weakness of this study is that for those that did not attend the hospital for

follow-up at 28 days there was no full clinical assessment. However, all these

children were followed up in the community by a non-clinical fieldworker and

the death of one child (WG014) was identified in this way though as this

number was small it is unlikely that it unduly influenced the findings.

The rise in haemoglobin observed 1 day after UC-RBC transfusion seen in

this study is consistent with estimations based upon the haemoglobin content

of the transfused blood and the circulating volume of children: for a child with

a circulating volume of 80mL/kg, the transfusion of 2.2g/kg of haemoglobin

might be expected to raise the haemoglobin by 2.8g/dL. However, although

cord blood units were selected for transfusion based upon an estimation of

the unit haemoglobin content, it is not possible to ascertain from these data

how much haemoglobin was actually issued and transfused to each child.

16

The significant rise in haemoglobin 1 month after transfusion in children aged

greater than three months is consistent with previous data from Kilifi and other

sites in East Africa (3, 4, 21, 22).3,4,21,22 Increases in haemoglobin over a

similar time period are also observed in children with severe anaemia who do

not receive a transfusion and survive (3, 21, 22).3,21,22 This highlights the

importance of other therapies in the management of severe anaemia such as

treatment of infection, anti-helminthics, haematinics and diet. The relative

importance of these will depend on the aetiology of the anaemia and this was

not investigated here.

The children aged less than 3 months in this study were likely to have a very

different aetiology for their anaemia than the older children and many of them

were presumed to have anaemia of prematurity. Several of these children

required further transfusion and in those that did not the effect of a single UC-

RBC transfusion at one month was much more modest. Of note, however, is

the number of young infants that were eligible for UC-RBC transfusion. This is

a group of patients that carry a high burden of mortality in sub-Saharan Africa

and who may potentially benefit substantially from more evidence about the

role of transfusion in preventing the high death rates (1).1 These young

children might particularly benefit from the availability of cord blood for

transfusion because they only require small volumes of blood for transfusion.

The microbiological safety of cord blood provided by the donation programme

that we have established at Coast Provincial General Hospital in Mombasa

compares favourably to that of conventional blood from the same setting

(15).15 Mothers who donate their infants’ umbilical cord blood are rigorously

selected (including self-reporting of antenatal testing for syphilis and HIV), and

aseptic cord blood collection undertaken by trained fieldworkers and not the

midwives managing the deliveries (13, 15).13,15 Furthermore, all cord blood

donations in this study were screened for bacterial contamination. These

rigorous techniquesconditions may be difficult to replicate outside of a

research setting without considerable additional resources.

17

Our findings suggest that further trials of umbilical cord blood transfusions are

warranted but the challenges of conducting such trials and the barriers to

potential scale up of such an intervention should not be underestimated.

Attributing effects to the intervention is difficult in such a sick group of

children. Despite this, further clinical trials should also include children with

signs of critical illness who potentially have the most to gain from an improved

blood supply. The infrastructure and training required to set up collection and

administration of umbilical cord blood is complex and such trials would require

meticulous monitoring during and after the transfusion. Poor haemovigilance

systems in these settings means that very little is known about the harms

associated with conventional blood transfusion which would be the

comparator group in such trials (7).7

Other improvements and additions to the design of future trials include: better

characterisation of anaemia aetiology and assessment of any correlation with

benefits and harms of cord blood transfusion; immunological and genetic

testing to compare rates of alloimmunisation and microchimerism; and

operational analyses comparing the availability of cord blood and adult-

donated blood for urgent transfusion in children, and the impact of cord blood

transfusion for children on the blood supply for adults requiring larger volumes

of conventional blood.

In this study, we have demonstrated that where demand for low volume

transfusions for children is high and supplies of conventional blood are limited,

umbilical cord blood may be a safe and efficacious supplementary source of

blood for transfusion. Further trials comparing cord and conventional blood

transfusion are merited.

Nevertheless our data have demonstrated a low frequency of adverse

reactions associated with UC-RBC transfusion which would merit carefully

designed randomised clinical trials of cord and conventional blood transfusion.

These should take place in low-income settings where conventional blood is

in short supply and paediatric blood use is high.

18

Conflicts of interestNone of the authors have any conflicts of interest.

AcknowledgementThe authors would like to thank the following people without whom the study

could not have been undertaken: Jay Berkley, Victor Bandika, Michael Boele

van Hensbroek, Mike English, Trudie Lang, Kevin Marsh, Jennifer Othigo,

Norbert Peshu, Sophie Uyoga, Tom Williams. Also many thanks to the Wazo

Geni team, the midwives and mothers at Coast Provincial General Hospital,

and the clinical staff at Kilifi District Hospital. We are grateful to the Wellcome

Trust for funding the study.

Role of the funding sourceThe study funders had no role in study design; in the collection, analysis, and

interpretation of data; in the writing of the report; and in the decision to submit

the paper for publication.

Research in contextSystematic review

A review of the literature relating to cord blood transfusion was conducted

prior to this study. Allogeneic cord blood transfusion was first reported in the

1930’s before the advent of modern blood transfusion services. Subsequently,

most research and clinical activity relating to cord blood transfusion has

concerned autologous cord blood transfusion in pre-term neonates. A series

of about 200 mainly elderly patients with chronic and/or terminal disease has

been transfused allogeneic cord blood in India. No adverse reactions were

reported. To our knowledge, tThere has been no previous clinical trial of

allogeneic cord blood transfusion in African children.

Interpretation

This trial demonstrates that there is a low probability of adverse events

associated with the transfusion of sedimented red blood cells from umbilical

cord blood donations (UC-RBC) to children with severe anaemia in sub-

Saharan Africa. Haemoglobin recovery after UC-RBC transfusion is within

19

expected limits. To our knowledge, tThis is the first time such a trial has been

undertaken and umbilical cord blood may be a safe and efficacious

supplementary source of blood for transfusion where demand for low volume

transfusions for children is high and supplies of conventional blood are limited.

Further work needs to be undertaken by clinical researchers to establish the

safety and efficacy of cord blood transfusion compared to conventional blood

transfusion. Additional research is also required on the operational aspects of

cord blood collection including costs, impact on the conventional blood supply

and scalability.

References

1. United Nations Children's Fund. Levels and Trends in Child Mortality. 2013.2. WHO. The prevention and management of severe anaemia in children in malaria-endemic regions of Africa: A review of research. Geneva: WHO; 2001.3. Lackritz EM, Hightower AW, Zucker JR, Ruebush TK, 2nd, Onudi CO, Steketee RW, et al. Longitudinal evaluation of severely anemic children in Kenya: the effect of transfusion on mortality and hematologic recovery. AIDS (London, England). 1997 Oct;11(12):1487-94.4. English M, Ahmed M, Ngando C, Berkley J, Ross A. Blood transfusion for severe anaemia in children in a Kenyan hospital. Lancet. 2002 Feb 9;359(9305):494-5.5. Lackritz EM, Campbell CC, Ruebush TK, 2nd, Hightower AW, Wakube W, Steketee RW, et al. Effect of blood transfusion on survival among children in a Kenyan hospital. Lancet. 1992 Aug 29;340(8818):524-8.6. Allain JP, Owusu-Ofori S, Bates I. Blood transfusion in sub-Saharan Africa Transfusion Alternatives in Transfusion Medicine. 2004;6(1):16-23.7. Tagny CT, Mbanya D, Tapko JB, Lefrere JJ. Blood safety in Sub-Saharan Africa: a multi-factorial problem. Transfusion. 2008 Jun;48(6):1256-61.8. Tapko JB, Sam O, Diarra-Nama AJ. Status of Blood Safety in the WHO African Region: Report of the 2004 Survey WHO Regional Office for Africa; 2007.9. Bhattacharya N. Placental umbilical cord whole blood transfusion: a safe and genuine blood substitute for patients of the under-resourced world at emergency. J Am Coll Surg. 2005 Apr;200(4):557-63.10. Hassall O, Bedu-Addo G, Adarkwa M, Danso K, Bates I. Umbilical-cord blood for transfusion in children with severe anaemia in under-resourced countries. The Lancet. 2003;361:678-9.

20

11. Khodabux CM, Brand A. The use of cord blood for transfusion purposes: current status. Vox sanguinis. 2009 Nov;97(4):281-93.12. Bates I, Chapotera GK, McKew S, van den Broek N. Maternal mortality in sub-Saharan Africa: the contribution of ineffective blood transfusion services. BJOG 2008; 115: 1331-9.13. Hassall O, Maitland K, Fegan G, Thitiri J, Pole L, Mwakesi R, et al. The quality of stored umbilical cord and adult-donated whole blood in Mombasa, Kenya. Transfusion. 2010 Nov 13;50(3):611-6.14. Hassall O, Ngina L, Kongo W, Othigo J, Mandaliya K, Maitland K, et al. The acceptability to women in Mombasa, Kenya, of the donation and transfusion of umbilical cord blood for severe anaemia in young children. Vox sanguinis. 2008 Feb;94(2):125-31.15. Hassall OW, Thitiri J, Fegan G, Pole L, Mwarumba S, Denje D, et al. The microbiologic safety of umbilical cord blood transfusion for children with severe anemia in Mombasa, Kenya. Transfusion. 2012 Jul;52(7):1542-51.16. WHO. Pocket book of hospital care for children: guidelines for the management of common illnesses with limited resources. Geneva: WHO; 2005.17. Hassall O, Maitland K, Pole L, Mwarumba S, Denje D, Wambua K, et al. Bacterial contamination of pediatric whole blood transfusions in a Kenyan hospital. Transfusion. 2009 Aug 4;49(12):2594-8.18. EMEA. ICH Guideline for Good Clinical Practice. London: The European Agency for the Evaluation of Medicinal Products; 2002.19. MHRA. UK Blood Safety and Quality Regulations. Implementation of the EU Blood Safety Directive. Background and Guidance on reporting Serious Adverse Events & Serious Adverse Reactions. London: MHRA.20. FDA. COSTART: Coding Symbols for Thesaurus of Adverse Reaction Terms. Rockville, MD: Public Health Service, FDA; 1995.21. Akech SO, Hassall O, Pamba A, Idro R, Williams TN, Newton CR, et al. Survival and haematological recovery of children with severe malaria transfused in accordance to WHO guidelines in Kilifi, Kenya. Malaria journal. 2008;7:256.22. Holzer BR, Egger M, Teuscher T, Koch S, Mboya DM, Davey Smith G. Childhood anemia in Africa: to transfuse or not transfuse? Acta Trop. 1993;55:47-51.

21

Figure 1 Schematic representation of monitoring of harm for participants in

the trial. Not shown here is the active follow-up in the community of children

who did not attend for outpatient follow-up.

22

Figure 12 Trial participant flow

23

Transfusions to children413

Age <= 3m105

Age > 3m308

Hb > 10g/dL12

Hb <= 10g/dL93

Hb <= 4g/dL114

Hb > 4g/dL294

Children eligible by Hb207

Children eligibleby clinical criteria

87Cord blood unavailable

24

Declined consent

6Children enrolled

57

Children transfused

55

Withdrawnbefore transfusion

2

Children ineligibleby clinical criteria

120

Outpatient follow-up

44

Community follow-up

11

Table 1 The number and frequency (with confidence intervals) of adverse

reactions at the minimum and maximum predicted sample sizes

Children transfused40 80

Serio

us a

dver

se re

actio

ns

*00.0

0.0-8.8

0.0

0.0-4.5

12.5

0.1-13.2

1.3

0.0-6.8

25.0

0.6-16.9

2.5

0.3-8.7

37.5

1.6-20.4

3.8

0.8-10.6

410.0

2.8-23.7

5.0

1.4-12.3

512.5

4.2-26.8

6.3

2.1-14.0

* One-sided, 97.5% confidence intervals (with a lower limit of zero) are given for event

frequencies of zero. All others are 95% confidence intervals.

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Table 12 Selected admission characteristics of trial participants (SS=

homozygous for sickle cell genotype; MPs= malaria parasites; WHZ= weight-

for-height Z-score; Hb= pre-transfusion haemoglobin)

N Male SS MPs WHZ < -3

Pre-term Weight (kg)* Hb (g/dL)*§

Aged < 3 months

24 12 - 0 - 14 1.6 8.7(5.5 to 10.0)

‍Aged > 3 months

31 16 6 6 7 - 8.6 3.2(1.9 to 4.0)

All 55 28 6 6 7 14 5.3(1.1 to 14.5) -

* Median (range)§ Pre-transfusion

Aged <= 3 months

Aged > 3 months

All

N 24 31 55

Male 12 16 28

SS - 6 6

MPs 0 6 6

WHZ< -3 - 7 7

Pre-term 14 - 14

Weight (kg)* 1.6 8.65.3

(1.1 to 14.5)

Hb (g/dL)* §8.7

(5.5 to 10.0)

3.2

(1.9 to 4.0)-

* Median (range)§ Pre-transfusion

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Table 23 Serious adverse events and adverse events experienced by children

receiving umbilical cord red cell transfusion.

Serious Adverse Events Adverse Events

N 10 94No. of children 10 43

TimingBefore transfusion 4 4Transfusion + 24h 1 1224h to 28d follow-up 5 78

Indicator of severityFatal 1 N/ALife-threatening 7 N/AHospitalisation 2 N/A

Imputability level*Not assessable 0 0Excluded (0) 4 15Unlikely (0) 3 76Possible (1) 3 3Likely/Probable (2) 0 0Certain (3) 0 0

* Numbers in parentheses refer to 4-point imputability score described in the text

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Table 4 Description of Serious Adverse Events occurring after umbilical cord red cell transfusion

ID No.

Age* Sex Description (time after CBT start)

Indicator of severity

Comments Imputability level (score)

14 1y 1m F Diagnosis of an acute leukaemia- probably acute lymphoblastic leukaemia (N/A)

Fatal Diagnosis by microscopic examination of a peripheral blood film; film taken before UC-RBC transfusion but reported afterwards.

Child was referred to regional hospital but discharged against medical advice and taken by bus to mother’s marital home in western Kenya (Kisumu).

Child was noted to be ‘pale, sick-looking and febrile’ at discharge

Child reported by grandfather to have died the day after arriving in Kisumu, approximately 1 week after UC-RBC transfusion.

Unlikely (0)

44 2d M Abdominal distension, respiratory distress, jaundice (7d)

Life-threatening Pre-term infant (estimated gestation, 30 weeks; birth weight, 1440g)

Probable neonatal sepsis Full recovery after intervention with

broad-spectrum antibiotics, nil by mouth, intravenous fluid and phototherapy.

Unlikely (0)

54 4y M Anaemia (28d) Hospitalisation Sickle cell disease Discharged well 3 days after UC-

RBC transfusion (Hb 6.2) with haematinics

Returned early (21d) for follow-up (mistakenly) (Hb 6.6)

Hb 4.1 at 28d follow-up; admitted for conventional blood transfusion; discharged next day (Hb 6.1).

Unlikely (0)

34 24d M Diarrhoea, dehydration and metabolic acidosis(26h)

Life-threatening Pre-term infant (estimated gestation, 28 weeks; birth weight, 1080g)

Probable neonatal sepsis Full recovery after intervention with

oxygen, broad-spectrum antibiotics and intravenous fluid.

Possible (1)

49 9d F Sepsis, peumonia, apnoeas (14h)

Life-threatening Pre-term infant (estimated gestation, 30 weeks; birth weight, 1200g)

Radiologically confirmed pneumonia

Full recovery after intervention with broad-spectrum antibiotics, oxygen, aminophylline and blood transfusion.

Possible (1)

56 5d F Anaemia (28d) Hospitalisation Low birth weight (probable prematurity), neonatal jaundice and possible sepsis

Discharged well 3 days after cord blood transfusion (Hb 11.5)

Hb 6.2 at 28d follow-up; offered admission, declined but re-presented 1 week later; admitted; Hb 8.4 and transfused conventional blood once donor found (initially no blood available)

Discharged next day with haematinics

Possible (1)

* At enrolment to the study

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Table 5 Haemoglobin concentrations (median, IQR) of children receiving UC-

RBC stratified by age

Pre-transfusio

n1 day 1 month

Aged <= 3 months

N 24 24 13

Hb 8.7(7.8 – 9.2)

11.4(10.5 – 12.4)

9.2(8.4 – 9.9)

Hb change - +2.7(2.2 – 3.8)

+0.5(0.2 – 1.2)

Aged > 3 months

N 31 31 20

Hb 3.2(2.7 – 3.8)

5.8(5.3 – 6.2)

9.3(8.3 – 11.0)

Hb change - +2.6(2.1 – 3.1)

+6.1(5.3 – 8.2)

AllN 55 54* 33§

Hb change - +2.6(2.1 – 3.1)

+5.0(1.0 – 6.8)

* Excludes 1 child for whom consent was declined for a blood test§ Excludes 22 children: 10 received further transfusions, 11 were followed up in the community, 1 was not bled in error.

28