2 Liverpool School of Tropical Medicine, Liverpool L1 5QA, United Kingdom

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, Kenya

3Centre for Clinical Vaccinology & Tropical Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.

4Coast Provincial General Hospital, Mombasa 80100, Kenya

5Regional Blood Transfusion Centre, Mombasa 80100, Kenya

6Department of Paediatrics, Imperial College London, London SW7 2AZ, United Kingdom

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

+ 44 (0) 7774 354716

Abstract

Background

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

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)

Introduction

Background

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).1Severe 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).13,14We 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 of conventional adult blooddonated to the Regional Blood Transfusion Centre in Mombasa (15).15Here 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.

Methods

Trial 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, numberwith 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 andat 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 ablood culture. Haemoglobin electrophoresis to detect haemoglobin S 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 2-6C 50km by road to Kilifi, sedimented by storing vertically in racks at 2-6C and quarantined until screened for bacterial contamination. This was done by incubation of a 4ml sample in 40ml of brain heart infusion at 37C 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 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 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 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

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.

Results

Participant 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)

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).