Packed red blood cell transfusion in preterm infants

Prévia do material em texto

www.thelancet.com/haematology Vol 9 August 2022 e615
Review
Lancet Haematol 2022; 
9: e615–26
*Contributed equally
Department of Pediatrics and 
Adolescent Medicine, 
Comprehensive Center for 
Pediatrics (L Bellach Cand Med, 
A Hassanein Cand Med, 
Prof A Repa MD, 
Prof K Klebermass-Schrehof MD, 
L Wisgrill MD, V Giordano PhD, 
Prof A Berger MD), Center for 
Medical Biochemistry, 
Max Perutz Labs 
(M Eigenschink Cand Med, 
D Savran Cand Med, 
Prof U Salzer PhD, 
Prof E W Müllner PhD), Medical 
University of Vienna, Vienna, 
Austria
Correspondence to: 
Prof Angelika Berger, 
Department of Pediatrics and 
Adolescent Medicine, 
Comprehensive Center for 
Pediatrics, Medical University of 
Vienna, Vienna 1090, Austria 
angelika.berger@meduniwien.
ac.at
Packed red blood cell transfusion in preterm infants
Luise Bellach*, Michael Eigenschink*, Abtin Hassanein, Danylo Savran, Ulrich Salzer, Ernst W Müllner, Andreas Repa, Katrin Klebermass-Schrehof, 
Lukas Wisgrill, Vito Giordano, Angelika Berger
Premature infants commonly receive adult packed red blood cells (pRBCs) during their hospital stay. As adult 
erythrocytes differ substantially from those of preterm infants, transfusion of adult pRBCs into preterm infants can 
be considered inappropriate for the physiology of a preterm infant. An absence of standardisation of transfusion 
protocols makes it difficult to compare and interpret pertinent clinical data, as reflected by unclear associations 
between pRBC transfusion and complications related to prematurity, such as bronchopulmonary dysplasia, 
neurodevelopmental impairment, retinopathy of prematurity, or necrotising enterocolitis. The difficulty in 
interpreting clinical data is further increased by differences in study designs that either overestimate pRBC-associated 
complications of prematurity or have not yet been designed to directly link pRBC transfusions to their respective 
complications. Thus, neonatal transfusion practice has become an ongoing difficulty, in which differences in 
transfusion guidelines hinder the ability to generate comparable clinical data, and heterogeneity in clinical data 
prevents the implementation of standardised transfusion protocols. To overcome these issues, novel approaches 
with biochemical-clinical translational designs could enable clinicians to gather causal evidence instead of 
circumstantial correlation.
See Online for appendix
Introduction 
According to WHO, about 15 million babies are born 
prematurely every year, with a ratio varying between 5% 
and 18%, depending on the country of origin.1 Considering 
the amount of physiological, haemodynamic, and 
respiratory immaturity, prematurity is an acute and 
potentially life-threatening condition that implies specific 
complications of multiple organ systems. Broncho-
pulmonary dysplasia,2 intraventricular haemor rhage, 
post-haemorrhagic hydrocephalus, periventricular leuko-
malacia,3 retinopathy of prematurity,4 necrotising entero-
colitis,5 and hypoxaemia and anaemia of pre maturity6 are 
only a few examples of such complications. Although 
advancements in neonatal intensive care have reduced 
mortality to a great extent, preterm birth still represents 
one of the most prevalent causes of death for children 
younger than 5 years. Infants with a birthweight of less 
than 1500g, referred to as very low birthweight, or infants 
with a birthweight less than 1000g, referred to as 
extremely low birthweight,6 are especially likely to face 
lifelong disabilities, including visual, hearing, motor, 
cognitive, and behavioral impairments,1 showing the 
need for optimisation of the clinical procedures used in 
the care of premature infants.
Anaemia of prematurity is an issue that commonly 
arises in the management of premature infants,6 and is 
facilitated by routine blood drawing and low iron 
reservoirs. Since erythropoietin is mainly produced in 
tissues less sensitive to hypoxia in preterm infants, such as 
the liver, and overall erythropoietin clearance is accelerated, 
preterm infants show a reduced erythropoietin response 
in states of hypoxaemia. Moreover, the lifespan of fetal 
erythrocytes is lower than adult erythrocytes, which might 
facilitate the development of anaemia of prematurity.6 
Consequently, one of two preterm infants receive red 
blood cell (RBC) transfusions during their hospital stay,7 
the most susceptible group being infants with extremely 
low birthweight, with up to 90% receiving adult packed 
RBCs (pRBCs).6 Possible strategies to prevent the 
development of anaemia of prematurity constitute 
implementing a prudent approach to phlebotomy 
indications, delayed cord clamping to ensure maximal 
salvage of cord blood, and iron and erythropoietin 
supplementation to boost endogenous erythropoiesis.7
The rationale behind pRBC transfusion is to increase 
haematocrit and haemoglobin concentrations to offset 
episodes of apnea and reduce hypoxaemia7 and, 
consequently, to ameliorate the potential deleterious 
effects of anaemia of prematurity on cerebral oxygen 
supply.6 However, unrestrained pRBC transfusion holds 
some caveats. An increased incidence of adverse clinical 
outcomes with the transfusion of pRBCs, such as 
bronchopulmonary dysplasia, retinopathy of prematurity, 
and necrotising enterocolitis, has been a topic of scientific 
discourse. To our knowledge, no satisfactory conclusions 
have been drawn because data between most studies 
cannot be compared because of differences in design, 
quality, and heterogeneity in international and national 
transfusion guidelines (appendix p 2).7
We aim to summarise current evidence on the effect of 
pRBC transfusion on necrotising enterocolitis, broncho-
pulmonary dysplasia, retinopathy of prematurity, and 
neurological outcomes through a comprehensive 
literature search (appendix p 15); elucidate patho-
physiological caveats that should be considered upon 
transfusion of adult pRBCs into preterm infants; and 
discuss alternatives to previous study designs unsuitable 
to infer causality between pRBC transfusion and adverse 
clinical outcomes.
Bronchopulmonary dysplasia
Acute and long-term pulmonary dysfunction is a common 
issue in neonatology. Because of immaturity of lung tissue 
and impaired repair mechanisms, the pulmonary 
vasculature and alveoli are damaged, thus causing 
the development of bronchopulmonary dysplasia.2 
http://crossmark.crossref.org/dialog/?doi=10.1016/S2352-3026(22)00207-1&domain=pdf
e616 www.thelancet.com/haematology Vol 9 August 2022
Review
Approx imately two in five infants born prematurely 
develop bronchopulmonary dysplasia. This number 
increases with decreasing gestational age, as up to 
68% of infants born before a gestational age of 28 weeks 
are diagnosed with bronchopulmonary dysplasia. Well 
researched risk factors for bronchopulmonary dysplasia 
include prematurity, low birthweight, postnatal infections, 
and mechanical ventilation. It is diagnosed by a need for 
additional oxygen for at least 28 days postnatal and can be 
further classified as mild, moderate, and severe, according 
to the extent of the need for additional oxygen, more 
invasive means to deliver oxygen, and age (ie, post-
menstrual age of 36 weeks [gestational ageexperience of a tertiary neonatal center in UK. 
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of life in preterm neonates. Pediatr Res 2008; 64: 567–71.
56 Genzel-Boroviczény O, Christ F, Glas V. Blood transfusion 
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65 Heaton A, Keegan T, Holme S. In vivo regeneration of red cell 
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Copyright © 2022 Elsevier Ltd. All rights reservedlung injury, leading to the 
corrosion of pulmonary capillary endothelium, and 
resulting in rapidly progressing pulmonary oedema.12
Whether transfusion-associated lung injury seen in 
adults can also be observed in preterm infants has not 
been clarified. Nevertheless, the concept of neonatal post-
transfusion lung injury has been investigated and 
discussed by Rashid and colleagues.12 Furthermore, both 
prospective8 and retrospective studies9–11 were able to find 
associations between pRBC transfusion and the 
development of bronchopulmonary dysplasia. Timing 
and volume of transfusions were also associated with the 
development of bronchopulmonary dysplasia.10
For example, in one study (appendix p 4), the group 
receiving transfusion before 3 weeks of age had a 
significantly higher incidence of bronchopulmonary 
dysplasia than the group receiving late transfusion (ie, at 
3 weeks of age or later). Another study (appendix p 4) 
found an association between the use of multiple pRBC 
transfusions and increased incidence of broncho-
pulmonary dysplasia; however, the authors did not 
disclose the criteria according to which they initiated 
transfusions (appendix p 8).
Concerning the number of transfusions, in a 
retrospective study on infants with extremely low 
birthweight, the authors showed an association between 
pRBC transfusions, and number of pRBC transfusions, 
and the diagnosis of bronchopulmonary dysplasia11 at 
28 days of age. Furthermore, Patel and colleagues found a 
connection between total volume of administered pRBCs 
and incidence of bronchopulmonary dysplasia in infants 
with very low birthweight. The study was also able to show 
an association between oral intake of iron supplementation 
and incidence of bronchopulmonary dysplasia, and the 
authors discuss a possible connection to reactive oxygen 
species formation; however, there are not enough data 
available to sufficiently support this association. As for the 
pRBC transfusion, again, transfusion criteria used in the 
study population were not specified.10 The scarcity of 
disclosure concerning transfusion practices in studies 
evaluating the effect of pRBC transfusion on the risk of 
bronchopulmonary dysplasia is both common and 
hinders the comparison of published data. Most studies 
do not properly correct for known risk factors of broncho-
pulmonary dysplasia, such as oxygen supplementation, 
nor do they sufficiently report their method in the 
manuscript, which increases the risk for random 
effects.10,11 Compounding this issue, the definition of 
broncho pulmonary dysplasia also slightly deviates 
between studies, as exemplified by Valieva and colleagues,11 
since the authors defined bronchopulmonary dysplasia as 
the need for oxygen at a postnatal age of 28 days and at a 
corrected gestational age of 36 weeks. By contrast, another 
study defined broncho pulmonary dysplasia solely by the 
need for oxygen supplementation at a postmenstrual age 
of 36 weeks.9
Regarding transfusion thresholds used in studies 
that disclosed details on transfusion criteria, further 
discrepancies can be noted (table 1, appendix p 4). Valieva 
and colleagues11 used haematocrit thresholds of 20%, 30%, 
and 35%, depending on clinical features, such as how 
much oxygen the patient needed, or whether the patient 
was mechanically ventilated or showed cardiorespiratory 
stability. By contrast, other studies allowed transfusion to 
be initiated at the discretion of the neonatologist,9 or did 
not disclose any transfusion criteria at all in their 
methods.10 There are also differences concerning the age 
and composition of blood bags, as one study allowed the 
blood aliquots to be as old as 42 days when transfused.11 
Another study did not use pRBCs that had been divided 
into aliquots at all.9 Moreover, only a few studies provided 
information on composition and preparation of 
pRBCs,8,10,11 and inclusion criteria varied considerably 
across different studies. For example, whereas most 
studies had 1500 g as the upper weight limit,8–10 one study 
exclusively included infants with extremely low 
birthweight with a weight ranging from 500 g to 1000 g.11 
Furthermore, other studies defined the developmental 
stage suitable for inclusion with the weight at birth rather 
than gestational age.8–10 Altogether, these factors make it 
difficult to merge the available data into a comprehensive 
summary on the effect of pRBC transfusion on the 
incidence of bronchopulmonary dysplasia.
Adverse neurological outcome
Prematurity commonly leads to neurological deficits. 
Up to 30% of all preterm infants who survive show 
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e618 www.thelancet.com/haematology Vol 9 August 2022
Review
neurosensory or neuromotor impairments.16 Neuro-
development is most frequently assessed with the third 
edition of the Bayley Scales of Infant and Toddler 
Development (BSID),15,17 and cerebral palsy and gross 
motor functions are quantified by the Gross Motor 
Function Classification System.16 Clinical trials that 
assessed the effect of pRBC transfusion on various 
neurodevelopmental outcomes have provided conflicting 
results. Most of the trials compared different transfusion 
thresholds, either favouring a liberal18 or restrictive19 
approach to transfusion, depending on higher or lower 
haematocrit threshold levels triggering transfusion, or 
identifying no differences between the two strategies.3,16,17,20 
Only a few studies evaluated the overall effect of pRBC 
transfusion on neurodevelopmental outcomes.15,21,22
Although a randomised controlled trial by Bell and 
colleagues18 found a higher incidence of periventricular 
leukomalacia or intraparenchymal brain haemorrhage in 
the restrictive transfusion group of infants with very low 
birthweight and extremely low birthweight, Whyte and 
colleagues16 showed no beneficial effect of maintaining 
high or low haemoglobin thresholds. Only after adjusting 
the Mental Development Index score cutoff for cognitive 
delay from less than 70 to less than 85, they were able to 
show a significant improvement in the liberal transfusion 
group, thus favouring higher transfusion thresholds. 
However, a study that followed up 44 children for more 
than a decade, who were initially included in the study 
collective of Bell and colleagues,18 compared intracranial 
volume with healthy controls. The study found that 
children given pRBC transfusion showed a reduction in 
brain volume, with a tendency for the highest reduction 
in children transfused under a liberal regimen. Nonethe-
less, it should be mentioned that the number of total 
transfusions did not differ between the groups (p=0·15),19 
challenging the interpretability of the results.19 However, 
a third study with the same collective showed reductions 
in neurocognitive outcomes, such as associative verbal 
fluency, visual memory, and reading in infants transfused 
under liberal guidelines 13 years after the start of the first 
study.23 Moreover, two studies identified neurocognitive 
development and outcome at the age 18–26 month follow-
up to be inversely correlated with total transfusion count, 
transfusion donor exposure, and transfusion volume.15,24
The heterogeneity in results is further exacerbated by a 
retrospective analysis showing a positive correlation 
between the number of transfusions in the first 7 days of 
life and a more favourable neurocognitive outcome at 
the age 18–24 month follow-up in infants with extremely 
low birthweight;21 a Cochrane review with low risk of bias 
showing no difference in neurocognitive outcome 
between liberal and restrictive threshold transfusion in 
infants with very low birthweight after 18–22 months of 
follow-up;25 and a systematic review and meta-analysis 
showing no advantage of any transfusion threshold for 
neurodevelopmental outcome in infants with very low 
birthweight (table 2).26
Transfusion thresholds varied between trials (table 1, 
appendix p 4). For example, whereas some trials defined 
thresholds based solely on the method of 
O2 supplementation,18 others additionally used distinct 
numeric values for fractional concentration of oxygen in 
inspired air (FiO2)15,21 and positive expiratory pressure15 to 
guide their interventions. In intubated patients, Bell and 
colleagues18 used 46% haematocrit in blood as the liberal 
threshold and 34% as the restrictive threshold, whereas 
Shah and colleagues transfused patients under 
respiratory support at 31% haematocrit.15 Transfusion 
volume was also not standardised, varying between 
10 mL/kg and 20 mL/kg.15,18,21 Moreover, some studies 
chose transfusion volume according to the patient’s 
respiratory status and clinical presentation.15 Regarding 
blood handling, studies did not consistently report 
storage conditions, storage solution, or whether 
irradiation was done. For example, whereas Bell and 
colleagues18 included no information about pretreatments 
and storage, Wang and colleagues21 simply mentioned to 
have used leukocyte-poor concentrates, and Shah and 
colleagues15 reported the use of irradiated and leukocyte-
depleted erythrocyte concentrates treated with citrate-
phosphate-dextrose adenine.
In addition to the studies on infants with very low 
birthweight, several randomised controlled trials 
investigating the effect of transfusion thresholds on 
neurological or neurocognitive outcomes in infants with 
extremely low birthweight have been done, of which none 
showed a significant association.3,16,17,20 Although these 
trials are of high methodological quality, their 
comparability is limited by the absence of an international 
consensus on optimal transfusion guidelines. Although 
the cutoff for transfusion in the liberal group of the 
ETTNO trial20 for infants up to 7 days of postnatal age was 
set at 41% haematocrit for critical individuals and 
35% haematocrit for non-critical individuals, the TOP 
trial17 by Kirpalani and colleagues included haematocrit 
thresholds of 38% and 35%, depending on the use of 
respiratory support. Differences are also observable in the 
restrictive group, with transfusion thresholds set at 
34% and 28% haematocrit in the ETTNO trial20 versus 
32% and 29% haematocrit in the TOP trial.17 Moreover, 
follow-up length varied between18 months and 
26 months.16,17,20 Studies also differed in the volume of 
pRBCs transfused, with the ETTNO trial20 using a 
transfusion volume of 20 mL/kg, and Kirpalani and 
colleagues using 15 mL/kg bodyweight.17 The chosen 
neurodevelopmental scores also differed between the 
studies; some studies used the second edition of 
the BSID,16,21 other studies used the third edition of the 
BSID,15,17 and one study combined the second and third 
edition of the BSID.20
Of note, the quality of methods and statistical power of 
the ETTNO trial (n=1013)20 and TOP trial (n=1824)17 
provide robust evidence for equivalency of low and high 
transfusion threshold strategies in infants with extremely 
www.thelancet.com/haematology Vol 9 August 2022 e619
Review
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actually differed in 
transfusion protocols, and only one study explicitly 
reported the number of transfusions received within the 
first 10 days of life.3,17,20 Considering the pathophysiology 
of retinopathy of prematurity and the data collated by 
Lust and colleagues,27 the number of immediate postnatal 
transfusions might influence retinopathy of prematurity 
development and be distorted by the time needed for 
patient allocation.
Storage conditions and the handling of pRBCs were 
poorly documented in terms of pretreatments (irradiation, 
leukoreduction, storage solution).11,27,30,32 Moreover, hetero-
geneity also arises from patient selection. Although some 
groups characterised retinopathy of prematurity in infants 
with extremely low birthweight,26 other studies focused on 
infants with very low birthweight,30 or disregarded 
common classifications altogether,29 which is particularly 
problematic because both birthweight and gestational age 
are well known risk factors for the development of 
retinopathy of prematurity.4
Because of low comparability between retrospective 
studies, reduced focus on retinopathy of prematurity in 
large prospective trials that disregard early transfusion 
and focus on infants with extremely low birthweight, and 
low sample size of prospective trials that evaluated the 
relationship between retinopathy of prematurity and 
transfusion, the association between pRBC transfusion 
and retinopathy of prematurity remains unclear. Since 
the molecular pathophysiology of retinopathy of 
prematurity and the effects of pRBC transfusion are 
highly intertwined, however, cause–effect correlation 
seems plausible. Thus, future prospective studies 
focusing on retinopathy of prematurity with standardised 
methods are warranted.
Necrotising enterocolitis
Another complication associated with prematurity, 
especially concerning infants with very low birthweight, is 
necrotising enterocolitis. Up to 7% of preterm infants in 
the USA and Canada are diagnosed with necrotising 
enterocolitis, up to 30% of whom do not survive.5 
Symptoms are food intolerance, abdominal distension, 
and haemato chezia. The pathophysiology of necrotising 
enterocolitis revolves around immaturity of the 
gastrointestinal tract and concomitant inadequacy of 
intestinal barrier function. Gastrointestinal microbial 
dysbiosis and dysregulated immune response also 
contribute to the risk of necrotising enterocolitis.5 It has 
been postulated that necrotising enter ocolitis is triggered 
as a consequence of pRBC transfusions. Referred to as 
transfusion-associated necrotising entero colitis (TANEC), 
possible patho physio logical mech anisms include hypoxia-
induced intestinal damage exacerbated by pRBC 
transfusion, also termed hypo perfusion-reperfusion 
injury.6 There is also accumu lating evidence for a possible 
connection between trans fusion and the development of 
necrotising entero colitis,13,33–35 and worse clinical outcomes 
in TANEC.36
Blau and colleagues13 found an association between the 
timing of pRBC transfusion and necrotising enterocolitis 
in some infants with very low birthweight. They reported 
that patients with TANEC tended to have lower haema-
tocrit values and weighed less than the non-transfusion-
associated necrotising enterocolitis group. By contrast, 
Mally and colleagues35 found that preterm births in the 
TANEC group were better developed and healthier than 
in the non-transfusion-associated group, suggesting that 
pRBC transfusion might have induced development of 
necrotising enterocolitis. The two studies also differed in 
reported duration to the onset of TANEC (5 h [SD 1·0] vs 
22 h [SD 5·0]).13,35 Observational studies did not find any 
association between transfusion and the development of 
necrotising enterocolitis.21,37 Thus, the association remains 
unclear, as supported by a meta-analysis of observational 
studies that remained inconclusive on the relation 
between transfusion and necrotising enterocolitis.37
Moreover, there are considerable differences concerning 
inclusion, exclusion, and transfusion criteria between 
individual studies. For example, some studies chose an 
upper weight limit of 1500 g instead of an upper age 
limit.32 Regarding the definition of necrotising 
enterocolitis, most chose Bell’s stage II as threshold,34,36 
whereas other studies used a slightly different definition 
(Bell’s stage IIb).13 Another point in which study protocols 
differed was enteral feeding. A randomised trial 
comparing doppler ultrasound flow examinations of the 
superior mesenteric artery between fed and fasting infants 
during transfusion showed that transfusions reduce 
postprandial increase in mesenteric blood flow velocity, 
especially in infants weighing more than 1250 g.14 The 
results correspond with the findings of Mally and 
colleagues,35 who concluded that TANEC is associated 
with the intake of full enteral meals. Nevertheless, a meta-
analysis remained inconclusive because of the scarcity of 
sufficient high-quality data.38 Furthermore, in some 
studies, enteral feeding was stopped during transfusion,33 
but was not stopped,34 or not disclosed in other studies.21
Indications to initiate pRBC transfusion differed 
notably between studies (appendix p 4). Blau and 
colleagues13 initiated transfusion upon five or six episodes 
of apnea within 8 h, or if preterm infants did not gain 
e622 www.thelancet.com/haematology Vol 9 August 2022
Review
10–15 g/kg bodyweight per day within a week, or had 
lethargy, unexplained tachycardia or tachypnea, 
insufficient reticulocyte count increase, or a haematocrit 
less than 25%. Patients recruited in a study by Wang and 
colleagues,21 however, were transfused at a haematocrit 
less than 21% and no reticulocyte response if 
asymptomatic; at a haematocrit less than 31% if 
symptoms of inadequate oxygenation, such as the need 
for oxygen or ventilation and not gaining weight, were 
present; and at a haematocrit less than 36% upon need 
for more than 35% oxygen or ventilator assisted 
breathing, or upon phlebotomy blood loss of 15% of total 
blood volume within the first 7 days of life. One study 
prescribed concomitant furosemide infusion,14 whereas 
in other studies no such procedure was mentioned.13 
Moreover, both rate and duration of transfusions 
differed, with one study transfusing 10–15 mL/kg within 
2–3 h,21 and a set of studies transfusing 15 mL/kg within 
4 h.13,34 By contrast, another study provided detailed 
transfusion criteria and a formula to calculate 
transfusion volume. The timeframe in which pRBCs 
were administered, however, remained unspecified.33 Of 
note, only a few studies provided information on the 
blood bag itself,13,21,24 such as its age or expiration date.13
In accordance with a systematic review by Hay and 
colleagues,39 the differences in study protocols impede data 
comparison. More transparency and conscientiousness 
concerning handling and storage of pRBC packs and 
transfusion practices are warranted. This need holds 
especially true considering hypoperfusion–reperfusion 
injury as a possible cause of necrotising enterocolitis. Of 
note, it is severe anaemia, not transfusions, that is 
associated with the development of necrotising 
enterocolitis.40 Genetic background and microbial 
composition5 substantially influence the risk for 
necrotising enterocolitis. Carefully designed protocols in 
future should also consider the potential association 
between donor characteristics and the occurrence of 
necrotising enterocolitis.
Physiological aspects: adult cells in a premature 
body
Approximately 90% of infants with extremely low 
birthweight and 40% of infants with very low birthweight6 
receive at least one pRBC transfusion. Since top-up 
transfusions for non-bleeding individuals are usually 
done at 15 mL/kg,7 and blood volume of preterm infants 
weighing 800–1499 g approximates 83 mL/kg,41 15–20% 
of total blood volume is added with each pRBC 
transfusion. Consideringboth, heterogeneity in 
biophysical and biochemical properties between different 
blood packages (interdonor variability and inherent 
pRBC quality) and differences in neonatal vasculature 
and erythrocyte physiology between adults and neonates, 
the complexity of blood transfusion for preterm infants 
exceeds the scope of conventional clinical studies, calling 
for additional translational and basic research.
Fetal haemoglobin differs from that in adults. The first 
developmental switch, the replacement of embryonic 
haemoglobin with fetal haemoglobin occurs within the 
first trimester when erythropoiesis relocates from the yolk 
sack to the liver.42 The second switch, a process unique to 
higher primates,42 takes place after birth and is completed 
within the first 6 months of life. Fetal haemoglobin shows 
higher affinity to oxygen and lower affinity to 2,3-dipho-
sphoglycerate than adult haemoglobin, resulting in a left 
shift of the oxygen dissociation curve and a reduced ability 
to release oxygen in the periphery.43 Therefore, transfusion 
of adult pRBCs containing adult haemoglobin might lead 
to a sudden increase in the availability of oxygen, thus 
facilitating oxygen delivery to peripheral tissues.44 This 
process might be of pathophysiological relevance because 
of increased formation of ROS, potentially resulting in 
subsequent tissue damage.45 This hypothesis is 
complemented by the findings that (1) fetal haemoglobin 
concentrations of extremely low gestational age preterm 
infants transiently drop by 30% after one pRBC 
transfusion and by 60% after two pRBC transfusions, 
possibly because of dilution;46 (2) the activity of antioxidant 
enzymes, such as catalase, glutathione peroxidase, or 
superoxide dismutase, is reduced in preterm infants, as 
measured in cord blood hemolysates;47 (3) erythrocytes of 
neonates have higher glutathione turnover-rates than 
adult erythrocytes;48 and (4) low fetal haemoglobin concen-
trations were associated with the development of broncho-
pulmonary dysplasia49 and retino pathy of prematurity.50 To 
counteract these findings, more physiological approaches, 
such as cord-blood-derived pRBC transfusion, have been 
proposed. One study, although limited by small sample 
size, showed cord blood pRBC transfusion to circumvent 
sudden drops in fetal haemoglobin.51
RBCs of premature infants not only differ in 
haemoglobin composition but also in morphology and 
flow behaviour. Erythrocytes of preterm infants born with 
a gestational age of between 22 weeks and 32 weeks 
present with a mean corpuscular volume between 110 fL 
and 120 fL and a mean corpuscular haemoglobin 
concentration between 37 pg and 40·5 pg compared with 
adult values of 80–94 fL for mean corpuscular volume and 
27–31 pg for mean corpuscular haemoglobin 
concentration.52 This increased volume could account for 
the enhanced probability of aggregation and rouleaux 
formation, as measured in plasma free medium in vitro.53 
Moreover, the increased cell volume of about 120 fL and 
the concomitant need for additional deformability54 might 
necessitate the larger lumen of capillaries, arterioles, and 
venules in preterm neonates, with diameters of 7–24 µm 
being reported.55,56 However, rouleaux formation and 
overall blood viscosity is reduced in neonates, especially 
under low shear stress.57 This finding is accounted for by 
differences in plasma composition and reduced protein 
content, which counteract the enhanced probability of 
aggregation and rouleaux formation and decreases the 
risk of capillary obstruction.54 In summary, the 
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physiological challenges induced by the transfusion of 
adult pRBCs into preterm infants and how they affect the 
development of unfavourable outcomes remain unclear, 
showing the need for additional studies (figure).
Blood of premature infants contains a high abundance 
of so-called pitted (or pocked) erythrocytes (30–87%) when 
compared to adults (3%).58 Their number is inversely 
correlated to gestational age and birthweight and has been 
shown to be a reliable marker of splenic hypofunction or 
immaturity. These findings are suggestive of impaired 
splenic cell clearance in premature infants,59 which is of 
particular interest, since the life span of pRBCs transfused 
to preterm infants is approximately 60 days,60 compared 
with 120 days in adults.7 Therefore, extrasplenic clearance 
or erythrolysis might account for the accelerated cell loss 
after transfusion.60
The differences in erythrocyte and circulatory 
physiology between adults and preterm infants warrant 
further selection criteria for pRBCs used in neonatal 
transfusion. Biochemical and biophysical erythrocyte 
functionality declines over time during cold storage, 
resulting in collectively termed storage lesions.61 Large 
differences in erythrocyte stress resistance and 
functionality already become apparent immediately after 
blood donation and processing,62 but are not considered 
in blood banking. Storage lesions have a negative effect 
on the number of cells remaining in circulation 24 h 
after transfusion. A loss of 25% is currently deemed 
acceptable,63 and the mean average loss of cells 24 h after 
transfusion is 15%.63 Erythrocytes undergoing lysis or 
retained in the spleen not only delay reaching the desired 
haematocrit as calculated from the volume of transfused 
pRBCs, but are also a burden to the patient because of 
the release of deleterious cell components, such as 
microvesicles and free or oxidised haemoglobin.64 During 
routine cold storage, metabolites in pRBCs undergo 
changes in abundance, as extensively documented in a 
review incorporating data obtained from omics 
technologies, in addition to available literature.61 All 
approved storage solutions contain adenine as a building 
block for ATP and glucose to maintain chemical energy 
production. The decline of adenine and glucose, both 
intracellularly and in the storage medium, is rapidly 
reverted once pRBCs re-enter the blood, thus deemed 
unproblematic at first glance.65 However, other storage 
lesions, such as reduced shear stress and oxidative stress 
resistance and deformability, are irreversible.66 Most of 
these storage lesions are caused by (oxidative) damage to 
membrane components, since defence against reactive 
oxygen species becomes compromised during storage.67 
Because of pleiotropic deleterious effects, free 
haemoglobin, haem, and iron might be of concern,68 
particularly in preterm infants. Moreover, ageing pRBCs 
increasingly shed microvesicles that continue to be 
suspected as being problematic in vivo, especially if their 
concentration in the blood is suddenly increased after 
transfusion.64
At least three storage lesions have been connected 
to post-transfusion erythrocyte recovery so far: cell 
morphology, intracellular ATP content, and deformability.63 
Evaluation of all three parameters is within the limits of 
standard technology, thus their measurement could be 
implemented at various degrees of effort. Analysis of 
morphology, especially for echinocytes and spherocytes, 
requires simple cell type counting in a microscope; ATP 
concentration can be measured with commercially 
available test kits; and the elongation index, the most 
important rheological property related to cell 
deformability,69 could be measured with a microfluidic 
slit-flow ektacytometer.70 Ektacytometry would be a larger 
investment and require more qualified personnel to 
operate than other options, however, studies have shown 
that new methods for evaluating deformability of red 
blood cells with microfluidic capillary networks are more 
sensitive than ektacytometry for detecting storage induced 
changes in red blood cell theological properties, thus 
enabling faster data acquisition and routine monitoring of 
pRBC quality in clinical routine.71 Contrary to transfusion 
practice in adults, in which more than one pRBC unit is 
applied most of thetime, more extensive characterisation 
of erythrocytes seems feasible and warranted in preterm 
infants, in whom less than one unit is needed.
Besides the option to evaluate outcome related cell 
properties ahead of transfusion, erythrocytes from 
different donors vary in a plethora of biological parameters 
that can make them more suitable or less suitable for 
transfusion.72 The variability between donors, although 
adequately evaluated in biochemical studies, has not been 
evaluated in clinical trials and is, therefore, not routinely 
recorded with standard clinical tests in blood banks. For 
example, storage haemolysis, and osmotic and oxidative 
Figure: Adult erythrocytes in an immature body
Adult red blood cells differ substantially from the red blood cells of premature infants. 
0
25
50
75
100
O
2 s
at
ur
at
io
n 
of
 h
ae
m
og
lo
bi
n 
(%
)
Partial pressure of O2
Mean
corpuscular
volume
80–94 fL 110–120 fL
Adult haemoglobin Fetal haemoglobin
Adults Premature infants Adults Premature infants
4–6 μm 8–14 μm
Haemoglobin
Reference viscosity
Plasma proteins: 60–80 g/L
Fibrinogen: 300–400 mg/dL
Reduced viscosity
Plasma proteins: 30-50 g/L
Fibrinogen: 120-250 mg/dL
Life span of adult packed
red blood cells: 120 days
Normal clearance:
3% pitted red blood cells
Life span of adult packed
red blood cells: 56 days
Extrasplenic clearance:
30–87% pitted red blood cells
Capillary diameter
Plasma composition
Clearance
15–20%
of blood
volume
HbF
HbA
?
e624 www.thelancet.com/haematology Vol 9 August 2022
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stress resistance of pRBCs have been associated with 
donor age, sex, and ethnicity.73 Moreover, osmotic and 
mechanical stress resistance, as well as oxidative 
haemolysis to some extent, have been reported to be 
donor specific.74 Compounding the findings on donor 
variability, identical twin studies have shown that 
glycolysis (both enzyme activity and metabolite 
abundance), pentose–phosphate pathway activity, and in 
consequence, glutathione concentrations, are heritable.75,76 
Importantly, these results were also reproduced in stored 
pRBCs according to the work by van ’t Erve and 
colleagues.77 Thus, erythrocyte function seems to underly 
interindividual and heritable differences even on a 
molecular scale.
Interindividual differences in pRBC quality and 
clinical studies
Although a plethora of retrospective analyses, cohort 
studies, and case-control studies have reported pRBC 
transfusion to be associated with adverse outcomes in 
preterm infants, high-quality RCTs focusing on 
differences between liberal versus restrictive transfusion 
thresholds have not reproduced such results. Paradox at 
first glance, the controversy between the results of RCTs 
and cohort studies, supported by absence of 
standardisation and limited comparability even between 
similarly designed studies, might be an overall 
consequence of not accounting for interindividual pRBC 
quality in clinical trials. As retrospective, cohort, and 
case-control studies compare transfused individuals with 
non-transfused individuals, they have an inherent bias: 
transfused individuals are a-priori sicker than non-
transfused. Although studies account for this bias by 
applying multivariate analyses to correct correlations for 
known risk factors, they tend to overestimate effect sizes 
(ie, adverse association between pRBC transfusion and 
outcome). The problem of a-priori sickness has been 
circumvented with the introduction of liberal versus 
restrictive transfusion studies, as preterm infants are 
randomly assigned into either one of the groups. Usually, 
infants randomly assigned to the liberal group are more 
likely to be transfused and receive an overall higher 
volume of pRBCs. Yet, considering sample sizes, 
differences in transfusion incidence between groups 
might not be large enough to account for the 
overestimated effects observed in cohort studies, leaving 
observable adverse clinical outcomes dependent on 
differences in overall transfusion number and volume. 
Therefore, the analysis of a group of mostly transfused 
individuals holds caveats because donor exposure usually 
is not conclusively reported or does not differ between 
groups; donor data are neither recorded nor reported; 
and interindividual differences in pRBC quality are not 
quantified ahead of transfusion. Molecular erythrocyte 
properties are heritable, making pRBCs more suitable or 
less suitable for transfusion. The tolerability of pRBC 
transfusion might be shaped by differences in donor 
RBC quality rather than volume or number of 
transfusions. Thus, adverse clinical outcomes might be 
insufficiently recorded in liberal versus restrictive 
transfusion studies because overall erythrocyte quality 
equilibrates between large treatment groups that consist 
mainly of transfused individuals. The hypotheses that we 
have discussed would offer an explanation as to why 
studies evaluating the effect of transfusion versus no 
transfusion find associations between administration of 
pRBCs and adverse clinical outcomes in preterm infants, 
whereas restrictive versus liberal transfusion threshold 
studies do not find any association.
Conclusions
Differences in neonatal transfusion guidelines hinder 
the generation of comparable clinical data, and 
heterogeneity in clinical data prevents the implementation 
of standardised transfusion protocols in preterm infants. 
Although cohort studies have reported associations 
between pRBC transfusion and adverse outcomes in 
preterm infants, RCTs investigating different transfusion 
thresholds did not find such correlations. To provide 
Search strategy and selection criteria
This is a comprehensive Review of clinical studies on packed 
red blood cell (pRBC) transfusion, neurocognitive outcomes, 
and the risk for bronchopulmonary dysplasia, necrotising 
enterocolitis, and retinopathy of prematurity. For 
comparisons between studies, we included and 
comprehensively discussed original articles, systematic 
reviews, and meta-analyses in the text. We only further 
screened and summarised full articles in English with 
relevance to the topic in descriptive tables. We also retrieved 
numeric values, such as means, standard deviations, and 
sample sizes, and presented them in tables. A post-hoc 
cross-validation systematic research was done by the 
information retrieval office of the university library of the 
Medical University of Vienna, and 578 articles published in 
Ovid MEDLINE until April 27, 2022, were screened. From these 
articles, 224 were marginally related to the topic of pRBC 
transfusion and its association with necrotising enterocolitis, 
bronchopulmonary dysplasia, retinopathy of prematurity, 
and neurodevelopmental outcomes. Of these articles, 
we considered 19 studies relevant. We divided the research 
strategy into sections and their combinations. The main 
sections considered relevant words referring to: population of 
interest, procedure, outcomes, and type of study. We chose 
articles to be included into full-text analysis according to their 
overall methodological (sample acquisition and selection, 
randomisation, and if applicable, sample size) and statistical 
quality (correction for risk factors, multivariate models), or 
according to their representativeness for methodological 
flaws (if cited). We organised the literature according to 
prospective and retrospective designs and favoured 
prospective trials for inclusion.
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causal rather than correlational evidence for the 
association between pRBC transfusion and adverse 
outcomes in the clinically vulnerable group of preterm 
infants, novel study designs that consider interindividual 
differences in pRBC quality are warranted. Therefore, 
longitudinal prospective studies with a clinical–
biochemical transla tional design might finally circum-
vent this causality problem.
Contributors
ME had the idea for the manuscript. ME, LB, US,EWM, VG, and AB 
conceptualised the manuscript. LB, ME, VG, EWM, and AB prepared 
the original draft. LB, ME, VG, EWM, US, DS, AH, and AB wrote the 
manuscript and LB, ME, VG, EWM, US, LW, AB, AR, and KK-S reviewed 
and edited it. VG, LW, and AB were project supervisors for the 
neonatology section. US and EWM were project supervisors for the 
physiology section. All authors have read and agreed to the submitted 
version of the manuscript.
Declaration of interests
We declare no competing interests.
Acknowledgments
We thank the mentoring programme of the Medical University of 
Vienna (Vienna, Austria), for facilitating cooperation between different 
departments involved in this manuscript. We also thank 
Brigitte Wildner, from the information retrieval office at the university 
library of the Medical University of Vienna, for supporting the research 
strategy of this Review with extreme professionality and competence.
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