Impact of red blood cell transfusions on intestinal barrier function in preterm infants

Abstract

OBJECTIVE:

To determine the relationships of red blood cell (RBC) transfusion and enteral feeding to changes in intestinal permeability (IP) measured by the relative intestinal uptake of lactulose (La) and rhamnose (Rh) in preterm infants <33 wk gestation.

DESIGN/METHODS:

Infants 24⁰–32⁶wk gestation received La/Rh solution enterally on study days 1, 8 and 15.Urinary La/Rh ratio was measured by HPLC. Hematocrit preceding transfusion, total RBC transfusion volume, volume/kg, and feeding status during each study interval (birth-d1; d1-d8, and d8-d15) were determined.

RESULTS:

Of the seventeen (40.5%) subjects who received≥1 transfusion during the study period, 12 (70.6%) infants were <28 wk gestation and 5 (29.4%) infants were≥28 wk gestation, p < 0.0001. Lower pre-transfusion hematocrit was observed in intervals preceding high IP (La/Rh > 0.05) than in intervals preceding low IP (La/Rh≤0.05) measurements (33 vs 35.8, p = 0.1051). RBC transfusions occurred more frequently in intervals preceding high IP than in intervals preceding low IP (26.8%; vs 8.3%, p = 0.0275) with 5-fold higher total RBC volume and volume/kg in intervals preceding any time point with high IP. RBC transfusion during an interval was associated with a three-fold increased risk of high IP (aOR 2.7; 95% C.I 0.564–12.814; p = 0.2143). Exclusive breast milk exposure and post-menstrual age reduced the risk for high IP following RBC transfusion.

CONCLUSIONS:

Both RBC transfusion number and volume was associated with subsequent high IP measurements in preterm infants <33 weeks gestation and potentially may contribute to impairment of the preterm intestinal barrier.

Keywords

Red blood cell transfusion premature infants intestinal permeability enteral feeding

1. Introduction

Premature neonates born before 33 weeks gestation frequently receive multiple red blood cell (RBC) transfusions [1, 2]. Controversy remains regarding the contribution of RBC transfusions to necrotizing enterocolitis (NEC), a life-threatening gastrointestinal emergency [3 –7] and the practice of withholding enteral feeds during RBC transfusion to prevent NEC [8]. The mechanisms underlying RBC transfusion-induced intestinal injury are currently unknown.

The intestinal epithelium forms a tight, but selective barrier to microbes and most environmental substances, but nutrients are absorbed efficiently [9, 10]. Environmental antigen(s) or bacteria that gain access to the intestinal submucosa via paracellular passage from the intestinal lumen across an aberrant mucosal barrier trigger a host inflammatory response and subsequent intestinal wall injury and necrosis [11 –13]. Intestinal barrier function is developmentally regulated [14, 15], but barrier function maturation is delayed in some preterm infants, particularly with delayed onset of feeding and prolonged antibiotic exposure [16] that may increase the risk for NEC. Recent evidence suggests that breast milk exposure is associated with maturation of the intestinal barrier and may be protective against NEC [16 –19].

Despite the observed association between NEC, anemia and RBC transfusion, the impact of RBC transfusion on intestinal permeability (IP) is currently unknown. We hypothesized that exposure to RBC transfusions in preterm infants <33 wks gestation during the first 2 weeks of life is associated with increased IP and that enteral feeding may impact the preterm IP response to RBC transfusion. We analyzed the relationship of pre-transfusion hematocrit, timing and volume of RBC transfusion and IP with the modulating effect of enteral feeding during transfusion on intestinal barrier function in a cohort of preterm infants.

2. Methods

The current study is a secondary analysis of our previously published prospective study of the relationship of feeding and antibiotic exposure on intestinal barrier maturation during the first 2 weeks of life in very low birthweight infants (NCT01756040) [16]. Forty-three infants 24^0/7–32^6/7 weeks gestation and <4 days of age were enrolled at the University of Maryland Medical Center and Mercy Medical Center NICUs (Baltimore, MD) with institutional review board approval and parental consent. Exclusion criteria included non-viability or planned withdrawal of care, triplet or higher order multiple births, severe asphyxia (Apgar <3 at 5 min and cord pH <7.0), lethal chromosome abnormalities, cyanotic congenital heart disease, intestinal atresia or perforation, abdominal wall defects, significant gastrointestinal dysfunction (e.g., heme-positive stools, abdominal distension with girth >2cm baseline, bilious emesis/aspirates), and infants with galactosemia or other forms of galactose intolerance.

2.1. Sugar absorption test

On each of the three study days (days 1, 8±2 and 15±2), participants received 1mL/kg La/Rh solution [8.6 g of lactulose (La) (Cumberland Pharmaceuticals, Nashville, TN)+140 mg of rhamnose (Rh) (Saccharides, Inc., Calgary, Alberta, Canada)/100mL] by nipple or by gavage via a clinically indicated orogastric tube [20]. A minimum of 2 mL of urine was collected over a 4-hour period after the La/Rh dose was administered. The test was repeated the following day if less than 2 mL urine was collected. If there were signs of feeding intolerance (increased abdominal girth >2 cm, heme-positive stools, or gastric residuals), the sugar solution administration was either delayed until resolution of symptoms within the study 4 day window for each timepoint or not done. The amount of lactulose and rhamnose in each sample was measured using high-performance liquid chromatography (HPLC) and adjusted for urine volume [21]. The fractional urinary excretion of lactulose and rhamnose was calculated as the ratio of the total urinary excretion of each sugar probe to the total oral dose of the probe. For each subject, the La/Rh ratio was calculated as the fractional excretion of lactulose divided by that of rhamnose [22]. A La/Rh ratio >0.05 was considered indicative of increased IP [23].

2.2. RBC transfusions

For the present study, the total volume of RBC and volume per weight (ml/kg) between and within 48 hrs preceding La/Rh administrations and hematocrit preceding transfusion during each study interval (birth-day1; day1-day8, and day8-day15) was recorded. Enteral feeding status and type of feeds (exclusive breast milk, combined breast milk/formula vs. exclusive formula) during each interval and whether feeds were administered during RBC transfusions were determined.

2.3. Statistical analysis

Characteristics of subjects who did vs. did not receive RBC transfusions during the study period were compared using T-Test, Chi-square, and Fisher Exact Testing as appropriate. In addition, transfusion parameters and feeding status in intervals preceding increased IP (La/Rh > 0.05) were compared to those in intervals preceding normal IP (La/Rh≤0.05). Logistic regression was performed to assess predictors of abnormal/increased IP ratios. Data were analyzed using SAS 9.3 (SAS Institute, Carey NC).

3. Results

Forty-three subjects were enrolled in the original study [16]. One subject who had no IP measurement data recorded was excluded from the current analysis, giving a total of 42 subjects with a total of 118 IP measurements recorded. Subjects who received any RBC transfusion during the study had lower birth gestational ages (GA) and lower birth weights than those who were not transfused (Table 1). None of the study subjects developed NEC during their NICU stay. Of the seventeen subjects (40.5%) who received≥1 RBC transfusion during the study period, 12 (70.6%) infants were <28 wkGA and 5 (29.4%) infants were≥28 wkGA, p < 0.0001. Each of the 12 infants <28 wk GA (100%) received at least one RBC transfusion compared to only 5 of the 30 infants≥28 wk GA (20%), p < 0.0001. As shown in Fig. 1, the majority of the RBC transfusions including those within 48 hours preceding IP measurements occurred in the second interval (study d1-8). As previously reported [16], intestinal barrier maturation was gestational and postnatal-age dependent.

Fig. 1.

Distribution of RBC transfusion during each study interval in infants who received≥1 RBC transfusion at any time during the interval and those who received transfusion within 48 hrs of sugar test within the study interval compared to non-transfused infants.

Table 1

Demographic characteristics of study cohort based on RBC transfusion status

Variable	Total cohort (n = 42)	RBCT (n = 17)	No RBCT (n = 25)	p-value
Gender (male)	23 (54.8%)	10 (58.8%)	13 (52%)	0.6628
Race (African-American)	23 (54.8%)	13 (76.5%)	10 (40%)	0.0828
Gestational age (wk)	29.9±2.3	27. 6±1.8	31.5±0.8	<0.001 ^*
Birth weight (g)	1355±427	985±237	1573±357	<0.001 ^*
PTL	17 (40.5%)	9 (53%)	8 (32%)	0.1747
PPROM	13 (31%)	5 (29.4%)	8 (32%)	0.8586
Pre-eclampsia	8(19%)	4 (23.5%)	4 (16%)	0.6939
Antenatal Steroids	35 (83.3%)	15 (88.2%)	20 (80%)	0.4821
Maternal Antibiotics	28 (66.7%)	11 (64.7%)	17 (68%)	0.8241
Cesarean section	30 (71.4%)	11 (64.7%)	19 (76%)	0.4265

^*P < 0.05. Preterm labor-PTL; Preterm premature rupture of membranes-PPROM.

IP measurements were then analyzed by interval to evaluate the relationship between post-menstrual age (PMA) at the time of IP measurement, RBC transfusion, pre-transfusion hematocrit, type of enteral feeds and IP (Table 2). As previously reported, intervals associated with high IP measurements were found in subjects with significantly younger PMA (30.7 wks) than those associated with low IP measurements (32.5 wks, p = 0.0002) [16]. RBC transfusion occurred 3 times more frequently in intervals preceding high IP than in intervals preceding low IP measurements. RBC transfusion total volume and volume/kg were 5-fold higher in the intervals preceding high IP measurements. Lower pre-transfusion hematocrit was observed in intervals preceding high IP than in intervals preceding low IP measurements though this was not statistically significant (33 vs 35.8, p = 0.1051). Enteral feeds occurred frequently during transfusion in intervals preceding both low and high IP measurements (100% vs 77% respectively, p = 0.3559). In our cohort, all subjects (100%) who had both RBC transfusion and a low IP measurement vs. 55% of subjects who had both RBC transfusion and a high IP measurement were fed exclusive breast milk during the previous interval, though this did not reach statistical significance (p = 0.25).

Table 2

Relationship between post-menstrual age, RBC transfusion, pre-transfusion hematocrit, enteral feeds, and intestinal permeability (IP) measurements

Outcome	La/Rh≤0.05 N = 36 intervals Mean (SD) or N (%)	La/Rh > 0.05 N = 82 intervals Mean (SD) or N (%)	p-value
PMA at the time of IP measurement, wk	32.48±2.1	30.67±2.4	0.0002
Hematocrit preceding transfusion	35.8±1.8	33±4.7	0.1051
Number of intervals with RBCT preceding IP measurement	3 (8.3%)	22 (26.8%)	0.0275
Total RBC /interval (mL)	1.4±4.7	6.4±2.4	0.0226
Total RBC/interval (ml/kg)	1.5±5.0	7.6±15.4	0.0207
Total RBC within 48hrs of sugar test/interval (ml)	0.78 (3.4)	2.7 (6.5)	0.0902
Total RBC within 48hrs of sugar test/interval (ml/kg)	0.83 (3.7)	2.98 (7)	0.0884
Number of intervals with RBCT and any enteral feeds	3/3 (100%)	17/22 (77%)	0.3559
Number of intervals with RBCT and Exclusive Breastmilk	3/3 (100%)	12/22 (55%)	0.2500

PMA-post menstrual age; RBCT- red blood cell transfusion.

We performed logistic regression analysis to identify predictors of high IP measurements (Table 3). When adjusting for sex, advancing PMA at the time of IP measurement and exclusive breast milk feeding prior to IP measurement significantly decreased the odds of high IP measurement. In our cohort, those subjects who underwent RBC transfusion during the interval prior to IP measurement had an almost 3x the adjusted odds of having a subsequent high IP measurement, but this was not statistically significant.

Table 3

Logistic regression: Predictors of having high intestinal permeability (IP) measurements

Predictors	Odds ratio ^* of La/Rh > 0.05	95% CI	p-value
PMA (weeks) at the time of IP measurement	0.77	0.596–0.993	0.0443
Feeding exclusive BM prior to IP measurement	0.047	0.010–0.215	<0.0001
RBCT during interval	2.7	0.564–12.814	0.2143

^*Adjusted for sex of subject. PMA-post menstrual age; BM-breast milk; RBCT-red blood cell transfusion.

4. Discussion

To our knowledge, ours is the first study to highlight an observation of a possible association between RBC transfusion and high IP (La/Rh≥0.05) in pre term infants. RBC transfusion occurred more frequently in the interval preceding high IP, and total volume and volume/kg of RBC transfusion were five fold higher in the interval preceding any time point with high IP. Our analysis suggests that advancing PMA and exclusive breast milk feeds reduce the impact of RBC transfusion on IP.

Significant knowledge gaps exist regarding the effects of RBC transfusion on IP. Premature infants remain a heavily transfused population and multiple studies have examined the association and time interval between RBC transfusion and NEC [3 , 25]. Despite close temporal association observed between NEC and RBC transfusion within 48 hrs, underlying mechanism(s) by which RBC transfusion alters intestinal barrier function remains unknown. It is likely to involve abnormal intestinal perfusion in the immediate post-transfusion state. Anemia is a known risk factor for impaired splanchnic perfusion [26]. There is accumulating evidence that the risk of transfusion-associated NEC may be higher in infants transfused with the greatest severity of anemia [27 –29], but the underlying mechanism of interaction between severe anemia and RBC transfusion is currently unknown. Underlying anemia could predispose to ischemic injury secondary to tissue hypoxia, anaerobic metabolism and possibly NEC [30]. Other possible mechanisms include ischemia-reperfusion injury [31], entrapment of poorly deformable red cells and residual allogenic white blood cells that trigger an immunomodulating response in the host similar to transfusion associated lung injury (TRALI) [4 , 33]. Postoperative systemic inflammatory response syndrome has been observed following intraoperative blood transfusion, suggesting that inflammatory mediators and leukocyte-related activation triggered by RBC transfusion could potentially modulate an inflammatory reaction [34]. We report a lower pre-transfusion hematocrit in infants with high IP compared to those with low IP measurements among the transfused cohort, although there was no statistical evidence of heterogeneity (test for subgroup difference p = 0.1051). We speculate that impaired intestinal barrier function as a result of transfusion-mediated microvascular ischemia and re-oxygenation injury may explain, in part, the association of anemia, RBC transfusion and NEC. Alternatively, since the most immature infants have high IP due to intestinal barrier immaturity, and are more likely to develop iatrogenic anemia and subsequent tissue hypoxia, they may be more susceptible to transfusion-mediated intestinal epithelial/microvascular injury.

An immature gut barrier is a predisposing factorin NEC pathogenesis [35]. We have previously reported that exclusive breastmilk feeds are associated with improved intestinal barrier function [16]. Several studies have investigated the relationship between enteral feeds, RBC transfusion and NEC [36 –39]. Whether holding feeds and various types of enteral feeding regimens at the time of RBC transfusion alters the risk for subsequent NEC has been a subject of major debate [38 , 41]. Enteral feeding failed to stimulate a post-prandial hyperaemic response to the mesenteric blood flow in the immediate post-transfusion period in a small group of <1250g infants [32]. The lack of hyperemic response to feeding in the immediate post-transfusion state has been speculated to increase the susceptibility to NEC from intestinal hypoperfusion. On the contrary, type of enteral feeds, especially breast milk has been associated with a much lower incidence of NEC compared to formula [42 –44]. Postnatal introduction of feeding accelerates intestinal maturation [16, 19]. In the current study, feeding during transfusion did not appear to be a factor associated with high IP, but exclusive breast milk appeared protective.

Study limitations include the small sample size and retrospective data collection of RBC transfusion data since the original prospective study was not designed to evaluate the effect of transfusion on IP. The earlier timing prior to the postnatal age of around four weeks when preterm infants are at highest risk for NEC to study pathogenesis of transfusion associated NEC could be considered as an additional limitation. Nevertheless, we observed that our cohort had almost 3x the odds of an abnormal IP measurement following RBC transfusion. Though this was not statistically significant, this finding is compelling and further larger studies should continue to evaluate the effect of both anemia and RBC transfusion on IP in preterm neonates. Using the dual sugar probe test as a research tool to compare intestinal permeability before and after RBC transfusion in the presence or absence of feeding may provide additional insights into the pathogenesis of transfusion-associated NEC.

Funding source

Funded by NCCIH R34AT006945.

Financial disclosure

The authors have no financial relationships relevant to this article to disclose.

Conflict of interest and ethics

The authors have no conflicts of interest to disclose. The institutional review board at University Of Maryland reviewed and approved the study. Parental consent was obtained for the study.

Footnotes

Acknowledgments

We thank Dr Jonathan Meddings, University of Calgary, Calgary, Alberta, Canada for the HPLC analysis of urine samples and Ashley Bathgate, Kirsty L. Chesko, and Elise Janofsky for research assistance (funded by NCCIH R34AT006945).

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