Readmission for neonatal hyperbilirubinemia in an area with a high prevalence of glucose-6-phosphate dehydrogenase deficiency: A hospital-based retrospective study

Abstract

BACKGROUND: Hyperbilirubinemia is one of the most common causes of neonatal readmission to hospital.

AIMS: To assess risk factors for hyperbilirubinemia among neonates readmitted for this condition and the ratio of the mean corpuscular hemoglobin concentration (MCHC) to the mean corpuscular volume (MCV).

METHODS: We retrospectively studied the clinical and laboratory findings, management and possible risk factors for hyperbilirubinemia in 301 neonates born at ≥35 weeks gestation and readmitted to hospital owing to hyperbilirubinemia over five years.

RESULTS: No risk factors for hyperbilirubinemia were identified in 64 (21.3%) neonates, and one or more risk factors were found in 237 neonates (78.7%). The most prevalent risk factor (41.9%) was G6PD deficiency, which occurred in 11 of the 15 neonates with a serum bilirubin level ≥427 μmol/l. A double-volume exchange blood transfusion was performed in two neonate boys in whom G6PD deficiency was the single risk factor for hyperbilirubinemia. One of them developed kernicterus later. The MCHC/MCV ratio of neonates with idiopathic hyperbilirubinemia, unexplained hemolysis, or other risk factors overlapped.

CONCLUSIONS: This study confirmed that in an area where G6PD deficiency is prevalent, it is the most common and most severe risk factor for hyperbilirubinemia. This finding supports routine neonatal screening for G6PD deficiency in such areas. The usefulness of determining the MCHC/MCV ratio in the management of hyperbilirubinemia is uncertain.

Keywords

Neonatal hyperbilirubinemia glucose-6-phosphate dehydrogenase deficiency hereditary spherocytosis phototherapy erythrocyte indices

1 Introduction

Indirect hyperbilirubinemia is considered to be one of the most common causes of neonatal readmission to hospital during the first week of life [1 –5]. The prevalence and ranking of the risk factors for this condition vary geographically depending on the genetic background of the population [1–3 , 6–9]. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a hereditary condition caused by X-linked recessive mutations, is very common in the Al-Ahsa area in the Eastern Province, Saudi Arabia, with an overall prevalence of 23% in men and 13% in women [10]. Most cases of G6PD deficiency are severe in this region, as the G6PD-Mediterranean variant constitutes 84% of G6PD mutations [11]. In 1999, a study from a public hospital in Al-Ahsa area showed that G6PD deficiency, diagnosed by the fluorescent spot test [12], was the most prevalent risk factor for hospital readmission among neonates with hyperbilirubinemia [1]. However, most hospitals in Al-Ahsa do not routinely screen neonates for G6PD deficiency [13, 14]. The lack of screening might be due to the fact that the 1999 study was conducted in a single public hospital, evaluated only six risk factors, and is the only study that has addressed readmission for neonatal hyperbilirubinemia in Al-Ahsa area.

The prevalence of idiopathic hyperbilirubinemia is high, and unidentified hemolysis has been proposed as the underlying risk factor [15, 16]. Recently, a research group showed that the ratio of the mean corpuscular hemoglobin concentration (MCHC) to the mean corpuscular volume (MCV) was substantially greater in neonates with hereditary spherocytosis compared with a group of neonates who were born at 35–41 weeks gestation, had no anemia and did not require blood transfusion [17]. The researchers suggested that an MCHC/MCV ratio greater than 0.36 is a simple method of identifying hereditary spherocytosis among neonates with idiopathic hyperbilirubinemia with a 97% sensitivity,>99% specificity, and >99% negative predictivevalue [17].

In this study, we aimed to assess 1) the replicability of the 1999 study by evaluating more risk factors for hyperbilirubinemia in a hospital in Al-Ahsa that performs transcutaneous bilirubin measurement and G6PD deficiency screening (with the hope that the results will boost routine neonatal screening for G6PD deficiency in Al-Ahsa) and 2) the MCHC/MCV ratio in neonates with hyperbilirubinemia.

2 Methods

2.1 Study design

This retrospective study was performed in Almana General Hospital (AGH) in Al-Ahsa area. We included infants that were born at ≥35 weeks gestation and were readmitted to the pediatric ward after discharge from the neonatal ward between January 2009 and December 2013. Clinical and laboratory data, as well as information about risk factors and management of hyperbilirubinemia, were extracted from the medical records of neonates. Demographic data and information about the blood groups of the mothers were also obtained. We evaluated 12 established risk factors for indirect neonatal hyperbilirubinemia [18 –20]. Six of these risk factors had also been evaluated in the 1999 study (shared risk factors): 1) G6PD deficiency, 2) ABO blood group incompatibility, 3) breast milk jaundice, 4) Rhesus factor (Rh) incompatibility, 5) sepsis, and 6) red blood cell (RBC) membrane disorders [1]. Six additional risk factors had not been evaluated in the 1999 study: 1) birth at 35–36 week’s gestation, 2) dehydration/inadequate breastfeeding, 3) cephalohematoma, 4) trisomy 21, 5) unexplained hemolysis, and 6) mother with any types of diabetes. Unexplained hemolysis was defined as the presence of either reticulocytosis or a hematocrit <40% in the absence of G6PD deficiency, ABO incompatibility, Rh incompatibility, or RBC membrane disorders. Reticulocytosis was defined as an elevated reticulocyte count for the individual’s age as follows: >8.0% at 24–71 h of age;>4.0% at 72–168 h of age; and >2.0% at ≥168 h of age [21]. If none of these 12 risk factors was identified, the hyperbilirubinemia was labelled as idiopathic [20]. The study was approved by AGH (03/12) and consent was waived as s the study was a retrospective chart review. The ethical principles outlined in the Declaration of Helsinki were followed.

2.2 Routine care for neonatal indirect hyperbilirubinemia at AGH

The standard care of neonates at AGH includes screening for G6PD deficiency and blood grouping using cord blood samples. The levels of G6PD are measured quantitatively using an automated commercial kit (Udilipse Auto Analyzer from United Diagnostics Industry, Dammam, Saudi Arabia). The level of 60 mU/10⁹ RBCs is used as the lower reference limit. This level was extrapolated from the reference range among the adult Saudi population (60–130 mU/10⁹ RBCs) [10]. However, it is stated in the operation leaflet for the reagents of the Udilipse Auto Analyzer that 135 mU/10⁹ RBCs was the 2.5th percentile of the G6PD level among 150 normal adults of both sexes from the Dammam area in Eastern Province, Saudi Arabia.

Transcutaneous bilirubin (TcB) levels are measured by a MBJ20 transcutaneous bilirubinometer (Beijing Electronic Instruments Co Ltd, Beijing Bioengineering and Medicine Industry Base, Haungoun Town, Daxing Area, Beijing, China). These measurements are performed every 8 h in all neonates born at ≥35 weeks gestation until they are discharged from the neonatal ward or at the commencement of phototherapy. Healthy neonates born at >38 weeks gestation are discharged 1-2 days after vaginal delivery and 3–5 days after Cesarean delivery. At discharge, the parents are given special health education including a leaflet written in Arabic about neonatal hyperbilirubinemia and its possible complications. All neonates are given outpatient follow-up appointments within 1–3 days post-discharge according to their clinical status and the presence or absence of known risk factors for developing indirect hyperbilirubinemia that required phototherapy treatment as per graph issued by American Association of Pediatrics (AAP), which includes bilirubin threshold levels for treatment recommendations [18, 19].

During an outpatient or emergency room visit, TcB levels are measured if the infant looks jaundiced. Every TcB measurement is plotted on a phototherapy and exchange transfusion graph issued by the AAP [18, 19]. Each TcB level that approaches or crosses the phototherapy threshold curve is confirmed by a serum bilirubin (SBR) measurement. As uniform categorizing severity of hyperbilirubinemia is lacking [22], hyperbilirubinemia is categorized arbitrarily as following: 1) mild when SBR lies below phototherapy threshold line of AAP graph, 2) moderate when SBR lies within the halfway between phototherapy and exchange transfusion threshold lines, 3) severe when SBR lies above the halfway between phototherapy and exchange transfusion threshold lines, and 4) extreme when SBR lies at/above exchange transfusion line.

If the SBR level lies within the phototherapy zone, the neonate is admitted with the mother to the pediatric ward for phototherapy. A standard single-surface phototherapy unit (Choongwae Medical Corporation, Korea) is used for the treatment of moderate hyperbilirubinemia. A double-surface phototherapy unit consisting of a standard phototherapy unit on top of the baby, with the baby lying on a phototherapy bed (Medela Bilibed, Switzerland), was used for treatment of severe hyperbilirubinemia until June 2013, when it was replaced by a 360° exposure cylindrical intensive phototherapy unit (Tosan Manufacturer & Supply of Neonatal Systems, Iran). Generally, phototherapy is discontinued when the SBR level decreases to approximately 100 μmol/l below the phototherapy threshold. Supplemental oral or intravenous fluid is administered according to the assessment and at the discretion of the treating physician. If the SBR level lies within the exchange transfusion zone (extreme hyperbilirubinemia), the neonate is admitted to the neonatal intensive care unit.

Other investigations that are performed to determine the presence or absence of risk factors for hyperbilirubinemia include serial complete blood cell count, reticulocyte count, direct antiglobulin test (DAT), serum electrolyte analysis, and renal function analysis. The complete blood cell count and reticulocyte count are performed using CELL-DYN 3700 (Abbott Diagnostics, Santa Clara, CA, USA). RBC morphology is performed sporadically when a hematopathologist is available. Blood grouping is performed for all pregnant women antenatally or during delivery. ABO incompatibility is considered when the mother’s blood group is O and her neonate’s blood group is either A or B, regardless of neonatal DAT results [23]. A positive result in the neonatal DAT test is a prerequisite in diagnosing Rh incompatibility [23]. Breast milk jaundice is considered when an exclusively breastfed healthy infant has hyperbilirubinemia in the second week of life with no other identifiable risk factors [24, 25]. Dehydration or inadequate breastfeeding is defined as losing >10% of birth weight, having serum sodium levels ≥150 mmol/l, or according to the treating physician’s assessment among exclusively breastfed neonates [19, 26].

2.3 Statistical analysis

Laboratory and baseline scale variables were categorized at clinically relevant cutoff points for hyperbilirubinemia (Table 1) [17 , 27–29]. A binomial test with an expected proportion of 0.5 was performed to compare the observed proportions of each sex, maternal age, and parity among the studied cohort. Breast milk jaundice was excluded from the analysis because no case fit the aforementioned definition of this condition [24, 25]. Therefore, only 11 risk factors were included in the analysis. Bootstrapping based on 1000 bootstrap samples was used to calculate a 95% confidence interval (CI) for percentages (proportions) [30].

Table 1
Baseline characteristics and laboratory findings of 301 neonates readmitted to hospital

Characteristics and findings n Percentage (95% confidence interval)

Gestational age

35–37 weeks 92 31% (25–26%)

38–42 weeks 209 69% (64–75%)

Birth weight

<2500 g 36 12% (9–16%)

2500–4000 g 257 85% (81–89%)

>4000 g 8 3% (1–5%)

Sex of neonate^a

Boys 198 66% (60–72%)

Girls 103 34% (28–40%)

Born in the study hospital 291 97% (95–99%)

Born on other hospitals 10 3% (1–5%)

Delivery mode

Spontaneous vaginal 272 90.4% (87–94%)

Vacuum-assisted 3 1.0% (0–2%)

Forceps-assisted 1 0.3% (0–1%)

Cesarean 25 8.3% (4–13%)

Maternal age^b

<25 years 126 42% (36–48%)

≥25 years 175 58% (52–64%)

Parity^c

Primiparous 122 41% (35–46%)

Multiparous 179 59% (54–65%)

Age of neonate at readmission

36–72 h 47 15.6% (11.6–19.6%)

73–168 h 206 68.4% (63.5–73.4%)

169–336 h 46 15.3% (11.3–19.6%)

337–550 h 2 0.7% (0.0–1.7%)

Hematocrit

31–39% 21

40–64% 280 7.0% (4.3–9.6%)

Not measured 2 93.0% (90.4–95.7%)

Mean corpuscular volume (MCV)

71–94 fl 189 63.2% (57.9–68.6%)

95–105 fl 105 35.1% (29.8–40.5%)

106–113 fl 5 1.7% (0.3–3.3%)

Not measured 2

Mean corpuscular hemoglobin concentration (MCHC)

31–35 g/dL 176 59.9% (54.1–65.3%)

36–39 g/dL 118 40.1% (34.7–45.9%)

Not measured 2

Missing data 5

MCHC/MCV ratio

0.31–0.36 61 20.7% (16.3–25.2%)

0.37–0.40 125 42.5% (36.7–48.6%)

0.41–0.45 93 31.6% (26.2–37.1%)

0.46–0.51 15 5.1% (2.7–7.8%)

Reticulocytosis

Not present 238 90.8% (87.4–94.3%)

Present 24 9.2% (5.7–12.6%)

Reticulocyte count not measured 39

Maximum serum bilirubin level

198–290 μmol/l 78 25.9% (20.9–31.2%)

291–341 μmol/l 138 45.8% (40.5–51.5%)

342–426 μmol/l 70 23.3% (18.6–27.9)

427–512 μmol/l 12 4.0% (2.0–6.3%)

513–686 μmol/l 3 1.0% (0.0–2.3%)

Characteristics and findings	n	Percentage (95% confidence interval)
Gestational age
35–37 weeks	92	31% (25–26%)
38–42 weeks	209	69% (64–75%)
Birth weight
<2500 g	36	12% (9–16%)
2500–4000 g	257	85% (81–89%)
>4000 g	8	3% (1–5%)
Sex of neonate^a
Boys	198	66% (60–72%)
Girls	103	34% (28–40%)
Born in the study hospital	291	97% (95–99%)
Born on other hospitals	10	3% (1–5%)
Delivery mode
Spontaneous vaginal	272	90.4% (87–94%)
Vacuum-assisted	3	1.0% (0–2%)
Forceps-assisted	1	0.3% (0–1%)
Cesarean	25	8.3% (4–13%)
Maternal age^b
<25 years	126	42% (36–48%)
≥25 years	175	58% (52–64%)
Parity^c
Primiparous	122	41% (35–46%)
Multiparous	179	59% (54–65%)
Age of neonate at readmission
36–72 h	47	15.6% (11.6–19.6%)
73–168 h	206	68.4% (63.5–73.4%)
169–336 h	46	15.3% (11.3–19.6%)
337–550 h	2	0.7% (0.0–1.7%)
Hematocrit
31–39%	21
40–64%	280	7.0% (4.3–9.6%)
Not measured	2	93.0% (90.4–95.7%)
Mean corpuscular volume (MCV)
71–94 fl	189	63.2% (57.9–68.6%)
95–105 fl	105	35.1% (29.8–40.5%)
106–113 fl	5	1.7% (0.3–3.3%)
Not measured	2
Mean corpuscular hemoglobin concentration (MCHC)
31–35 g/dL	176	59.9% (54.1–65.3%)
36–39 g/dL	118	40.1% (34.7–45.9%)
Not measured	2
Missing data	5
MCHC/MCV ratio
0.31–0.36	61	20.7% (16.3–25.2%)
0.37–0.40	125	42.5% (36.7–48.6%)
0.41–0.45	93	31.6% (26.2–37.1%)
0.46–0.51	15	5.1% (2.7–7.8%)
Reticulocytosis
Not present	238	90.8% (87.4–94.3%)
Present	24	9.2% (5.7–12.6%)
Reticulocyte count not measured	39
Maximum serum bilirubin level
198–290 μmol/l	78	25.9% (20.9–31.2%)
291–341 μmol/l	138	45.8% (40.5–51.5%)
342–426 μmol/l	70	23.3% (18.6–27.9)
427–512 μmol/l	12	4.0% (2.0–6.3%)
513–686 μmol/l	3	1.0% (0.0–2.3%)

^aBinomial P < 0.001. ^bBinomial P = 0.006. ^cBinomial P = 0.001.

Risk factors for hyperbilirubinemia occur as a single risk factor or coexisting with other risk factor(s) [20]. In this respect, three types of risk factor can be defined: those with no predominance of single or coexisting occurrence; those that predominantly coexist with other risk factor(s); and those that predominantly occur as a single risk factor. For the purpose of the study, single or coexisting predominance was considered when ≥75% of the risk factors occurred as such.

A one-sided test for binomial variables and two-sided tests for all other variables were considered statistically significant at P < 0.05. The analysis was performed using IBM SPSS Statistics 20 (Chicago, IL, USA) and GraphPad Prism 6 (San Diego, CA, USA) [31].

3 Results

A total of 307 neonates born at ≥35 weeks gestation were readmitted to hospital owing to indirect hyperbilirubinemia during the 5-year study period. Of these, 25 neonates (8%) were readmitted more than once. Six neonates were excluded from further analysis because some of their data were incomplete, including their G6PD levels. Table 1 depicts the baseline characteristics and laboratory findings of the 301 neonates included in this study.

Of the original cohort, 237 neonates (78.8% [95% CI: 74.1–83.1%]) had one or more risk factor(s) for hyperbilirubinemia. Of these, 151 (63.7% [95% CI: 57.2–69.3%]) neonates had a single risk factor, 76 (32.1% [26.7–38.3%]) had two co-occurring risk factors, 9 (3.8% [95% CI: 1.6–6.3%]) had three co-occurring risk factors, and one (0.4% [0.0–1.3%]) had four co-occurring risk factors. Of the 11 studied risk factors, seven had no predominance of single occurrence or co-occurrence and four predominantly co-occurred with other risk factors (Table 2).

Table 2
Distribution and prevalence of identified risk factors for hyperbilirubinemia

Risk factors for hyperbilirubinemia As a single As a coexisting Total Prevalence^b

risk factor^a risk factor^a (95% confidence interval)

Risk factors with no predominance of single occurrence or co-occurrence

G6PD deficiency 76 (60.3%) 50 (39.7%) 126^c 41.9% (36.2–47.2%)

ABO incompatibility 27 (47.4%) 30 (52.6%) 57^d 18.9% (14.6–22.9%)

Unexplained hemolysis 18 (56.2%) 14 (43.8%) 32 11.1% ^e (7.6–14.5%)

Mother with diabetes 8 (44.4%) 10 (55.6%) 18 6.0% (3.3–9.0%)

Rh incompatibility 3 (60.0%) 2 (40.0%) 5 1.7% (0.3–3.0%)

RBC membrane disorders 2 (66.7%) 1 (33.3%) 3 2.7% ^f (0.0–5.4%)

Trisomy 21 1 (50.0%) 1 (50.0%) 2 0.7% (0.0–1.7%)

Risk factors predominantly co-occurring

35–36 weeks gestation 7 (17.5%) 33 (82.5%) 40 13.3% (9.6–17.3%)

Dehydration/inadequate breastfeeding 6 (15.4%) 33 (84.6%) 39 13.0% (9.3–16.9%)

Cephalohematoma 2 (25.0%) 6 (75.0%) 8 2.7% (1.0–4.7%)

Suspected sepsis 1 (25.0%) 3 (75.0%) 4 1.3% (0.3–2.7%)

Risk factors for hyperbilirubinemia	As a single	As a coexisting	Total	Prevalence^b
Risk factors with no predominance of single occurrence or co-occurrence
G6PD deficiency	76 (60.3%)	50 (39.7%)	126^c	41.9% (36.2–47.2%)
ABO incompatibility	27 (47.4%)	30 (52.6%)	57^d	18.9% (14.6–22.9%)
Unexplained hemolysis	18 (56.2%)	14 (43.8%)	32	11.1% ^e (7.6–14.5%)
Mother with diabetes	8 (44.4%)	10 (55.6%)	18	6.0% (3.3–9.0%)
Rh incompatibility	3 (60.0%)	2 (40.0%)	5	1.7% (0.3–3.0%)
RBC membrane disorders	2 (66.7%)	1 (33.3%)	3	2.7% ^f (0.0–5.4%)
Trisomy 21	1 (50.0%)	1 (50.0%)	2	0.7% (0.0–1.7%)
Risk factors predominantly co-occurring
35–36 weeks gestation	7 (17.5%)	33 (82.5%)	40	13.3% (9.6–17.3%)
Dehydration/inadequate breastfeeding	6 (15.4%)	33 (84.6%)	39	13.0% (9.3–16.9%)
Cephalohematoma	2 (25.0%)	6 (75.0%)	8	2.7% (1.0–4.7%)
Suspected sepsis	1 (25.0%)	3 (75.0%)	4	1.3% (0.3–2.7%)

^aThe numbers and percentage values represent the number and ratio of patients with each risk factor (% of total). ^bDenominator = 301 unless otherwise noted. ^cDirect antiglobulin test was positive in 7 (12.3%) neonates. ^dMedian of G6PD level (mU/10⁹ RBCs) = 11 (interquartile range: 5–20). ^eDenominator = 289 because the reticulocyte count and/or hematocrit were not measured in 12 neonates who had no other hemolytic diseases. ^fDenominator = 111, the number of RBC morphology analyses performed. Two cases of hereditary elliptocytosis and one case of hereditary ovalocytosis were diagnosed.

Of the identified risk factors, G6PD deficiency was the most prevalent (Table 2). This risk was more prevalent in neonates with SBR levels ≥427 μmol/l (Table 3). It was the only risk factor in all three neonates with SBR levels 513–686 μmol/l (Table 3). In total, 108 neonates had G6PD deficiency without other hemolytic diseases. Of these, 107 neonates had hematocrit measurements, and 90 neonates had reticulocyte count measurements. Of the 107 neonates with hematocrit measurements, 10 (9.3% [95% CI: 4.1–15.0%]) had a hematocrit <40%. Of the 90 neonates with reticulocyte count measurements, 16 (17.8% [95% CI: 10.1–26.7%]) developed reticulocytosis. However, only three neonates (3.3% [95% CI: 0.0–7.6%]) had both a hematocrit <40% and reticulocytosis.

Table 3

Risk factors in neonates with a maximum serum bilirubin level ≥427 μmol/l

Risk factor	Maximum serum bilirubin level
	427–512 μmol/l (n = 12)	513–686 μmol/l (n = 3)
Idiopathic hyperbilirubinemia	2 neonates	0 neonates
Single risk factor	3 neonates: G6PD deficiency	2 neonates: G6PD deficiency
	1 neonate: 35–36 weeks gestation
Two coexisting risk factors	3 neonates: G6PD deficiency and 35–36 weeks gestation	1 neonate: G6PD deficiency and suspected sepsis
	2 neonates: G6PD deficiency and dehydration/inadequate breastfeeding
	1 neonate: ABO incompatibility and dehydration/inadequate breastfeeding

Of the 18 neonates with unexplained hemolysis as a single risk factor, seven had G6PD levels <135 mU/10⁹ RBCs (range: 72–125 mU/10⁹ RBCs); six of them were girls. No risk factors were identified (idiopathic hyperbilirubinemia) in 64 neonates (21.3% [95% CI: 16.6–26.2%]). Eleven neonates had G6PD levels <135 mU/10⁹ RBCs (range: 74–27 mU/10⁹ RBCs).

Of the original study cohort, 294 neonates had MCHC and MCV measurements (Table 1 and Fig. 1). Of these, 233 (79%) neonates had a MCHC/MCV ratio >0.36 including 46 (16.5%) neonates with idiopathic hyperbilirubinemia, 26 (8.8%) neonates with unexplained hemolysis, and 161 (54.8%) neonates with other identified risk factors. The ratio of unexplained hemolysis, idiopathic hyperbilirubinemia, and other risk factors overlapped (Fig. 1).

Fig.1

Distribution of the ratio of mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV).

All neonates included in the study cohort required phototherapy treatment. A double-volume exchange blood transfusion was performed in two neonate boys in whom G6PD deficiency was the single risk factor for hyperbilirubinemia. The level of G6PD was 3 mU/10⁹ RBCs in one case and 19 mU/10⁹ RBCs in the other. The neonate with the G6PD level of 3 mU/10⁹ RBCs developed kernicterus. He was discharged from the neonatal ward with a SBR of 82 μmol/l; his SBR was within the phototherapy zone during the follow-up visit, but his parents refused admission for phototherapy. He was readmitted on day six of life with symptoms and signs of acute bilirubin encephalopathy and a SBR of 686 μmol/l and albumin level of 3.5 g/dl. He was found to have kernicterus at the age of two years. A summary of various treatment measures is shown in the (Fig. 2).

Fig.2

Summary of treatment measures.

4 Discussion

The present study is the second of its kind to be performed in Al-Ahsa area, a region with a very high prevalence of G6PD deficiency. We evaluated six risk factors in addition to the six risk factors that were evaluated in a previous study [1]. It confirms the results of the previous study, namely that G6PD deficiency is the most prevalent risk factor for hyperbilirubinemia among neonates readmitted to hospital owing to this condition [1]. Our results indicate that G6PD deficiency becomes the most severe risk factor for hyperbilirubinemia with the widespread use of Rho(D) immunoglobulin in a geographic region with a very high prevalence of G6PD deficiency. We found that four risk factors predominantly co-occurred with other risk factors: 35–36 weeks gestation, dehydration/inadequate breastfeeding, cephalohematoma, and suspected sepsis. This finding supports the concept that these risk factors are secondary to or are co-morbidity risk factors for hyperbilirubinemia [20]. Logically, the presence of one of these risk factors should not preclude searching for other risk factors.

The World Health Organization (WHO) and other researchers endorsed screening cord blood samples from all neonates in populations with a prevalence of G6PD deficiency of 3–5% or more in males [32, 33]. Our finding that G6PD deficiency is the most common and most severe risk factor for hyperbilirubinemia in an area where G6PD deficiency is highly prevalent supports this endorsement. However, exact mechanism of this hyperbilirubinemia is still to be elucidated [34].

The prevalence of idiopathic hyperbilirubinemia in the present study was less than half of that reported in the 1999 study (21.3% versus 51%) [1]. This difference is attributable to the fact that the present study evaluated six additional risk factors. It could also be due to the finding that the prevalences of six shared risk factors were higher in the present study than in the 1999 study, except for breast milk jaundice (0% versus 5.2%) [1]. Nonetheless, the ranking of the remaining five shared risk factors is comparable between these two studies [1]. The difference in the prevalence of the six shared risk factors is attributable to the fact that each study measured or defined these risk factors differently [1]. For instance, G6PD activity was measured quantitatively in cord blood in the present study, whereas it was measured qualitatively (using the fluorescent spot test) in neonates’ blood in the 1999 study [1]. The fluorescent spot test can diagnose only severe G6PD deficiency, as its cutoff point is 2.1 U/g hemoglobin [12].

Of the 15 neonates with SBR ≥427 μmol/l, only two (16.7%) had idiopathic hyperbilirubinemia. This prevalence is much lower than the prevalence reported by others; for example, the prevalence of idiopathic hyperbilirubinemia was 73.6% among 114 neonates with SBR levels ≥427 μmol/l in a US study [15]. The authors of the US study hypothesized that the risk factors for having SBR levels ≥427 μmol/l are associated with hemolysis, including G6PD deficiency. For that reason, those studies endorsed screening for hemolytic disorders in all neonates with SBR levels ≥427 μmol/l in whom the cause of these levels is unclear [15, 16]. Therefore, the low prevalence of idiopathic hyperbilirubinemia that we found among neonates with SBR levels ≥427 μmol/l could be due to the high prevalence of G6PD deficiency in our region and the routine performance of cord blood screening for G6PD deficiency.

The findings of the present study challenge the recently proposed concept that hereditary spherocytosis is the most likely risk factor for idiopathic hyperbilirubinemia when the MCHC/MCV ratio is greater than 0.36 [17, 35]. First, the results of our study show no clear separation between the upper limit of the ratio among neonates with or without identified risk factors (Fig. 1). Rather, the upper limit of this ratio was lower in neonates with unexplained hemolysis than in other neonates (0.45 versus 0.51, respectively). Second, 26 (8.8%) neonates with unexplained hemolysis had MCHC/MCV ratio is greater than 0.36. Despite the fact that the prevalence of hereditary spherocytosis in Saudi Arabia is unknown [36], we do not think that as many as 8.8% of hyperbilirubinemia cases are due to hereditary spherocytosis. A prospective study performed in Paris, France showed that the incidence of hereditary spherocytosis was 1% (n = 4) among 402 neonates treated with phototherapy at a SBR threshold lower than that recommended by the AAP guidelines [37]. The MCHC/MCV ratios in these four neonates with spherocytosis were 0.33, 0.35, 0.36, and 0.40 which would yield a sensitivity of 25% for MCHC/MCV ratio >0.36. We agree with the recommendation that the cutoff point of >0.36 should not be transferred to other settings because pre-analytical factors and measurement methods differ between institutions and laboratories [17]. Possible hereditary spherocytosis still would be unexpectedly high at a ratio of >0.40 (36.7%) or >0.45 (5.1%) among the studied cohort (Table 1). Unfortunately, we were not able to perform statistical measures for the accuracy of diagnostic tests as no gold standard test was performed to confirm the hereditary spherocytosis diagnosis. Nonetheless, it is obvious we would not get the same sensitivity and specificity as the other investigators did (97% sensitivity,>99% specificity, and >99% negative predictive value) [17].

In addition to being a retrospective study, several limitations of the present study should be noted. First, RBC morphology was only performed in 111 (37%) neonates. Second, the definition of G6PD deficiency was based on adult data, but G6PD levels in cord blood are known to be higher than in adult samples [13]. Therefore, it is possible that the prevalence of G6PD deficiency was underestimated. Third, strict criteria for phototherapy discontinuation were lacking. Fifth, lack of more sensitive and specific laboratory tests for diagnosis of hemolysis and its etiology including carboxyhemoglobin, end-tidal carbon monoxide, eosin-5-maleimide, and next-generation sequencing of genes known to be associated with hemolysis andhyperbilirubinemia [38].

5 Conclusions

The present study supports the WHO recommendation of screening cord blood samples from all neonates in populations with a prevalence of G6PD deficiency of 3–5% or higher in males [32]. However, hyperbilirubinemia might be present in neonates with normal G6PD levels, which supports the AAP recommendation of a combined clinical and/or laboratory assessment to predict subsequent hyperbilirubinemia [19]. The prevalence of idiopathic hyperbilirubinemia is still high, which necessitates more clinical and laboratory investigations. Unfortunately, the present study suggests that a high MCHC/MCV ratio might not be a reliable marker of hereditary spherocytosis and thus calculation of this ratio might not reduce the prevalence of idiopathic hyperbilirubinemia.

Disclosure statement

All authors have no financial or non-financial competing interests to disclose. No funding has been received. However, the language editing of this manuscript by Macmillan Science Communication was financed by King Abdullah International Medical Research Center.

Human research statement

This study was conducted in accordance with the ethical standards of institutional committee and the World Medical Association’s Helsinki Declaration.

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