Material and Method: Complete blood count parameters obtained on the first day of life of neonates born between January 2015 and December 2019 were analyzed retrospectively. The distribution and correlation of hematological indices according to gestational age and birth-weight were examined.

Results: The mean gestational age of 340 newborns was 32.0±4.9 weeks and birth-weight was 1821.68±923.97 g. There was no difference between the two genders in terms of hematological indices, except that RDW value was higher in girls than in boys. There was a weak positive correlation between gestational age and leukocyte, platelet and erythrocyte counts (r =0.245, r =0.347 and r =0.310, respectively; p <0.05 in all comparisons). A moderate negative correlation was found between gestational age and MCV and MCH (r =-0.611, r =-0.564, respectively; p <0.05 in all comparisons). When hematological indices were compared according to gestational weeks, leukocyte and platelet counts were also different, similar to erythrocyte indices (p <0.05). When these hematological indices were analyzed according to birth-weight, similar results were obtained to those obtained accord-ing to gestational age.

Conclusion: The reference ranges for various hematologic indices in the neonatal period are not constant and change significantly with advancing gestational age. To identify abnormal results, normal reference values for CBC parameters must be determined for each neonatal population.

The various parameters of CBC analyzed in this way include erythrocyte indices such as RBC count, hema-tocrit (HCT), hemoglobin (HGB) concentration, mean corpuscular volume (MCV), mean corpuscular hemo-globin (MCH), mean corpuscular hemoglobin concentration (MCHC), and PLT count, mean platelet volume (MPV), and WBC count¹.

Recognizing low or high blood concentrations of eryth-rocytes, platelets, or leukocytes, or abnormally small or large erythrocytes or platelets, may affect clinical decisions. These cannot be interpreted with normal reference ranges established in healthy children, but rather with reference ranges derived from neonatal datasets. Unfortunately, reference ranges for various CBC pa-rameters in the neonatal period are not fixed and change significantly with advancing gestation or birth-weight. Therefore, results are interpreted according to high-quality reference ranges based on gestational - and postnatal age^2,3.

Accepting that a laboratory test result is abnormal is an integral part of medical practice. Hematologic reference ranges are used as medical decision tools to inter-pret numerical test results in newborn patients. Estab-lishing reference ranges for various CBC parameters in newborn infants is of great importance to neonatolo-gists or provides much information regarding the diag-nosis and treatment of many diseases^4,5. It is widely known that neonatal hematologic parameters differ from those in infants and adults. Therefore, hematologic reference ranges established for neonates are of great importance⁶. However, reference ranges for various parameters in the CBC are often based on small studies and are obtained with laboratory equipment that is now considered obsolete. Recent studies at Intermountain Healthcare have established new reference ranges for neonatal hematologic parameters based on very large sample sizes obtained with modern hematologic analyzers^7-10.

Therefore, this study aimed to determine the normal reference ranges of CBC parameters according to different gestational ages and birth-weights in the Turkish newborn population.

Newborn infants with maternal gestational diabetes and/or hypertensive disorder, intrauterine growth retar-dation (IUGR), blood group incompatibility, perinatal asphyxia, cyanotic congenital heart disease and con-genital anomalies (congenital diaphragmatic hernia, neural tube defects, etc.) were excluded from the study. Similarly, newborn infants with conditions such as premature rupture of membranes (PROM), preterm PROM (PPROM) and early neonatal sepsis were also excluded from the study. Additionally, newborns with gestational ages <24 weeks and >40 weeks were ex-cluded from the study due to insufficient number of cases for statistical analysis. Recalling the last men-strual period (LMP, Naegele formula) and new Ballard scoring were used to determine gestational age. Cases with discordance between LPM and the new Ballard scoring were excluded from the study.

Newborn infants were divided into 5 groups according to their gestational age: i) extremely preterm (<28 weeks of gestation), ii) very preterm (28+^0/7-31+^6/7 weeks of gestation), iii) moderately preterm (32+^0/7-33+^6/7 weeks of gestation), iv) late preterm (34+^0/7-36+^6/7 weeks of gestation) and v) full-term (37+^0/7-42+^0/7 weeks of gestation). Prematurity refers to births occur-ring before 37^0/7 weeks of gestation. Newborns were also divided into 4 groups according to birth-weight: i) extremely low birth-weight (ELBW, <1000 g), ii) very low birth-weight (VLBW, 1000-1500 g), iii) low birth-weight (LBW, 1500-2500 g) and iv) normal birth-weight (NBW, >2500 g) (11,12).

Statistical analysis
Data were analyzed using SPSS for Windows, version 25.0 (SPSS Inc., Chicago, IL). Data were expressed as mean±standard deviation (SD) for normally distributed data, as median (minimum-maximum) for non-normally distributed continuous variables, and as num-ber of cases (%) for nominal variables. Since the sam-ple size was >50, the conformity of continuous varia-bles to normal distribution was determined by the Kolmogorov-Smirnov test. Student’s t test was used for comparing means, and Mann Whitney-U test was used for comparing medians. ANOVA test or Kruskal-Wallis test was used for comparing continuous varia-bles belonging to two or more independent sample groups. According to the ANOVA test results, Post Hoc test was performed using Tukey and Bonferronni when the variances were homogeneous, and Tamhane’s T2 test was performed when the variances were not homogeneous. After the Kruskal-Wallis test, Kruskal Wallis One-Way ANOVA test was used for Post Hoc tests. According to the Kruskal-Wallis test results, Post Hoc test was performed using Kruskal-Wallis One-Way ANOVA. Spearman correlation analysis and Pearson correlation analysis were used to evaluate the relationships between quantitative variables. Accord-ingly, correlation coefficient r value <0.5 was evaluated as weak, 0.5-0.7 as moderate, and ≥0.7 as strong. A two-tailed p-value of <0.05 was accepted as significant.

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Table 1: Demographic characteristics of cases

The general distribution of hematological indices was given in table 2. When these hematological indices were evaluated according to gender, only the RDW values were found to show a statistically significant difference between female (17.5±1.4) and male (16.9±1.3) infants (p <0.05) (Table 2).

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Table 2: General distribution of hematological parameters.

Table 3 showed comparisons of newborn infants’ he-matological indices on the first day of life based on the weeks of their gestation. In table 3, there was a statisti-cally significant difference between the hematologic indices that had the same letter (p <0.05).

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Table 3: Comparisons of hematological indices of newborns on the first day of life according to their gestational ages.

To illustrate the situation described above with an example, in table 3, the letter “a” for the WBC value is found in both extremely preterm infants and term in-fants. This situation will be interpreted as a statistically significant difference in WBC values between extreme-ly preterm infants and term infants (p <0.05). Similarly, the letter “b” is present in both very preterm infants and term infants. This situation will be interpreted as indicating a statistically significant difference in WBC values between very preterm infants and term infants (p <0.05).

Correlation between gestational age and hematolog-ical indices
A weak positive correlation was found between gesta-tional age and WBC, PLT and RBC counts (r =0.245, r =0.347, r =0.310, respectively) (p <0.05 for all com-parisons) (Figure 1).

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Figure 1: Correlation between gestational age and WBC (a), PLT (b) and RBC (c).

There was no correlation between gestational age and HGB, HCT, MCHC and RDW values (p >0.05 for all comparisons).

There was a moderate negative correlation between gestational age and MCV and MCHC values (r =-0.611, r =-0.564, respectively), (p <0.05 for all comparisons) (Figure 2).

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Figure 2: Correlation between gestational age and MCV (a) and MCH (b).

Comparisons of hematological indices of newborn infants on the first day of life according to their birth-weights were shown in table 4. The difference between the hematologic indices with the same letter in table 4 was statistically significant (p <0.05).

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Table 4: Comparisons of hematological indices of newborns on the first day of life according to their birth-weights.

To illustrate the above situation with an example, in table 4, the letter “a” is found in the WBC values of both extremely low birth weight infants and normal birth weight infants, the letter “b’ is found in the values of both very low birth weight infants and normal birth weight infants, the letter “c” is used for both extremely low birth weight infants and low birth weight infants, and finally, the letter “d” is used for both very low birth weight infants and low birth weight infants. These situations will be interpreted as follows:

• There is a statistically significant difference in WBC values between normal birth weight infants and extremely low and very low birth weight infants (p <0.05).

• There is a statistically significant difference in WBC values between low birth weight infants and extremely low and very low birth weight infants (p <0.05).

Correlation between birth-weight and hematologi-cal indices
A weak positive correlation was found between birth-weight and WBC, PLT and RBC counts (r =0.299, r =0.416, r =0.237, respectively) (p <0.05 for all com-parisons) (Figure 3).

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Figure 3: Correlation between birth-weight and WBC (a), PLT (b) and RBC (c).

There was no correlation between birth-weight and HGB, HCT and RDW values (p >0.05 for all comparisons).

There was a moderate negative correlation between birth-weight and MCV and MCHC values (r =-0.675, r =-0.586, respectively), (p <0.05 for all comparisons). A weak positive correlation was found between birth-weight and MCHC value (r =0.132, p <0.05) (Figure 4).

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Figure 4: Correlation between birth-weight and MCV (a), MCH (b) and MCHC (c).

In our study, a weak positive correlation was found between gestational age and WBC count. In other words, a slight increase was seen in WBC count as gestational age increased. In a study examining the effect of gestational age and birth-weight on WBC count in newborns, it was reported that WBC counts were significantly lower in premature and small-for-gestational-age infants¹⁵. Although this finding is consistent with our results, a weaker positive correla-tion was found in our study compared to that study. Chetana and Xiangying¹⁶ reported that neutropenia was more common in premature infants due to mater-nal and antenatal conditions, congenital syndromes, immune-mediated processes, hospital infections, and idiopathic causes. Although some pathological causes that could affect hematological parameters were ex-cluded from our study, the above-mentioned causes may help explain the low WBC count in low-birth-weight premature infants.

In our study, a weak positive correlation was found between gestational age and PLT count. In other words, as gestational age increases, PLT count also increases to some extent. However, in a single-center study conducted in Türkiye by Cantürk^17, it was determined that there was no significant difference in PLT count between full-term and preterm newborns. The difference between those findings and our results may be due to the characteristics of the study popula-tion, the exclusion criteria from the study, and the dif-ference in sample size. In a study conducted on mice, similar to our results, it was shown that the PLT count was low and functions were hyporeactive in the early fetal period¹⁸. Accordingly, Cremer¹⁹ reported a statistically significant but low correlation between gestational age and PLT count.

In our study, a weak positive correlation was observed between gestational age and RBC count. In other words, erythrocyte count slightly increased as gesta-tional age increased. However, no correlation was found between gestational age and HGB and HCT values. On the other hand, a moderate negative correla-tion was found between gestational age and MCV and MCH values. Accordingly, MCV and MCH values decreased moderately as gestational age increased. However, no correlation was found between gestational age and MCHC and RDW values. In a study investigat-ing the effects of race and gestational age on erythro-cyte indices in very low birth-weight infants in the USA, it was shown that HGB and HCT values increased and MCV values decreased with advancing gestational age²⁰. Rasmussen and Oian²¹ reported that the hemoglobin value of fetuses decreased as ges-tational age and weight increased. Morshed et al.²² found that RBC count, HGB and HCT levels were higher in full-term infants than in preterm infants. However, they showed that MCV values were lower in full-term infants than in preterm infants. In our study, it was found that MCV values decreased similarly with advancing gestational age, while HGB and HCT values did not change differently. This difference may be due to the fact that our study was single-centered, the rela-tively low number of cases, differences in inclusion and exclusion criteria, and geographical and racial differences in our study population. In a multicenter study including 12,000 newborns in which erythrocyte indices were investigated, it was reported that MCV and MCH values decreased with increasing gestational age, but MCHC values did not change⁸. The results of that study are consistent with the results of our study.

When the correlation between birth-weight and hema-tological indices was evaluated, it was seen that there was a weak positive correlation between birth-weight and WBC, PLT and RBC counts. However, no correla-tion was found between birth-weight and HGB and HCT values. But, a moderate negative correlation was found between birth-weight and MCV and MCH val-ues. Contrary to the correlation between gestational age and MCHC, a weak positive correlation was found between birth-weight and MCHC. Consistent with the correlation between gestational age and RDW, no cor-relation was found between birth-weight and RDW value.

Factors limiting our study include the fact that our study was single-centered, the number of cases was relatively small, the number of babies with a gestation-al age below 24 weeks and above 40 weeks was not sufficient for statistical analysis, and the cases included in the study belonged to a single geographical region and a single race.

In conclusion, there was a weak to moderate correla-tion between gestational age and hematological indices. Since newborns with IUGR were excluded from the present study, the correlation between birth-weight and hematological parameters gave similar results to the correlations between gestational age and hematological parameters. The reference values of hematological parameters determined according to different gestational age and birth-weight groups would be useful in neonatal practice.

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