BLOOD CELLS PROFILE IN UMBILICAL CORD OF LATE PRETERM AND TERM NEWBORNS

ABSTRACT Objective: To describe the hematological profile in cord blood of late preterm and term newborns and compare blood indices according to sex, weight for gestational age and type of delivery. Methods: Cross-sectional study with late preterm and term newborns in a second-level maternity. Multiple gestation, chorioamnionitis, maternal or fetal hemorrhage, suspected congenital infection, 5-minute Apgar <6, congenital malformations, and Rh hemolytic disease were excluded. Percentiles 3, 5,10, 25, 50, 75, 90, 95 and 97 of blood indices were calculated for both groups. Results: 2,662 newborns were included in the sample, 51.1% males, 7.3% late preterms, 7.8% small for gestational age (SGA) and 81.2% adequate for gestational age (AGA). Mean gestational age was 35.6±1.9 and 39.3±1.0 weeks, respectively, for premature and term neonates. The erythrocytes indices and white blood cells increased from 34-36.9 to 37-41.9 weeks. Basophils and platelets remained constant during gestation. Premature neonates presented lower values of all blood cells, except for lymphocytes and eosinophils. SGA neonates presented higher values of hemoglobin, hematocrit and lower values of leukocytes, neutrophils, bands, segmented, eosinophils, monocytes and platelets. Male neonates presented similar values of erythrocytes and hemoglobin and lower leukocytes, neutrophils, segmented and platelets. Neonates delivered by C-section had lower values of red blood cells and platelets. Chronic or gestational hypertension induced lower number of platelets. Conclusions: Blood cells increased during gestation, except for platelets and basophils. SGA neonates had higher hemoglobin and hematocrit values and lower leukocytes. Number of platelets was smaller in male SGAs, born by C-section and whose mothers had hypertension.


INTRODUCTION
Currently, there are few publications on complete blood cell count from umbilical cord blood. 1,2 Ozurek et al. 1 have reported higher values of normoblasts, hemoglobin, hematocrit and erythrocytes in cord blood count of newborns small for gestational age (SGA) compared to infants adequate for gestational age (AGA). Among SGAs, 21.9% were reported to have neutropenia and 4.7% presented with neutrophils below 1,500 u/ μL on the first day.
Hemoglobin and hematocrit values from cord blood are useful for indications of red blood cell transfusions or, when associated with bilirubin values in the cord, for indication of intravenous immunoglobulin in Rh hemolytic disease. 3 Platelet count helps to monitor immune thrombocytopenia and preeclampsia. 4,5 Cord leukogram's reliability to identify risk of infections at birth is questionable. 6,7 Nevertheless, some authors suggest that leukogram analysis, along with C-reactive protein analysis, may help diagnose bacterial infections of maternal origin. 8,9 Thus, an analysis of umbilical cord blood cell count has the advantage of reducing blood spoliation, keeping the newborn (NB) from being exposed to vascular puncture and risk of infections, besides allowing clinical decision-making upon birth in some situations. 3,4,10 In this context, the objective of this study was to describe the hematological profile of late preterm and term newborns by analyzing their cord blood and to compare blood parameters according to gender, adequacy of weight and gestational age, and type of delivery at a maternity in the Metropolitan Region of São Paulo.

METHOD
This is a cross-sectional study conducted with live births of Hospital São Luiz Gonzaga from July 2007 to February 2009.
It is part of a research project entitled "Evaluation of frequency of neonatal alloimmune neutropenia in Brazilian newborns", with free and informed consent signed by the legal representatives of NBs included in the original project (project no. 0051/07). The current project was approved by the Research Ethics Committee of Universidade Federal de São Paulo (Unifesp) (project no. 1624/08), with the informed consent form of the original project being considered.
Hospital São Luís Gonzaga is a maternity affiliated to Santa Casa de São Paulo, where approximately 2,000 births happen every year. The maternity unit has 30 rooming-in beds, and the neonatal unit has eight intermediate care beds plus six intensive care beds.
Consecutive live births that occurred in the study period were included, and a sample of umbilical cord blood of each newborn was collected to perform complete blood count.
NBs with gestational age <34 weeks or gestational age >42 weeks, multiple gestations, mother with positive serology for human immunodeficiency virus (HIV), syphilis (venereal disease research laboratory -VDRL, and positive treponemal test), positive serology (immunoglobulin G -IgG -and immunoglobulin M -IgM) for toxoplasmosis and cytomegalovirus, signs of chorioamnionitis (premature amniorrexis plus at least two clinical or hematological signs of maternal-fetal infection), previous placental abruption, 5-minute Apgar <6, major congenital malformations, Rh hemolytic disease (direct and indirect positive Coombs), maternal or neonatal hemorrhage, clinical signs of congenital infection or early neonatal sepsis suspicion were excluded.
After birth, 5 mL of umbilical vein blood was collected in a tube containing ethylenediaminetetraacetic acid anticoagulant (EDTA) for complete blood count and analysis of hematological parameters of neonates. Blood was collected by the nursing team, along with the collection for blood typing of newborns, up to two minutes after placenta discharge. Late umbilical cord clamping routine was not being used at the service at the time of the study.
The complete blood count of all neonates was performed routinely at the hospital's birth laboratory. The device CellDyn 3700-Abbott ® was used by automated method with electronic count of red cells, red cell volume measurement, hemoglobinometry, complementary microscopy of blood extensions stained with Romanowsky dye. The parameters measured directly by the apparatus were: number of erythrocytes (u/mL), hemoglobin concentration (g/dL), hematocrit (%), mean corpuscular volume (MCV: fL), and Red Cell Distribution Width hematocrit values and lower leukocytes. Number of platelets was smaller in male SGAs, born by C-section and whose mothers had hypertension. (RDW: %). Parameters calculated were: mean corpuscular hemoglobin (MCH: pg) and mean corpuscular hemoglobin concentration (MCHC: mg/dL). In addition, the number of leukocyte (u/mL) and platelet series (u/μL) were evaluated.
In addition, blood parameters were compared between SGA and AGA newborns, male and female, C-section and vaginal delivery, and children of mothers with and without diabetes or arterial hypertension.
Demographic and clinical data of mothers were collected from medical reports described by the obstetrics team, and also based on records of infants with up to 48 hours of life. Birth weight was measured using a Filizola ® digital scale, with maximum capacity of 15 kg, minimum of 250 g, and precision level of 5 g.
The gestational age was attributed by the hospital's neonatology team, which routinely considered the best obstetric estimate based on the date of last menstrual period or ultrasound examination before 14 weeks. 11 In the absence of such data or in the presence of difference of two weeks or more between obstetric and pediatric evaluation, the evaluation by the pediatrician was considered. 12 Adequacy of weight for gestational age was assessed by the Intergrowth curve, 11 considering AGA the NBs with birth weight between the 10 th and 90 th percentiles; and SGA the NBs with birth weight below the 10 th percentile; NBs large for gestational age (LAG) were those weighing above the 90 th percentile.
The numerical variables were evaluated by the Kolmogorov-Smirnov test for the Gauss distribution and compared by the Student's t-test or the Mann-Whitney's test for variables with normal distribution (expressed as mean and standard deviation) or asymmetric distribution (expressed in median and minimum-maximum values), respectively. Categorical variables were described as number and percentage and compared by the chi-square or Fisher's exact test.
Statistical analyses were performed with the aid of the Statistical Package for Social Sciences (SPSS) version 17 (SPSS Inc., Chicago, IL, USA). Statistical significance level was set at p<0.05.
The  Tables 1 and 2. Comparison of blood parameters between late preterm and term NBs showed that mean erythrocytes (u/mL) ( The distribution of blood values was shown to have greater amplitude for all parameters of the hematological profile in late preterm compared to term infants, as evidenced by the widest 95% confidence interval (95%CI) (Figures 2 and 3).
Comparisons of blood indices between SGA and AGA newborns are shown in Table 3

DISCUSSION
We could see a progressive increase in blood cell count during pregnancy from 34 weeks of gestation, with the exception of  The hematological profile found in our study was similar to what other authors have reported, with slight variations in the number of cells. [13][14][15][16][17] In a retrospective study with blood collected in the first six hours of life in NBs born with 22-40 weeks of gestation, the hematocrit increased by 0.64% and hemoglobin by 0.21 g/dL for each week of gestation. 16 Another study showed that in NBs of 22 to 42 weeks of gestational age, the percentile 5 for hemoglobin level ranged from 12 to 14 g/dL, and the 95th percentile, from 18 to 22 g/dL. 15 Lee et al. 17 described no difference in the number of leukocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils between different gestational ages in samples collected from umbilical cord of healthy newborns. Christensen et al. 18 reported that the mean values of eosinophils and monocytes on the day of birth increased linearly between 22 and 42 weeks of gestational age.
In our study, MVC, MCH and RDW showed little variation in pregnancy, similarly to other studies. 14,15 MVC estimates the mean red cell size and has a direct correlation with MCH. High levels of MCHC may indicate that erythrocytes are spherical, assuming that MCHC increases in the presence of spherocytosis, but it is not known whether this index helps to identify infants with hemolysis from ABO incompatibility. 19 From the clinical point of view, although the blood parameters were reported to have small differences when each parameter related to red, white and platelet series were analyzed, 95%CI was much higher among late preterm NBs than among term infants, which shows the difficulty of interpreting blood count results in preterm infants.
Compared to AGA, SGA NBs had higher hemoglobin and hematocrit levels, lower white cells -except lymphocytes and Figure 3 Distribution of white blood cell series and platelet (mean and 95% confidence interval) in term and late preterm newborns. 15  basophils -and lower number of platelets. Studies suggest that increased erythropoiesis and reduced white cells are associated with prolonged exposure to hypoxia. 20,21 It is suggested that frequent placental infarctions with platelet consumption may lead to thrombocytopenia and polycythemia, and that preeclampsia and chronic hypertension may play a role in the genesis of thrombocytopenia. 5 Male NBs had similar erythrocyte parameters and lower leukocyte and platelet values, compared to the females. Data in the literature are controversial, but studies conducted with large samples did not report differences between the genders. 15,22 Schmutz et al. 23 reported an average of 2,000 more neutrophils in female NBs.
NBs delivered by vaginal birth presented higher red cell and platelet numbers compared to those born by C-section, possibly due to the higher body water content in cesarean births. 24 It is possible that the transfer of water from the intravascular to the extravascular system is triggered by labor and that the start of this process is intrauterine. 24 A meta-analysis showed mean difference in hematocrit levels of -2.91% (95%CI -4.16--1.6) and of -0.51 (95%CI -0,79--0,27) in hemoglobin levels between cesarean and vaginal delivery. 25 We found no differences in the white cell series as related to the type of delivery, but Schmutz et al. referred that neonates born after labor had a greater number of neutrophils in the first 72 hours than those born by elective cesarean section. 23 The reason is unknown, with some authors attributing a beneficial immunological effect for the stress of labor, 26 while a recent review has shown controversial results as for the influence of type of delivery on oxidative processes in newborns. 27 The increase in platelets would be explained by the increase of thrombopoietin and cortisol levels in vaginal delivery. 28,29 Late preterm NBs presented greater variability of blood parameters, in contrast to term NBs. This may represent a limitation of this research when it comes to sample size.
Nevertheless, this study has internal validity, especially for term newborns, and shows the possibility to collect blood from umbilical cord for blood cell count. Henry et al. reported that one of the factors that contributed to reduce the number of transfusions was having initial laboratory tests performed in cord blood samples. 10 Another multicenter study showed that collecting first test samples from umbilical cord was a feasible technique and that NBs tested for cord blood had an increase in hemoglobin in up to 12-24 hours and received fewer vasoactive drugs and transfusions compared to those who had peripheral blood tested. 30