The development of the human immune system begins early in the fetal period, but it is not completed at birth; post-natal maturation proceeds for months to years, even into adulthood. Neonates have little immunological memory and a developing immune system, which increases their vulnerability to infectious agents.1

Umbilical cord blood is the blood left over in the placenta and the umbilical cord after the birth of the baby. The cord blood is composed of all the elements found in whole blood; it contains red blood cells, white blood cells, plasma, platelets and is also rich in hematopoietic stem cells.2

The blood vessels are the part of the circulatory system, and microcirculation, that transports blood throughout the human body. Capillary blood sampling via a heel lance is the most common procedure performed in hospitalized neonates, capillary blood obtained by skin puncture is from a dynamic tissue fluidic system that contains circulating capillary blood, interstitial fluid and lymphatic fluid.3

Blood is a complex tissue consisting of a very specialized network of circulating immune cells. The compartments of first-line innate immune response and adaptative response are essential to protect from a wide variety of pathogens. Dysregulated immune response can lead to an increased susceptibility to infection, primary immunodeficiency, autoimmune diseases or cancer.4 Both genetic and non-genetic factors may contribute to variations in the number of human immune cells. It is also possible that different linages have different fluctuations in accordance with varied environmental and nutritional states.5 It is therefore important to have reference values for varied ethnic-geographic populations.

Immunophenotyping of blood lymphocyte subpopulations is an essential tool in the diagnosis and follow-up of children with immunodeficiencies, hematological diseases, and other disorders. Studies of leukocytes from preterm newborns have been limited, and there is little information available concerning the development and function of the innate immune system depending on the gestational age of newborns. Preterm infants, due to the immaturity of the immune system, are at a heightened risk of acquisition of recurrent bacterial infection during their first weeks of life, due to frequent exposure to the micro-organism, frequently, invasive procedures such as catheterization and intravascular or assisted ventilation need to be done in order to treat these infections.6,7

In this work, we investigated the counts of leukocytes subsets in two types of blood samples (cord blood and capillary blood) extracted from healthy newborns to establish reference ranges that may be helpful to the pediatricians to determine possible alterations in these cell subsets. To this, we have performed flow cytometric phenotyping to determine values reference and studied whether there are differences between genders.

Our data further show that the extent of dispersal between individuals varies widely for different blood leukocyte subsets.

Statistical analyses were performed using GraphPad Prism, version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Two-group comparisons were performed using the Mann–Whitney test. Stratified data were expressed as the mean value. The results were expressed as the median, and p-values <0.05 were considered to be significant. The normal reference values of cord and capillary blood were defined as 5–95 percentiles, and the median rank was shown at the same time.

A newborn represents the culmination of developmental events from conception, implantation, and organogenesis. Dramatic changes occur in the blood and bone marrow of the newborn infant during the first hours and days after birth, and there are rapid fluctuations in the quantities of all hematologic elements. Neonates process a developing immune system, which is different from adults, additionally, neonates are highly susceptible to infections since they change from a semi-allogeneic sterile to being exposed to a microbial environment.1,8

The umbilical cord provides the fetus with oxygen, glucose, amino acids, water, vitamins, and hormones. The flow of fetal blood from the umbilical cord ultimately derives in the chorionic hair space. The space between one hairy column and the other contains maternal blood, separated from the fetal blood within the villus by a membrane created by the trophoblast, composed of specialized epithelium called syncytiotrophoblast and – more internally – cytotrophoblast. This epithelial barrier allows the passage of nutrients into the fetus and the passage of wastes such as CO2, urea and creatinine into the maternal circulation but the passage of blood cells is not usual.9 The umbilical cord blood cells differ from peripheral blood in composition, number, and properties, in the cord blood there is the hematopoietic precursor of stroma.10,11

Fluctuations in the numbers of leukocytes are frequent at all ages but are greatest in infancy. Leukocytosis is typical at birth for full-term and preterm infants, with a wide range between the limits of normal values. However, at birth, neonates have an increased susceptibility to infectious agents due to the functional immaturity of their immune system. Immunophenotyping of blood cell populations to identify these cells is an essential tool in the diagnosis and follow-up of children with immunodeficiency and other immune disorders. A correct interpretation of the results obtained from patients requires knowledge of the normal development in the first hours of life.12 Cord blood is an ideal source for laboratory examination in just-born neonates; it reveals not only normal or abnormal development of human fetal hematopoiesis but also the clinical conditions of these babies.13 On the other hand, capillary blood is easily collected by a minimally invasive procedure and has excellent potential use in point-of-care (POC) health monitoring.3 Point-of-care testing enables rapid diagnosis or monitoring of chronic conditions that can facilitate clinical decisions. Such technologies have the potential to significantly change the healthcare and diagnostic pathways. Patients can obtain immediate results in the hospital, physician’s office, or even at home. This approach saves patients’ a tremendous amount of time, resulting in a faster application of efficient treatment or referral for further investigation.7 It is important to implement the analysis of leukocyte populations to diagnose primary and secondary immunodeficiencies or infections in the newborn in time. In these conditions, early detection and rapid initiation of treatment are key to reducing morbidity and mortality.

We determined reference values for the absolute and relative values of leukocyte subsets in newborns, by analyzing cord and capillary blood samples. Our data will contribute to the development of reference standards for the sampled ethnic-geographic population in this study. These data may also permit the development of clinical strategies to treat infections or diagnose diseases such as immunodeficiencies among others.

Neutropenia is defined as the reduction in the number of circulating neutrophils to less than 1.5×109/L. Neutropenia represents a decrease in neutrophil production or an increase in consumption. Neutropenia can result from infection, impaired bone marrow production or abnormal distribution. Sepsis in neonates is a common cause of morbidity, particularly in premature and low-birth-weight infants.14 Neutropenia is more predictive of neonatal sepsis than in neutrophilia, but it can also be associated with birth asphyxia, intrauterine growth restriction, Rh hemolytic disease or peri-ventricular hemorrhage. Neutrophilia can result from inflammation, certain malignancies.15

Leukopenia refers to decreased counts; it can be seen with viral or bacterial infections, as well as in infants born from women with pregnancy-induced hypertension (PHI). Neonatal patients with lymphopenia may be due to chronic infection with viruses such as cytomegalo virus or Epstein-Barr virus.16 Initiation of prophylactic treatment of opportunistic infections and monitoring responses to antiviral therapy are well established, so immunophenotyping of blood lymphocyte subpopulations is an important tool to evaluate the neonate’s immune system.

Leukocytosis refers to an elevated cell count and it might be observed in patients with infections and leukemias. Lymphocytes constitute about 30% of the leukocytes at birth and may be increased to 60% at four to six months. Immunophenotyping of the blood cell population to identify these cells is an essential tool in the diagnosis and follow-up of children with immunodeficiencies and other immune disorders.17,18

Primary Immunodeficiency (PID) is a heterogeneous group of inherited disorders of the immune system. Immunophenotypic evaluation of PIDs using flow cytometry provides important clues in the identification of diagnosis of these diseases. There are significant phenotypic variations in patients with certain PIDs.7,19

Quantitative assessment of leukocytes populations and subpopulations in neonates is useful for the diagnosis of X-linked agammaglobulinemia (XLA) characterized by the absence of B cells in the peripheral blood (<1%). Patients with severe combined immunodeficiency (SCID) lack T cells while the impact on B and NK cells is variable depending on the genetic defect. Most of the children with SCID die of infections very early in life unless they are treated with hematopoietic stem cell transplantation (HSCT); studies have clearly shown that affected infants diagnosed with SCID as neonates had better survival than patients with delayed diagnosis and this also results in a significant reduction in the cost of therapy.20,21

Patients with Combined Immunodeficiency (CID) have a defect in T lymphocytes in protein that does not affect their development, but affects their function. The patients with ZAP-70 deficiency and MHC I deficiency help guide the defects, when CD8+T lymphocytes are at least 7%. The patients with MHCII deficiency have CD4+T lymphocytes very low.22

NK cells play a major role in the resolution of severe acute respiratory viral infections caused by influenza or respiratory syncytial virus. Although the information on NK cells in bacterial infections in children is limited, it has been shown that young children with recurrent otitis media and sinusitis have NK cell genetic defects.23 The patients with profound monocytopenia, B lymphocytes and NK lymphocytes low, could guide a diagnosis to a Gata 2 deficiency.24 Therefore, establishing baseline values of leukocyte populations in neonates helps guide early diagnosis for some PIDs.

In the present work, we consistently observed differences in the absolute number of different leukocyte populations among cord and capillary blood neonates. We observed higher frequencies and absolute counts of total lymphocytes in capillary blood compared with cord blood. In absolute numbers, it was the capillary blood which had significant differences in all the leukocyte populations. In neonates, the cellular immune system matures rapidly in the first three months of life. The development of neonatal immunity is influenced by multiple factors including maternal cytokines, antigen exposure and precursor frequency of lymphocytes. Fluctuations in the number of leukocytes are common at all ages but are greatest in neonates.8 Our hypothesis as to why there are more lymphocytes, monocytes, and neutrophils in capillary blood is that the newborn needs to mobilize a higher number of cells to the periphery in order to face possible infectious agents and also start the activation and proliferate process to become a mature leukocyte. These data allow us to identify new interesting differences in certain specific leukocyte subsets between the cord and capillary blood of neonates.

Previous studies provided important information about reference values of cord blood for flow cytometry25–27; while reference values for the major immune cells categories that are necessary for clinical purposes have been generated for both adults and neonates.26 In this work, we compared frequencies and the absolute number of cord and capillary blood leukocytes; also establishing reference values in a Mexican population of neonates. Our data provide interesting differences between the blood and identify specific leukocyte subpopulations. Diversity in the phenotypes of leukocyte proportions and concentrations in blood is thought to have variable contributions from genetic, environmental and epigenetic factors.

The authors have no conflict of interest to declare.