**3. Platelets**

The first morphologically visible platelets appear in the fetal circulation at seven to nine weeks, and the platelet counts reach adult levels before 18th gestational week [2, 22]. The intrauterine thrombocytopenia could diagnose through fetal blood sampling after 18th ges‐ tational week [2]. The platelet counts are constant at birth and in neonatal period and com‐ patible to the count in adult. Neonatal thrombocytopenia has been defined traditionally as a platelet count less than 150 × 10<sup>9</sup> /L. This definition was challenged by recent studies. Large‐scale study presented that platelet counts of preterm neonates born before 35 weeks gestation were significantly lower than those that were of late‐preterm and preterm infants [23]. Wasiluk [24] reported the platelet count is found to be decreased in preterm and late‐ preterm newborns. The platelet counts were increasing with completed weeks of gestation and birth weight. Decreased platelet count in preterm could be considered as immaturity of thrombopoiesis and impaired process of megakaryopoiesis characterized by the rapid proliferation of megakaryocyte precursors and full cytoplasmic maturation of megakaryo‐ cytes leading to the production of high number of platelets. Levels of thrombopoietin and reticulated platelets (immature platelet fraction, IPF) could reveal the megakaryopoiesis of fetus and neonates.

Except iatrogenic anemia, thrombocytopenia is the most common hematological abnormal‐ ity in neonates [11]. Incidence of thrombocytopenia is 1–5% in newborns at birth [25–27]. Thrombocytopenia may be caused by feto‐maternal and neonatal conditions such as impaired platelet production, consumption and sequestration, and combined mechanisms [28–31]. Platelet transfusion is associated with several risks including infection, transfusion‐related acute lung injury, transfusion‐associated circulatory overload, alloimmunization, allergic reaction, and other complications. Therefore, platelet should be given when clearly clini‐ cally indicated [31, 32]. Reference values for normal platelet counts, especially lower limit, are important to diagnose thrombocytopenia. In particular, there is a need for supplementary parameters in order to evaluate the megakaryopoiesis and bleeding risk [31].

IPF is newly released from fetal liver or the bone marrow and containing high amount of ribonucleic acid (RNA). Thiazole orange, a fluorescent dye, is characterized by binding to nucleic acid, particularly RNA, and flow cytometric analysis of platelets after staining with


† Reference interval.

distribution of data. Furthermore, partitioning into subclasses for separate reference intervals should be considered if appropriate according to gestational age, gender of neonates, and

For difficulties of sample obtaining, it is not easy to establish reference intervals of parameters for neonates according to the CLSI guideline. Even published reference intervals using neo‐ nates' peripheral blood or UCB are very useful and informative for clinical laboratory tests, physicians should keep in mind that some of published reference intervals did not satisfy the

The first morphologically visible platelets appear in the fetal circulation at seven to nine weeks, and the platelet counts reach adult levels before 18th gestational week [2, 22]. The intrauterine thrombocytopenia could diagnose through fetal blood sampling after 18th ges‐ tational week [2]. The platelet counts are constant at birth and in neonatal period and com‐ patible to the count in adult. Neonatal thrombocytopenia has been defined traditionally

Large‐scale study presented that platelet counts of preterm neonates born before 35 weeks gestation were significantly lower than those that were of late‐preterm and preterm infants [23]. Wasiluk [24] reported the platelet count is found to be decreased in preterm and late‐ preterm newborns. The platelet counts were increasing with completed weeks of gestation and birth weight. Decreased platelet count in preterm could be considered as immaturity of thrombopoiesis and impaired process of megakaryopoiesis characterized by the rapid proliferation of megakaryocyte precursors and full cytoplasmic maturation of megakaryo‐ cytes leading to the production of high number of platelets. Levels of thrombopoietin and reticulated platelets (immature platelet fraction, IPF) could reveal the megakaryopoiesis of

Except iatrogenic anemia, thrombocytopenia is the most common hematological abnormal‐ ity in neonates [11]. Incidence of thrombocytopenia is 1–5% in newborns at birth [25–27]. Thrombocytopenia may be caused by feto‐maternal and neonatal conditions such as impaired platelet production, consumption and sequestration, and combined mechanisms [28–31]. Platelet transfusion is associated with several risks including infection, transfusion‐related acute lung injury, transfusion‐associated circulatory overload, alloimmunization, allergic reaction, and other complications. Therefore, platelet should be given when clearly clini‐ cally indicated [31, 32]. Reference values for normal platelet counts, especially lower limit, are important to diagnose thrombocytopenia. In particular, there is a need for supplementary

IPF is newly released from fetal liver or the bone marrow and containing high amount of ribonucleic acid (RNA). Thiazole orange, a fluorescent dye, is characterized by binding to nucleic acid, particularly RNA, and flow cytometric analysis of platelets after staining with

parameters in order to evaluate the megakaryopoiesis and bleeding risk [31].

/L. This definition was challenged by recent studies.

maternal age.

**3. Platelets**

fetus and neonates.

CLSI guideline for sample collection.

32 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

as a platelet count less than 150 × 10<sup>9</sup>

Note: Data were expressed as mean ± standard deviation, range, or median (range).

**Table 1.** Comparison of studies measuring reticulated platelets and immature platelet fraction in healthy newborns.

thiazole orange reflects the activity of megakaryopoiesis in the bone marrow [33]. Measuring IPF of the systemic circulation is a novel parameter to estimate the megakaryopoiesis and can be useful to recognize quickly as having platelet destruction or bone marrow failure in a neonate with low platelet count [34]. Measuring IPF may potentially avoid the need for bone marrow examination. Increased IPF% or normal IPF number (IPF#) is considered in the case of platelet consumption in thrombocytopenia, whereas normal or decreased IPF% and IPF# is considered in the case of bone marrow failure in thrombocytopenia. Today, IPF can be measured on fully automated routine hematology analyzers (XE‐2100 and XN modular system; Sysmex, Kobe, Japan) [35]. Establishment of reference intervals for platelet and IPF in neonates is essential for diagnosis of neonatal thrombocytopenia, for facilitating the clini‐ cal usefulness of IPF, and for clear indication of transfusion. **Table 1** shows the comparison of studies measuring IPF in healthy subjects. The new automated hematology analyzer, XN modular system, demonstrated remarkable higher and broader reference intervals for plate‐ lets and IPF compared with XE‐2100 [36]. For these differences, clinical laboratories should establish or verify reference intervals for platelets and IPF according to their own instrument.

#### **4. Lymphocytes**

Lymphocytes in UCB are naïve and immature, are enriched in double‐negative CD3+ cells, and produce fewer cytokines [19]. Lymphocyte counts, T cell and B cell, can reflect status of immune system in fetus and neonates. B‐ and/or T‐cell lymphocytopenia could be noted in some viral infection but also in Wiskott‐Aldrich syndrome, X‐linked agammaglobulinemia,

and severe combined immunodeficiency [37–40]. To define abnormality of lymphocyte counts, quantitation of the lymphocytes and their subtypes with flow cytometry and estab‐ lishment of reference interval are necessary.

Circulating T cells in the fetus and neonate are fundamentally different from naïve adult T cells such as containing high concentration of T‐cell receptor excision circles (TRECs), high cell turnover, increased susceptibility to apoptosis, and presence of CD25+ regulatory T lymphocytes (Tregs), and so on [41]. Natural Tregs originate in the thymus and are specific for self‐antigens presented by thymic epithelial cells [42]. Maternal cells commonly cross the placenta and engraft into fetal circulation and tissues in uterus, resulting in maternal microchimerism [17, 43]. Naturally acquired microchimerism can contribute to autoimmune diseases. In particular, maternal microchimerism has been studied in systemic sclerosis, der‐ matomyositis, and neonatal lupus [43]. Especially, Tregs in UCB may contribute to maintain the immune homeostasis in the feto‐maternal relationship, and the presence of Tregs would be essential to prevent immune dysregulation in fetus and neonates [17, 44]. Fetal Tregs are known to regulate fetal immune responses against noninherited maternal alloantigens. During labor, neonatal immune system faces big challenge. The tolerogenic immune state of the semi‐allogeneic fetus should switch over to prevent potentially damaging inflammation or infection. In immunosuppressive state, several cells such as helper T cells with a specific cytokine profile, neutrophilic myeloid‐derived suppressor cells, erythroid CD71<sup>+</sup> cells, and Tregs are potential mediators [45, 46].

Infection of newborn and infants is a major healthcare challenge with global mortality in excess of one million lives especially in very low‐birth‐weight preterm infants [47]. Preterm infants are highly susceptible to invasive infections, which are leading causes of mortality and long‐ term morbidity. Treg levels and gestational age inversely correlated in several studies [48–50]. Preterm infants have higher Treg levels than full‐term newborns. Tregs inhibit antimicrobial immune responses. The T cell immune response in preterm infants is supposed to be dys‐ regulated and affected by prenatal factors including intrauterine inflammation and maternal characteristics. This dysregulation of T cell immunity could lead to ineffective clearance of pathogens [49]. Tregs have two populations (CD31+ and CD31‐ ), and the ratio alteration of these populations is associated with different intra‐ and/or extra‐uterine milieu. The CD31‐ Treg lev‐ els are significantly higher in UCB of preterm pregnancies associated with inflammation and prenatal lipopolysaccharide exposure. The alteration of homeostatic composition of Tregs sub‐ sets related to reduced *de novo* generation of recent thymic emigrants. Tregs may contribute to premature delivery, and *vice versa*. Early‐onset septic infants have significantly higher Treg frequencies than infants without early‐onset sepsis. The increased Treg level may cause an uncontrolled immunosuppression and therefore results in an increased risk of sepsis for the preterm infants especially for the most vulnerable very low‐birth‐weight infants (**Figure 1**) [46].

In spite of the growing attention on the importance of Tregs in UCB and neonates, the dis‐ tribution of Tregs in normal UCB or healthy neonates was not well‐known. **Table 2** showed the comparison of studies measuring lymphocyte subsets and Tregs in healthy subjects. Each study showed different values for lymphocyte subsets and Tregs. For these differences, clinical laboratories should establish or verify reference intervals for lymphocyte subsets and Tregs.

Reference Intervals of Platelets, Lymphocytes and Cardiac Biomarkers in Umbilical Cord Blood http://dx.doi.org/10.5772/66458 35

**Figure 1.** The frequency of regulatory T cells (Tregs) is higher in preterm infants than in term infants. Box‐plots [median, interquartile range (IQR), 95% confidence interval (CI)] describe the frequencies of Tregs across groups of different gestational age. Adapted from [46] with permission of John Wiley and Sons, Inc.


† Reference interval.

‡ Median values with 10th and 90th percentiles.

§ CD4+/CD25+.

and severe combined immunodeficiency [37–40]. To define abnormality of lymphocyte counts, quantitation of the lymphocytes and their subtypes with flow cytometry and estab‐

Circulating T cells in the fetus and neonate are fundamentally different from naïve adult T cells such as containing high concentration of T‐cell receptor excision circles (TRECs), high cell turnover, increased susceptibility to apoptosis, and presence of CD25+ regulatory T lymphocytes (Tregs), and so on [41]. Natural Tregs originate in the thymus and are specific for self‐antigens presented by thymic epithelial cells [42]. Maternal cells commonly cross the placenta and engraft into fetal circulation and tissues in uterus, resulting in maternal microchimerism [17, 43]. Naturally acquired microchimerism can contribute to autoimmune diseases. In particular, maternal microchimerism has been studied in systemic sclerosis, der‐ matomyositis, and neonatal lupus [43]. Especially, Tregs in UCB may contribute to maintain the immune homeostasis in the feto‐maternal relationship, and the presence of Tregs would be essential to prevent immune dysregulation in fetus and neonates [17, 44]. Fetal Tregs are known to regulate fetal immune responses against noninherited maternal alloantigens. During labor, neonatal immune system faces big challenge. The tolerogenic immune state of the semi‐allogeneic fetus should switch over to prevent potentially damaging inflammation or infection. In immunosuppressive state, several cells such as helper T cells with a specific

cytokine profile, neutrophilic myeloid‐derived suppressor cells, erythroid CD71<sup>+</sup>

populations is associated with different intra‐ and/or extra‐uterine milieu. The CD31‐

els are significantly higher in UCB of preterm pregnancies associated with inflammation and prenatal lipopolysaccharide exposure. The alteration of homeostatic composition of Tregs sub‐ sets related to reduced *de novo* generation of recent thymic emigrants. Tregs may contribute to premature delivery, and *vice versa*. Early‐onset septic infants have significantly higher Treg frequencies than infants without early‐onset sepsis. The increased Treg level may cause an uncontrolled immunosuppression and therefore results in an increased risk of sepsis for the preterm infants especially for the most vulnerable very low‐birth‐weight infants (**Figure 1**) [46]. In spite of the growing attention on the importance of Tregs in UCB and neonates, the dis‐ tribution of Tregs in normal UCB or healthy neonates was not well‐known. **Table 2** showed the comparison of studies measuring lymphocyte subsets and Tregs in healthy subjects. Each study showed different values for lymphocyte subsets and Tregs. For these differences, clinical laboratories should establish or verify reference intervals for lymphocyte subsets and Tregs.

Infection of newborn and infants is a major healthcare challenge with global mortality in excess of one million lives especially in very low‐birth‐weight preterm infants [47]. Preterm infants are highly susceptible to invasive infections, which are leading causes of mortality and long‐ term morbidity. Treg levels and gestational age inversely correlated in several studies [48–50]. Preterm infants have higher Treg levels than full‐term newborns. Tregs inhibit antimicrobial immune responses. The T cell immune response in preterm infants is supposed to be dys‐ regulated and affected by prenatal factors including intrauterine inflammation and maternal characteristics. This dysregulation of T cell immunity could lead to ineffective clearance of

and CD31‐

cells, and

Treg lev‐

), and the ratio alteration of these

lishment of reference interval are necessary.

34 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

Tregs are potential mediators [45, 46].

pathogens [49]. Tregs have two populations (CD31+

¶ CD4+/CD25+/CD127‐.

Note: Data were expressed as mean ± standard deviation, range, or median (range).

**Table 2.** Comparision of studies measuring lymphocyte subsets and Tregs in healthy subjects.
