**2. Establishing reference intervals in umbilical cord blood**

blood volume per draw; thus, in neonates and infants, a less‐than‐optimal amount of blood may be available for testing. Approximately 1.5 – 4 mL of blood drawn for admission blood test may cause iatrogenic anemia to neonates especially in extremely low birth weight infants [1, 2]. Umbilical cord blood (UCB) is remained blood in the placenta and attached umbilical cord after the birth of baby. Several studies suggest that UCB could be an alternative source for admission of blood tests in neonates [1, 3, 4]. Especially, very low birth weight infants who typically have greater phlebotomy blood loss on the first day of life than any other day during their hospitalization would benefit most from admission laboratory studies being obtained

30 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

At birth, full‐term newborns show relative polycythemia, macrocytosis, and marked poly‐ chromasia with nucleated red blood cells (RBCs) [7, 8]. The red cell distribution width (RDW) is elevated, showing anisocytosis, compared with adult standards. Full‐term new‐ borns have a high white blood cell (WBC) count with relative transient neutrophilia at birth while soon after birth, neutrophils gradually decrease and lymphocytes become major population in neonate's peripheral blood. This neutrophilia may arise from bone marrow mobilization under stress during labor, and these WBCs show shift‐to‐left neu‐ trophils such as metamyelocytes, myelocytes, and even circulating blasts [9, 10]. The platelet counts are similar to the older children and adults. Neonatal thrombocytopenia is

Thrombocytopenia is one of the most common hematological abnormalities except iatro‐

During the pregnancy, human placenta forms an imperfect barrier, allowing bidirectional passage of soluble antigens and cells between a mother and a baby without any mixing between the two blood supplies [13]. This results in the presence of fetal cells in the maternal circulation, known as fetal microchimerism, and maternal cells in the fetal circulation, known as maternal microchimerism [14]. Maternal microchimerism was first described in 1963 by Rajendra G. Desai who identified maternal leukocytes and platelets in UCB [15]. This bidirec‐ tional trafficking of cells begins at seven to 16 weeks, increases steadily after 24 weeks, and reaches a peak at parturition [16]. At delivery, maternal microchimerism has been reported in 42% of normal pregnancies [13]. For this, microchimerism does not occur in all pregnan‐ cies, altered maternal‐fetal bidirectional passage has been associated with disruption of the feto‐maternal interface, and the biologic role of this bidirectional passage is unclear. This pas‐ sage is implicated in development of the fetal immune system [17]. Substantial numbers of maternal cells cross the placenta to reside in fetal lymph nodes, inducing the immune system, the development of CD4+CD25highFOXP3+ regulatory T cells (Tregs), which suppresses fetal

UCB is a rich source of hematopoietic cells or precursors to blood cells. Since the first UCB stem cell transplantation in 1988 to treat a child with Fanconi's anemia, UCB has been used as an important source of hematopoietic stem cell transplantation [18]. UCB could be collected at birth without any harm to the newborn infant. UCB cells have many theoretical advantages as grafts for stem cell transplantation because of the immaturity of newborn cells and immatu‐ rity of the immune system at birth. These properties should decrease the alloreactive potential

/L in any neonate of a viable gestational age.

from UCB [5, 6].

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

genic anemia in neonates [11, 12].

antimaternal immunity.

The production of health‐associated reference values must be implemented in accordance with a well‐defined protocol. The Clinical Laboratory Standards Institute (CLSI) offers a protocol for determining reference intervals that meet the minimum requirements for reliability and usefulness related to quantitative clinical laboratory tests [21]. The CLSI suggested a protocol outline for obtaining reference values and establishing reference intervals. First, researchers should establish a list of analytical interferences and sources of biological variability from medical and scientific literatures. Then, they must establish selection and partition criteria and an appropriate questionnaire designed to reveal these criteria in the potential reference individuals. An appropriate written consent should be signed by legal guardians of neonates. Researchers have to categorize the potential refer‐ ence individuals based on the results of questionnaire and health assessments and exclude individuals based on the exclusion criteria. For UCB, gestational age, birth weight, mater‐ nal age, maternal health, and maternal history of medical, smoking, and alcohol consump‐ tion are important.

The reference interval is defined as the internal between and including two numbers, an upper and lower reference limit, which are estimated to enclose a specified percentage (usually 95%). For most analytes, the lower and upper reference limits are estimated as the 2.5th and 97.5th percentiles of the distribution of test results for reference populations. To decide on an appro‐ priate number of reference individuals, in consideration of desire confidence limits, the CLSI suggests that a minimum of 120 reference values for 90% confidence limits, 146 observations for 95% confidence limits, and 210 reference values for 99% confidence limits. It is necessary to define whether the sample should be arterial or venous UCB in a manner consistent with the routine practice for patient specimens. Inspection of the reference value data, preparing a histogram, and identifying possible data errors and/or outliers are essential to evaluate the distribution of data. Furthermore, partitioning into subclasses for separate reference intervals should be considered if appropriate according to gestational age, gender of neonates, and maternal age.

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 CLSI guideline for sample collection.
