BASIC CONSIDERATIONS

Normal blood volume in children is ~80 ml/kg, of which ~45% is contributed by various cellular elements, i.e. red blood cells (RBCs), while blood cells (WBCs) and platelets, rest being the plasma.

Developmental hematopoiesis: While fetal hemato­poiesis begins as early as in the 3^rd week, normal hematological values in utero and in postnatal life vary according to the physiological needs of baby. Some important aspects of hematopoiesis in general and in different age groups, are as follows:

Site: Fetal hematopoiesis is mainly extramedullary till late gestation, i.e. in extra-embryonic yolk sac from 10-14^th day to 6-8 weeks and in liver and spleen from 6-8 weeks to 20-24 weeks. Bone marrow gradually takes over this function from 20-24^th weeks onwards. Virtually all marrow cavities in body are involved in hematopoiesis at birth, but gradually this function moves to central bones, e.g. sternum, pelvis, vertebrae and ribs. Remaining marrow in the extremities and skull is replaced by fat. However, hematopoietic cells continue to exist in liver, spleen and other medullary sites and get reactivated during the state of increased needs (extramedullary erythropoiesis in hemolytic anemia).

Regulators: Bone marrow is populated by hemangioblasts, i.e. pluripotent stem cells (PSC) and descendent cell-lines, i.e. myeloid, erythroid, and megakaryocytic lineage at various levels of maturity (Fig. 19.1). Predominance of a cell-line produced at any stage, depends on the needs of fetus/baby and guided by various cytokines (growth factors), e.g. erythropoietin (for RBCs), colony stimulating factors (G-CSF, GM-CSF and M-CSF) and interleukins IL1__U (for leukocytes) and thrombopoietin (for platelets).

Erythropoiesis: Important steps in marrow erythropoiesis include differentiation of PSC into erythroid colony forming units (CFU-E) and formation of erythroblasts (earliest

Fig. 19.1: Hematopoietic cell differentiation.

PSC: Pluripotent stem cell; BFU: Burst forming unit; CFU: Colony forming unit; EPO: Erythropoietin; TPO: Thrombopoietin; CSF: Colony stimulating factors; IL: Interleukin

in vivo cell of erythroid series), which mature through various stages into normoblast - the last nucleated cell of erythroid series. Normoblasts further mature with extrusion of nucleus to form reticulocytes (earliest non­nucleated cell of erythroid series), which retains its capacity for hemoglobin synthesis till released into circulation and matures as RBC.

Hematopoiesis is an enormously regulated process, with gt;10 billion erythroid and myeloid cells produced per hour, sustained throughout life by slowly cycling quiescent pool of hematopoietic stem cells. These stem cells have the capacity to self-renew and to commit to one of the 10 hematopoietic lineages by a stochastic process that induces a proliferation and differentiation program driven by expression of sets of transcription factors.

Mature RBCs, although devoid of nucleus, mito­chondria and hemoglobin synthesis capacity, continue to function for a normal life span of ~120 days, before elimination of senescent cells by liver and spleen. However, fetal and neonatal RBCs have a shorter life span of ~30-60 days and ~60-90 days respectively.

Rate of erythropoiesis is primarily controlled by erythropoietin (EPO)-a hormone produced by juxtaglomerular apparatus of kidney in response to hypoxia, which stimulates differentiation of PSC into erythroid precursors. During fetal life, EPO is produced in liver.

Fetal hematopoiesis is primarily erythroid in nature, due to higher oxygen requirements and lower oxygen tensions in transplacental blood (60-65%).

Hemoglobin (Hb) is a tetramer molecule with two pairs of polypeptide globin chains, each having a heme group attached to it. Human Hb contains different combinations of these polypeptide chains during various stages of development that differ from each other in their amino acid structure and designated as - #945;, #946;, #947;, #948;, #958; (zeta), #949; (epsilon) chains (Table 19.1). While #945; gene is located on chromosome 16, #946;, #947;, and #948; genes are closely linked to chromosome 11.

At birth, 70% of Hb is fetal Hb (HbF), which differs from adult Hb in terms of—(i) higher affinity to oxygen, and (ii) resistance to acid as well as alkali (used as a diagnostic test).

A switch-over from fetal to adult Hb synthesis in late intrauterine and early postnatal life leads to gradual decline in HbF levels during infancy and its replacement by adult Hb.

Adult Hb includes two different hemoglobin forms- predominant HbA and HbA2. HbA is detectable by 12-16 weeks and prenatal diagnosis of thalassemia is possible at this age. At birth, 30% of Hb is HbA and rapidly increases to contribute gt;95% of total Hb by late infancy. HbA₂ is lt;1% at birth and ~2.0-3.4% of total Hb after infancy.

Leukopoiesis involves differentiation of PSC into lymphocytes and granulocytes (polymorphs, eosiono- phils, basophils, monocytes), regulated by various cytokines, i.e. GM-CSF, G-CSF, M-CSF and interleukins (Fig. 19.1). Except T-lymphocytes, which leave bone marrow in early stages to mature in thymus, other leukocytes mature in marrow itself before entering the circulation and populating various tissues.

As fetus lives in relatively sterile environment, need for intrauterine leukopoiesis is limited and neutrophils are virtually absent till last trimester, when there is sudden surge in granulocyte formation. Consequently, preterms are more susceptible to infections than term newborns. While there is some drop in peripheral leukocyte and polymorph counts during first 1-3 months, postnatal bone marrow contains 2-5 times more cells of myeloid series than those of erythroid series. This includes a large pool of mature neutrophils, which may be released rapidly in circulation during acute infections.

Thrombopoiesis: Platelets develop from breakage of megakaryocytes-the large cells with abundant DNA, which differentiate from PSC and mature under the influence of Thrombopoietin. Normal platelet count remains unchanged from 18^th week of gestation onwards, between 1.5-4.5 lac platelets/mm³.

Normal Hematological Characteristics

Complete blood counts and peripheral smear examination are two most valuable investigations in hematology and correct interpretation of these reports is essential with due considerations to developmental and other physiological variations.

Complete blood count (CBC) usually include six basic parameters: (i) hemoglobin, (ii) hematocrit (or packed cell volume), (iii) RBC count, (iv) total and differential WBC counts, (v) platelet counts, and (vi) erythrocyte sedimentation rate. Normal values of some of them with age-wise variations are given in Table 19.2.

TABLE 19.2: Normal hematological values

Hct: Hematocrit; TLC: Total leukocyte count; PMN: Polymorpho­nuclear cells.

Peripheral smear (PS) is an extremely valuable screening test in hematology and infections. A freshly prepared and stained (hematoxylin-eosin staining) should be examined for:

• RBC morphology, e.g. shape, size, color and inclusion bodies, e.g. parasites,

• Differential WBC counts and their morphology, and

• Presence of platelet clumps. Absence of platelet clumps on PS is an indicator of thrombocytopenia, though should be confirmed by platelet count. Important PS changes in various hematological

disorders have been discussed in respective chapters.

19.3