HEMATOPOIESIS AND BLOOD CELLS
Hematopoietic stem cells are self-renewing cells that differentiate and become committed to different cell lineage.
Pluripotent stem cells and early progenitor cells can be cultured on culture media. In culture media, progenitor cells are defined as colony-forming units (CFUs). CFUs are earliest detectable progenitor cells that give rise to granulocytes, erythroblasts, monocytes and megakaryocytes, which together are termed CFUGEMM or CFUCMP (common myeloid progenitor). The more mature and specialized precursor cells are termed CFUGM (granulocytes and monocytes), CFUE (erythroblasts), and CFUEO (eosinophil). The burst-forming units (BFUE) of the erythroid lineage are early progenitor cells committed to erythrocyte differentiation and are the early ancestors of the CFUE. The BFUE has a limited capacity of proliferation and gives rise only to erythrocyte colonies. BFUE is insensitive to erythropoietin, and its progeny must go through as many as 12 divisions before they become mature erythrocytes.
The proliferation of stem cells and progenitor cells is under the control of cytokines. IL-3 and GM-CSF are nonlineage specific cytokines, which act on pluripotent and early progenitor cells; they are required for self-renewal and differentiation throughout the hematopoietic process. In contrast, cytokines such as granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-5, thrombopoietin and erythropoietin all act on more mature and specific hematopoietic lineage cells. Erythropoietin, a glycosylated peptide with its gene located on the chromosome 7, is predominantly derived from the kidney and with a small amount from the liver. It stimulates erythropoiesis at the stage of CFUE. Thrombopoietin, a polypeptide with its gene located on chromosome 3, is mainly formed in the liver and small portion in the kidney. It stimulates the formation of megakaryocytes and the release of platelets. It also works with erythropoietin to stimulate erythroid progenitor cells. The elimination half-life of thrombopoietin is approximately 30 hours. It is the longest half-life of the hematopoietic growth factors.
The Sequence of Hematopoietic Cell Maturation
- Erythroid lineage: Pluripotential stem cell → myeloid stem cell → BFUE → CFUE → pronormoblast → basophilic normoblast → polychromatic normoblast → orthochromic normoblast → polychromatic erythrocyte (reticulocyte) → erythrocyte.The last stage of erythroid lineage, which is still capable of division, is the orthochromic cell stage.
- Megakaryocyte lineage: Pluripotential stem cell → myeloid stem cell → CFUGEMM → CFUMEG → megakaryoblast → megakaryocyte → platelets.
- Myeloid lineage: Pluripotential stem cell → myeloid stem cell → CFUGEMM → CFUGM → myeloblast → promyelocyte → myelocyte → metamyelocyte → band → polymorphonuclear neutrophil.Monocyte maturation follows the following sequence: from CFUGM monoblast → promonocyte → monocyte then moves to circulation and tissue.The last stage of myeloid lineage, which is still capable of division, is the myelocyte stage. The earliest detectable, specific myeloid antigen is CD33.
- Eosinophil: Pluripotential stem cell → myeloid stem cell → CFUGEMM → CFUEO → CFUGM → myeloblast → promyelocyte → eosinophilic myelocyte → eosinophilic metamyelocyte → eosinophilic band → eosinophil.
- Basophil: Pluripotential stem cell → myeloid stem cell → CFUGEMM → CFUBASO → myeloblast → promyelocyte → basophilic myelocyte → basophilic metamyelocyte → basophilic band → basophil → mast cell.
- B-cell: Pluripotential stem cell → lymphoid stem cell → pre-B → B-lymphoblast → B-prolymphocyte → B-lymphocyte → plasma cell.Cytoplasmic CD22, CD10, CD19, TdT and HLA-DR are present on the very early B-cells.
- T-cell: Pluripotential stem cell → lymphoid stem cell → pre-T → T-lymphoblast → T-prolymphocyte → T-lymphocyte.CD7 is the earliest antigen present on the T-cell surface, and cCD3 (cytoplasmic CD3) is the earliest T-cell lineage specific antigen in the cytoplasm.
The main function of red blood cell is to carry oxygen to tissues and return carbon dioxide to the lung. Hemoglobin molecules present in the RBC contain four peptide chains. The lifespan of a normal RBC is 120 days.
When the oxygen is released, the hemoglobin β-chains are pulled apart so that 2,3 DPG can move in and lower the affinity of hemoglobin for oxygen, resulting in improved delivery of oxygen to the tissue.
Hemoglobin affinity for oxygen is increased by HbF, increased pH and decreased the level of 2,3 DPG. As the affinity for oxygen increases, the disassociation curve will shift to the left.
Hemoglobin affinity is decreased by HbS, decreased pH, increased the level of 2,3 DPG. As the affinity for oxygen decreases, the disassociation curve will shift to the right.
Neutrophil production and differentiation in the bone marrow take 6–10 days. Large numbers of band and segmented neutrophils are stored in the bone marrow as a reserve pool (10–15 times of the peripheral neutrophils). After being released from the bone marrow, neutrophils typically spend 6–12 hours in the peripheral blood circulation before migrating into tissues. Neutrophils then survive about 2–4 days in the tissue before being destroyed.
The Left and Right Shift of Granulocytes
The degree of nuclear lobulation of polymorphonuclear neutrophils (PMNs) is an indication of the cell's maturity. A predominance of hypolobulated cells is called a left shift. Conversely, a predominance of cells with four nuclear lobes is called a right shift. For practical purposes, a left shift is usually noted when more than 10–12% bands are seen on the CBC differential count, or when the total PMN count (segmented and band forms) is more than 80%.
Potential causes of left shift: Bacterial infection, toxemia, hemorrhage, and myeloproliferative disorders.
Potential causes of right shift: Liver disease, megaloblastic anemia, iron deficiency anemia, glucocorticoid use, and reaction to stress.
Eosinophils have receptors for IgE, histamine, the Fc portion of immuno- globulin, and complement. They are capable of phagocytosis. Their granules are membrane bound organelles with a “crystalloid” core. Eosinophils are particularly important in allergic and parasitic disease. Eosinophils release arylsulfatase and histaminase, which inactive histamine and SRS-A released from mast cells.
Fig. 1.1: Ultrastructure of an eosinophilA. Primary granules contain Charcot-Leyden crystals. B. Rectangular amorphous crystalloid bar in secondary granules contain major basic protein, eosinophil peroxidase, and other secondary/special granules.
A major constituent of the eosinophil is the Charcot-Leyden protein, which is composed of lysophospholipase. Massive infiltration of eosinophils often leads to the disintegration of cells and formation of Charcot-Leyden crystals. Charcot-Leyden crystals are bipyramidal, hexagonal crystals that may be seen in tissue and fluid specimen (Fig. 1.1).
Basophils and Mast Cells
They are both derived from the bone marrow; however, their relationship is not entirely clear.
Mature and immature basophils both contain coarse, densely stained blue-black granules with scattered red-purple granules of varying shapes and sizes.
Mast cells are larger than basophils, contain black, bluish-black, or reddish- purple metachromatic granules. The granules may overlay the cytoplasm and obscure the nuclear borders.
Monocytes share a common stem cell origin with myeloid cells. Mature monocytes have gray-blue cytoplasm and indented nuclei. The cytoplasm may contain vacuoles or fine granules. Monocytes spend only a short time in the bone marrow. After circulating in the blood for 12–24 hours, monocytes migrate into the tissue as antigen presenting cells (APC) or macrophages without dividing.
Mature T-cells comprise 65–80% of the circulating lymphocytes in peripheral blood. There are two major subsets, CD8+ suppressor (as known as cytotoxic) T-cells (predominantly in bone marrow), and CD4+ helper T-cells (predominantly in peripheral blood).
Maturation of T-cells occurs in the thymus. T-cells that have migrated from the bone marrow begin the thymic maturation process in the subcapsular region of the lobules of the thymus. As they mature, T-cells progress inward from the subcapsular region, to the cortical region, and finally to the medullary region where they are released into the peripheral blood. As the T-cells mature, cytoplasmic CD3, TdT, and CD7 are expressed first, followed by CD2, and then CD5. When the maturing T-cells have reached the cortex region, they express CD1a and co-express CD4/CD8. When they have reached full maturity in the medullary region, the T-cells express surface CD3, and either CD4 or CD8. Rearrangement of the T-cell receptor (TCR) occurs in a specific sequence following the order of δ, γ, β, and α. The delta (δ) gene (14q11) is rearranged first, followed by rearrangement of the gamma (γ) gene (7p14). This leads to expression of γ/δ T-cells (a minority of the circulating T-cell population), or in the case of the majority of T-cells, the T-cells progress on to rearrange the beta (β) gene (7q34), followed by deletion of the delta gene, and finally, rearrangement of the alpha (α) gene (14q11) to produce α/β T-cells (95% of the circulating T-cell population).
B-cells comprise 5–15% of the circulating lymphocyte population in the peripheral blood. B-cells express CD10, CD19, CD20, CD22, CD79a, PAX-5, and HLA-DR. Plasma cells are mature B-cells that secrete immunoglobulin.
B-cell maturation occurs in the bone marrow and follows a sequential B-cell gene rearrangement process. The first step in the production of a functional immunoglobulin involves recombination at the heavy chain locus (IgH) on chromosome 14q32 by joining a diversity (D) segment with a joining (J) segment to form a D-J fusion, followed by joining a IgH variable (V) region to the D-J fusion to form VDJ fusion. Following rearrangement of IgH, the next rearrangement is the light chain. The kappa light chain (located on chromosome 2p12) rearrangement occurs first, if unsuccessful, then lambda light chain (located on chromosome 22q11) rearrangement occurs.
Natural Killer Cells (NK Cells)
Natural killer cells are a minor population of cells, which do not carry either T- or B-cell markers. Large granular lymphocytes seen in peripheral blood smears comprise a major proportion of NK cells. Interferon-γ and IL-2 stimulate NK cell proliferation.
Anticoagulant Agents Used for Peripheral Blood Cell Counts
- EDTA is the anticoagulant of choice. EDTA can cause platelet clumping and/or satellitism in automated blood counters which is referred to as EDTA-pseudothrombocytopenia. EDTA is also the preferred anticoagulant agent for the extraction of DNA from plasma.
- Citrate causes dilution of the specimen, which requires mathematical correction.
- Heparin may cause WBC and platelet clumping; and therefore is unsatisfactory as an anticoagulant.
Terminology and Important Calculations
The hematology analyzer is a laboratory instrument that generates a histogram showing size distribution on the X-axis and relative number of particles on the Y-axis. RBC and MCV are directly measured. MCHC and MCH are calculated values.
Spurious analyzer readings may occur. The causes of spuriously increased WBC count include cryoglobulin, Heparin, monoclonal protein, nucleated red cells, platelet clumping, and unlysed red cells. The causes of spuriously decreased WBC count include clotting, smudge cells, and fragmented WBCs.
- Absolute WBC count: Total WBC × % (neutrophil, monocyte or lymphocytes)
- Red blood cell (RBC): The normal reference range for males is 4.6–6.0 × 106/µL, and for females is 4.1–5.4 × 106/µL. The presence of cryoglobulin, a WBC count >50,000/µL, or large platelets may lead to spuriously increased RBC count. Microcytic red blood cells (schistocytes, iron deficiency anemia, thalassemia) clotting, or agglutination may lead to spuriously decreased RBC counts.
- Mean corpuscular volume (MCV): The normal reference range is 80–100 fL. It is a measurement of the red blood cell volume or size. In cases of red blood cell clumping (warm or cold agglutinins), or osmotic abnormalities (hyperglycemia, hypernatremia), the MCV may spuriously elevate.
- Red blood cell distribution width (RDW): The normal reference range is 11.5–14.5%. It measured anisocytosis (difference in cell size). An increase RDW may indicate a mixed cell population.RDW is calculated as following:
- Hemoglobin concentration (Hb): The normal reference range for males is 14–18 g/dL, and for females is 12–16 g/dL. A commonly used method is to convert hemoglobin to cyanhemoglobin, and then measure the absorbance at a wavelength of 540 nm.Hgb is measured spectrophotometrically; therefore, causes of increased sample turbidity such as paraprotein, lipids, abnormal hemoglobins, or nucleated cells can lead to erroneous results.
- Hematocrit (Hct): The normal reference range for males is 40–50%, and for females is 37–47%. It is the percentage of blood volume occupied by RBCs. Errors can occur in centrifugation due to plasma trapping, over dilution by anticoagulant, or by prolonged tourniquet application. The hematocrit is calculated as following:
- Mean corpuscular hemoglobin (MCH): The normal reference range is 27–31 pg. It is the average amount of hemoglobin per cell.The MCH is calculated as follows:Mean corpuscular hemoglobin concentration (MCHC): The normal reference range is 32–36 g/dL. MCHC is increased in spherocytosis and decreased in microcytic or hypochromic anemia. Since this is a calculated value, an erroneous value for hemoglobin may affect the MCHC value.
- Reticulocyte count used as a general indicator of bone marrow erythropoiesis and release. A normal or decreased reticulocyte count associated with moderate or marked anemia is strong evidence that the bone marrow is not responding appropriately. An increased reticulocyte count indicates a rapid erythroid cells turnover, which may be associated with blood loss, or acute/chronic hemolysis. The lifespan of reticulocytes is approximately 3–4 days in the bone marrow and 24 hours in peripheral blood. Reticulocytes cannot be visualized with the regular Giemsa stain; therefore special stains such as new methylene blue and brilliant cresol blue are used to precipitate the residual RNA material in these cells to visualize reticulocytes. For automated or flow cytometry analysis, reticulocytes can be stained with auramine O and thiazole orange.The corrected reticulocytes count (CRC) is calculated as follows:The reticulocyte production index (RPI) is calculated as following:Correction factorHctCorrection factor40–451.035–391.525–342.015–242.5<153.0The normal range of RPI is 1 to 2. In an anemic patient, RPI < 1 indicates a decreased production of reticulocytes and red blood cells. RPI > 2 indicates an increased production of reticulocytes to compensate the loss of red blood cells (destruction or bleeding).
- Serum iron measures iron bounded to transferrin (ferric form). There is diurnal variation (highest level in the morning); therefore, serum iron levels should be drawn in the morning.
- Total iron-binding capacity (TIBC) measures the concentration of transferrin. It indicates the iron concentration needed to saturate all transferrin-binding sites.The normal percentage of transferrin saturation (PTS) is 30% and is calculated as follows:
- Serum ferritin: Ferritin is a storage complex of apoferritin and iron. It is a relatively sensitive and reliable indicator for iron-deficiency anemia. It is elevated in iron overload (sideroblastic anemia, hemochromatosis), Gaucher's disease, and inflammatory diseases (ferritin is also an acute phase reactant). If iron deficiency and inflammatory disease co-exist, the serum ferritin level may be normal.
Table 1.6 Potential causes of erroneous results of automated hematology analyzerFalsely increaseFalsely decreaseWBCCryoglobulinExtremely elevated proteinNucleated RBCUnlysed RBCMalaria parasites in RBCPlatelet clumpingWBC aggregatesFragmented WBCRBCCryoglobulinGiant plateletsHigh WBC countAutoagglutinationMicrocytic RBCPlateletCryoglobulinMicrocytic RBCFragmented WBCGiant plateletsPlatelet clumpingPlatelet satellitosisHemoglobinCryoglobulinHigh WBC countSevere lipidemiaHeparinMCVAutoagglutinationHyperglycemiaHigh WBC countCryoglobulinGiant platelets
- Soluble serum transferrin receptor (STFR): The transferrin receptor acts as an iron-transporting molecule, it present on the most cell surface. The expression of transferrin receptors is dependent on the concentration of iron in the cellular cytoplasm. STFR is increased in iron deficiency anemia and hemolytic anemia.
- Free erythrocyte protoporphyrin (FEP): FEP is useful in distinguishing iron deficiency anemia and thalassemia minor. FEP is elevated, when there is a failure of iron incorporation to heme (iron deficiency, sideroblastic anemia, anemia of chronic disease, lead poisoning). Thalassemia is associated with abnormal hemoglobin synthesis, but not abnormal heme synthesis, so the FEP level is normal.
- High performance liquid chromatography (HPLC): HPLC measures HbA2 (increased in most β-thalassemia) and globin chain ratio.
- Interfering substances of automated hematology analyzer (Table 1.6): Check for hemolysis and clotting before using the specimen.