Chapter 5 5 Nuclear Hematology Kshitish C. Das 5.1 Introduction 90 5.26.2 Bone Marrow Imaging by Scintigraphy 126 5.26.3 Leukocyte (WBC) or Granulocyte Labeling for 5.2 Hematopoiesis and Hematopoietic Tissues 91 Imaging Bone Marrow 127 5.3 The Sites and Distribution of Prenatal and 5.26.4 Blood Platelets 127 Postnatal Hematopoietic Tissue 96 5.26.5 Measurement of Platelet Survival 128 5.4 Hematopoietic Growth Factors 97 References 128 5.5 Hematopoiesis and Hematopoietic Stem Cells 98 5.6 Hematopoietic Cell Lineages 99 5.7 Erythropoiesis 100 5.1 5.8 Globin Chain Synthesis 100 Introduction 5.9 Heme Synthesis 101 Nuclear hematology deals with the use of radionuclides 5.10 Some Essential Hematopoietic Nutrients 101 or radiopharmaceutical agents in the study of the path- 5.11 Iron Metabolism and Erythropoiesis 101 ophysiology, diagnosis and therapy of hematological 5.12 Intracellular Regulation of Iron 103 diseases arising de novo in the hematopoietic tissues or 5.13 Investigations to Evaluate the Qualitative and as a consequence of some systemic diseases. This prac- Quantitative Aspects of Erythropoiesis 103 tice virtually began in 1940, when John Lawrence first 32 5.14 Iron Absorption 104 used P to treat a young patient with chronic myeloid 32 5.15 Ferrokinetics 104 leukemia [1]. This was followed by the use of Pasara- 5.15.1 Plasma Iron Clearance 105 dioactive label for red cells to measure blood volume 5.15.2 Plasma Iron Turnover 106 [2]. From these modest beginnings, nuclear hematolo- 5.16 Red Cell Utilization (RCU) of Radio-iron 106 gy has come a long way and evolved into a contempo- 5.17 Erythrocyte Iron Turnover 107 rary discipline as a very useful and often an essential investigative tool in many areas of hematology. Radio- 5.18 SurfaceCountsfor59Fe 107 5.19 nuclides are now widely used to label the formed ele- Imaging of Bone Marrow and Other Organs for ments of the blood (random labels) to trace their bio- Evidence of Erythropoietic Activity 108 logical distribution, function and life span in vivo as 5.20 TheLifeSpansofRedBloodCells 109 well as to study the proliferation and differentiation of 5.21 SurfaceCountstoDetermineSitesofRedCell hematopoietic progenitor and precursor cells in the Destruction Using 51Cr-Labeled Red Cells 112 bone marrow (cohort labels). The other major applica- 5.22 Use of Radionuclides in the Investigation of tions of nuclear hematology include the determination Patients with Megaloblastic Anemia 113 5.22.1 Etiopathogenetic Basis of Megaloblastic Anemia 113 of spleen size, splenic sequestration of blood cells and investigations relating to the absorption, metabolism 5.23 Vitamin B12 Radioassay 119 and utilization of hematopoietic nutrients such as iron, 5.24 Determination of Holo-Transcobalmin-II vitamin B and folate. (Holo-TC-II) 119 12 Many imaging techniques are being increasingly 5.25 Identification of the Cause of Vitamin B12 Deficiency 119 employed and explored in order to determine the ana- tomical distribution of hematopoietic tissues in the 5.25.1 Food Cobalamin (Vitamin B12)Malabsorption 120 5.25.2 DNA Synthesis and Deoxyuridine (dU) bone marrow and other organs, and to evaluate their Suppression Test in Megaloblastic Anemia 120 significance in the diagnosis and management of vari- 5.25.3 Deoxyuridine (dU) Suppression Test 121 ous hematological disorders. This chapter reviews the 5.26 Visualization and Imaging of the Spleen in Health pathophysiological basis of the important applications and Disease 122 of radiopharmaceuticals and radio-isotopes in the 5.26.1 Measurement of Splenic Activity 125 practice of hematology. 5.2 Hematopoiesis and Hematopoietic Tissues 91 5.2 tion. A dynamic equilibrium is maintained between Hematopoiesis and Hematopoietic Tissues cell death in peripheral circulation and compensatory production of these cells in the bone marrow, creating Hematopoiesis is a complex biological process which a steady state of “normal blood cell numbers” in physi- represents a unique paradigm of developmental biolo- ological conditions. gy and ontogeny in a replicating mesenchymal cell sys- The circulating blood cells, i.e., the red blood cells, tem – the hematopoietic system. The various blood leukocytes and platelets, are highly specialized cells cells develop from the stem cells by multiplication, dif- with distinctive morphology (structures) and func- ferentiation, orderly maturation and release of mature tions. They are end-stage cells of their respective line- cells from the bone marrow to the peripheral circula- ages and are destined to be lost from the circulation a b cd Fig. 5.1. a Neutrophilic polymorphonuclear leukocytes showing predominantly secondary or specific granules, which are fine and neither basophilic nor eosinophilic (neutral). Romanovsky stain. b Eosinophil, which is a polymorphonuclear leukocyte con- taining large eosinophilic prominent granules in the cytoplasm. Romanovsky stain. c Basophils, which are granulocytic leuko- cytes containing dark colored (basophilic) granules in the cytoplasm. Romanovsky stain. d Electron microscopic picture of an eosinophil showing a large number of intracytoplasmic granules containing electron dense crystaloid cores surrounded by lig- hter areas of matrix 92 5 Nuclear Hematology after a relatively brief time span of hours, days or plasm. The monocytes and all of these granulocytes weeks.Asteadystateisattained,however,andthe have variable degrees of phagocytic function. physiological range of their numbers is maintained by The mature neutrophils (Fig. 5.1a) contain several acontinuoussupplyofnewlyformedcells(regenera- types of granules and other subcellular organelles. tion) from the blood-forming (hematopoietic) tissues. These include the primary or azurophilic granules and The red blood cells (matureerythrocytes),eachap- specific granules.Theprimary granules appear at the proximately 8 µm in diameter, contain hemoglobin in a promyelocytic stage, gradually decrease as the cells reduced (ferrous) state for successful gaseous exchange mature and contain many antimicrobial compounds in the tissues. They circulate in the vascular system as which include myeloperoxidase, defensins, lysozyme, flexible biconcave discs maintaining osmotic equilibri- bactericidal permeability-increasing protein (BPI) and um against high intracellular hemoglobin concentra- several serine proteases such as elastase, cathepsin G, tion and differential concentration gradients of intra- proteinase 3 and others. The primary granules also and extracellular potassium and sodium. The energy contain organelles such as lysosomes, which fuse with required for this physicochemical stability is provided phagocytic vesicles and deliver antimicrobial contents by ATP generated by the anaerobic glycolytic (Emb- to the ingested organisms. Specific granules also called den-Meyerhoff) pathway and the hexose-monophos- secondary granules of the neutrophils may fuse with phate shunt pathway generating reduced coenzymes phagocytic vesicles in inflammatory processes. The NADH and NADPH respectively. contents of these granules include lactoferrin, vitamin- Thematureleukocytes(whitebloodcells)comprise B12-binding proteins (transcobalamins I and III), plas- two broad groups: the granulocytes and monocytes minogen activator, collagenase, etc. These granules al- (phagocytes), and the lymphocytes (immunocytes). so play an important part in promoting chemotaxis and Normally only the mature leukocytes are found in the antimicrobial activities. The main functions of neutro- circulating peripheral blood. These include mature phils consist in their mounting a protective response of granulocytes (neutrophilic polymorphonuclear leuko- the host to microbial infections. The neutrophils ad- cytes, eosinophils and basophils) (Fig. 5.1a–c), mono- here to the endothelial cells (marginate) and then ex- cytes (Fig. 5.2a,b) and lymphocytes (Fig. 5.3a,b). Both tend their cytoplasmic membrane (pseudopodia) into granulocytes and monocytes have phagocytic func- the endothelial cells lining the capillaries and thus emi- tions. With Romanovsky stain, polymorphonuclear grateintothetissuesatthesiteofinfection;theenergy neutrophils show faint, but fine granules (neutral) for this movement is generated by the activation of an- (Fig. 5.1a), eosinophils show larger spherical red or aerobic glycolysis in the granulocytes. The plasma pink granules (Fig. 5.1b), whereas basophils show membrane of the involved neutrophils envelops the in- many dark-staining granules (Fig. 5.1c) in their cyto- vading organisms or particles by its pseudopodia, ab Fig. 5.2a,b. Monocyte; these are generally larger than neutrophils, have delicate fine nuclear chromatin, indistinct nucleoli and thin membrane outline. The cytoplasm is abundant with irregular outline, light blue color and a ground glass appearance 5.2 Hematopoiesis and Hematopoietic Tissues 93 a b Fig. 5.3. a B-lymphocyte, which is a relatively small round mononuclear cell (8–10 µm in diameter), a nucleus occupying almost the whole of the cell usually with no nuclei, and a scanty cytoplasm with a bluish or moderately basophilic narrow rim of cytoplasm. b T-lymphocyte, which contains a dense, but uneven prominent chromatin structure with a shallow intranuclear indentation which fuse around the organisms (phagocytosis) form- entering the tissues. The dark cytoplasmic granules of ing phagosomes. The phagosomes fuse with the con- basophils contain heparin, histamine and minor quan- tents of the azurophilic (primary) and specific (second- tities of other biogenic amines.