Expanded when demand for blood cells increases (ex. Hemorrhage or infection)

Hematopoiesis - process by which blood cells are produced and released into the peripheral blood

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Each of the blood cell types in peripheral blood maintained within tightly controlled ranges via control mechanisms

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Hematopoietic progenitor cells

Hematopoietic growth factors

Bone marrow microenvironment

Three major components of hematopoietic system (expanded upon below):○

○ Normal adult blood cell counts:

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Hematopoiesis: A Brief Review•

All derived from pluripotent (multipotential) stem cell○

Hematopoietic Progenitor Cells •

Hematopoiesis, Bone Marrow Examination, and AnemiasMonday, October 12, 2015 11:46 AM

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Self-renewal□Differentiation□

Characterized by capacity for

Stem cells - earliest hematopoietic progenitor cells○

Progenitor cells become increasingly committed to differentiation along specific lineages -progressively lose ability to self-renew and increase ability to proliferate

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Earlier hematopoietic progenitor cells have morphological features of primitive cells (fine chromatin, nucleoli, little cytoplasm)

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Some stimulate production of certain blood cells□Others inhibit production□

Mediated through growth factor receptors on surface of progenitor cells

Soluble growth factors influence differentiation, maturation, and proliferation of hematopoietic progenitor cells

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Influences production of RBCs through negative feedback loop

Erythropoietin (EPO) - serves as prototype for how growth factors function ○

Hematopoietic Growth Factors•

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Produced by specialized peritubular cells in kidney (small amount in liver)

O2 delivery decreases (anemia), EPO production increases (normally) -> stimulates committed erythropoietic stem cells in bone marrow to mature and proliferate -> accelerated RBC production

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RBCs and O2 delivery normal -> EPO production down-regulated □

Amount produced based on delivery of oxygen to these cells

Fetal life - major sites of hematopoiesis shift from yolk sac -> liver and spleen -> bone marrow

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After birth - bone marrow unless disease ○

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Cellularity of bone marrow differs at different sites and at different ages

Increasing age - level of hematopoietic activity decrease in certain bones (long bones first -> eventually axial skeleton)

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Production of hematopoietic growth factors by osteoblasts, fibroblasts, and other bone marrow stromal cells

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Direct interactions between stromal and hematopoietic progenitor cells and between hematopoietic cells and ECM (mediated through adhesion molecules and receptors)

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Regulation occurs through

Microenvironment of bone marrow provides space and scaffold for hematopoiesis, plays a regulatory role in providing "niches" for hematopoietic stem cells, and controlling production and release of blood cells

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Bone Marrow Microenvironment•

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Rate of production, release, and senescence of each hematopoietic lineage varies○

Time to Release

# Progeny/Stem Cell Survival in Blood

Erythrocytic 5 days 16 120 days

Granulocytic 14 days 16-32 1-4 days

Megakaryocytic 5 days Hundreds 8-11 days

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Kinetics of Hematopoiesis •

Bone marrow examination is important in evaluation of many disorders of the hematopoietic system and other diseases that may involve the bone marrow cavity

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Aspirate (smear, clot)

Core biopsy

Two types of samples acquired:○

Differ in terms of how they can be used to evaluate the marrow ○

Specimens complement each other - both specimens acquired usually ○

Older children and adults - usually taken from posterior superior iliac spine ○

May be aspirated from sternum○

Young children - anterior tibia○

General •

Acquired by aspirating liquid marrow using syringe and special needle ○

Liquid smeared and stained for examination, lab studies, or allowed to clot and sectioned/stained like other tissue

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Bone Marrow Aspirate•

Take solid core sample of marrow using trephine needle ○

Core decalcified, sectioned, stained○

Bone Marrow Core Biopsy•

Bone Marrow Aspirate Bone Marrow Core Biopsy

Marrow cellularity Estimate 90% in children; 30% in elderly adults

Bone Marrow Examination

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adults

Foreign cells Metastatic neoplasm; biopsy better for some

Lymphoma, carcinoma; better than aspirate

Microorganism By morphology and/or culture Especially those found in granulomas

Maturation of hematopoietic lineages

Detailed cytologic evaluation Aspirate better

Myeloid: Erythroid ratio 3:1 -

Evaluation of "low frequency elements"

Plasma cells, lymphocytes, histiocytes

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Iron stores Histiocytic and sideroblastic iron stores

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Fibrosis - Biopsy only

Bone morphology - Evaluation of certain metabolic bone disorders

Granulomas - Biopsy only

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Reserved for evaluation of hematopoietic disorders after less invasive tests have not successfully established a definitive diagnosis

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Anemia, neutropenia, and/or thrombocytopenia

Leukemia

Metastatic neoplasms

Sealed infectious diseases (mycobacteria, fungi)

In general:○

Indications for Bone Marrow Examination•

Anemia - reduction in total circulating RBC mass -> reduction in oxygen carrying capacity of blood

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Definition: •

Reduction in hematocrit, hemoglobin concentration, and/or RBC count in peripheral blood○

Normal ranges (mean +- 2 S.D.):○

Men Women

Hematocrit (%) 46 (40-52) 41 (35-47)

Hemoglobin (g/dL) 15.5 (13.5-17.5) 13.5 (11.5-15.5)

RBC count (x10^6/uL) 5.2 (4.5-6.0) 4.5 (3.8-5.2)

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Gender

Age - higher in newborns; lower until puberty

Living at high altitude - lower O2 tension induces erythropoietin production

Posture - decreased if prolonged bed rest

Stress or extreme exercise - increases values

Dehydration - increases values (hemoconcentration)

Overhydration - as with IV fluids; decreases values (hemodilution)

Variables that may alter these values:○

Criteria for Anemia•

Pathophysiologic classification (superior - divides anemias according to mechanism of causation)

Morphologic classification (simpler - allows anemias to be categorized by determining the size of patient's RBCs)

Two different systems;○

Pathophysiologic Classifications of Anemia○

Classification of Anemia•

Introduction to Anemias

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○ Morphologic Classification of Anemias

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Clinical Features of Anemia•

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Fatigue

Dyspnea on exertion

Faintness

Vertigo

Angina pectoris

Claudication

Palpitation

Symptoms:○

Pallor

Tachycardia

Hypotension

Systolic murmur

Signs:○

Extreme tissue hypoxia -> MI, brain damage, shock, death

Severe:○

History of GI or GU bleeding - iron deficiency anemia□Pica - iron deficiency anemia □Jaundice - hemolysis□Dark urine - hemolysis□Neurologic complaints - B12 deficiency □

Symptoms:

Dry, atrophic skin and nail changes - iron deficiency anemia□Jaundice - hemolysis□Red, swollen tongue - B12 deficiency□Certain neurologic signs - B12 deficiency□

Signs:

Various anemias associated with more specific clinical features○

Rapidly developing - acute blood loss - hematocrit of 20% -> devastating effect□Slowly developing - B12 deficiency - hematocrit of 20% -> little clinical effect□

Rate at which anemia develops

Young, healthy patient can tolerate severe anemia better than an older person with cardiac/pulmonary disease

□Degree of cardiac or pulmonary compensation

Borderline anemic states significantly worsened when metabolic requirements are increased (ex. Pregnancy)

□Metabolic requirements

Certain underlying diseases (carcinomatosis, lymphoma, autoimmune disease) may play a role in causing anemia and contribute to severity of clinical impact on patient

□Underlying disease states

Severity may be affected by several factors:○

Clinical Features of Anemia•

Hematocrit, hemoglobin, RBC count○

Define RBC size, degree of variation in size, hemoglobin content as part of CBC

Measure of average volume of individual RBCs □Used to categorize anemia according to morphologic classification scheme □Degree of variation in size can be determined by examination of peripheral blood smear or analysis with blood cell counting instrument

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Normal adult rage: 80-99 um^3/RBC□Normal children rage: 70-90 um^3/RBC□

MCV (Mean Corpuscular Volume)

Measure of average total hemoglobin content of individual RBCs□Parallels MCV and MCHC closely - doesn't contribute much information□Normal adult range: 27-32 pg/RBC□

MCH (Mean Corpuscular Hemoglobin)

Mean Corpuscular Hemoglobin Concentration (MCHC)

RBC indices:○

Laboratory Evaluation of Anemia•

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Measure of average concentration of hemoglobin within individual RBCs□Normal adult range: 32-36 g/100ml RBC□

Mean Corpuscular Hemoglobin Concentration (MCHC)

Essential in evaluating any patient with anemia

Normocytic - normal size (7 MICRONS)

Microcytic - small size

Macrocytic - large size

Anisocytosis - significant variation in size

RBC size□

Normocytic RBCs area of central pallor measuring 1/3 to 1/2 diameter of cell

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Hypochromic - area of central pallor >1/2 RBC diameter ◊

Wright-Giemsa stain:

RBC Hemoglobin concentration□

Young/immature RBC will be slightly macrocytic and stain slightly more basophilic than mature RBC on Wright-Giemsa stained smear = basophilic

Number of immature RBCs□

Following features should be noted:

Peripheral blood smear examination○

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basophilic than mature RBC on Wright-Giemsa stained smear = basophilic RBCs

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Polychromasia/polychromatophilia = increase of basophilic RBCs in peripheral blood

Correspond with reticulocytes

Can be used to estimate rate of RBC production in and release from bone marrow

Nucleated RBCs (NRBCs) may be present in peripheral blood

Poikilocytosis - significant variation in RBC shape

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Target cell (codocyte) - structural hemoglobinopathies, thalassemias

Teardrop cell (dacryocyte) - myelophthisic anemia, myelofibrosis

RBC shape□

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Fragmented cell (schistocyte) - mechanical hemolysis

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Sickle cell (drepanocyte) - sickle cell anemia

Spherocyte - hereditary spherocytosis, autoimmune hemolysis

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Ovalocytes (elliptocytes) - hereditary elliptosytosis

Burr cell (echinocyte) - severe renal failure

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Young/immature RBCs have RNA in cytoplasm - detected using special stains = reticulocytes

Staining of reticulocytes more accurate than noting degree of polychromasia

Helpful in distinguishing hyperproliferative states (hemolytic anemias) from hypoproliferative states (aplastic anemia)

□Essential in determining rate of RBC production in and release from bone marrow

Expressed in percentage of total RBCs

Normal range: 0.5-1.5%; 25-75 x10^3/uL

Corrected reticulocyte count (CRC) (because reticulocyte count usually done in face of decreased RBC count or hematocrit) = raw retic. Count x (pt's hematocrit/mean normal hematocrit for age and gender)

Reticulocyte Count○

Normal adult range: 60-150 ug/Dl□Serum iron - measure of total iron concentration of serum

Reflection of serum transferrin concentration□Normal adult range: 300-400 ug/dL□

Total iron binding capacity (TIBC) - measure of total capacity of serum to bind iron

Normal adult range: 10-300 g/L (men); 5-100 ug/L (women)□Serum ferritin - allows estimation of total body iron stores

Iron studies○

Serum B12: 150-1,000 pg/mL (normal range)□Folate: Serum > 2.0 ng/mL; RBC > 150 ng/mL (normal range)□

Serum folate levels labile, depending on patient's recent diet - concentration within RBC is more reliable

Measurement of urine or serum methylmalonic acid and serum homocysteine levels may be useful in vitamin B12 deficiency

Vitamin B12 deficiency - useful test: assay for intrinsic factor blocking antibody -positive in 75% patients with pernicious anemia

Vitamin B12, Folate, and Related Studies○

General tests for hemolytic anemias○

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When extravascular or intravascular hemolysis occurs - hemoglobin released to plasma -> bound to haptoglobin -> removed from plasma by macrophates in reticuloendothelial system

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Significant hemolysis -> haptoglobin depleted and additional hemoglobin remains free in plasma

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Plasma hemoglobin

Serum haptoglobin

Intravascular hemolysis and increased plasma free hemoglobin -> excreted in urine

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Urine pink to dark brown (due to oxidation of hemoglobin)□

Urine hemoglobin

As hemoglobin is excreted in urine, some reabsorbed by renal tubular cells □Heme iron -> hemosider; when renal tubular cells sloughed, hemosiderin detected in urine by staining urine sediment for iron

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Urine hemosiderin

Hemoglobin broken down in macrophages with heme iron converted to hemosiderin and remaining heme moiety -> biliverdin -> bilirubin -> enters serum as unconjugated (indirect) bilirubin

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Significant extravascular/intravascular hemolysis - serum level of unconjugated (indirect) bilirubin increased until capacity of liver to clear is surpassed

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Bilirubin

Urine urobilinogen

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Some of the bilirubin that enters the gut in bile converted to urobilinogen -some absorbed in gut and excreted in urine

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Significant hemolysis - increase□

Urine urobilinogen

RBCs rich in LDH□Serum LDH level rises with hemolysis□

Serum lactate dehydrogenase (LDH)

Direct antiglobulin (Coombs) test: detects antibodies on surface of RBCs in immune-mediated hemolysis

RBC enzyme assays: used to detect presence of sickling hemoglobin (hemoglobin S)

Tests for sickling

Hemoglobin electrophoresis: detect structurally abnormal hemoglobins and in conjunction with quantitation of hemoglobins A2 and F may allow recognition of thalassemias

Sucrose hemolysis, Ham's, and specific red cell antigen tests: for paroxysmal nocturnal hemoglobinuria (PNH)

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Osmotic fragility, red cell membrane protein tests: hereditary spherocytosis□

Rarely used:

Specific tests for hemolytic anemias○

Once other studies fail to produce definite etiology

Aspiration and biopsy

M:E ratio - erythroid activity in marrow determined

Prussian blue stain - amount of storage iron and sideroblastic iron determined

Bone marrow examination○

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