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🩸 Blood - Complete Physiology Notes

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1. Overview

• Plasma - extracellular fluid rich in proteins
• Formed elements - red blood cells (RBCs), white blood cells (WBCs), and platelets

• Adult woman: ~70 mL/kg body weight
• Adult man: ~80 mL/kg body weight

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2. Blood Composition

Formed Elements (by centrifugation)

Hematocrit (packed cell volume)

• Fraction of total column occupied by RBCs
• Normal adult woman: ~40%
• Normal adult man: ~45%
• Newborn: ~55% (falls to ~35% at 2 months, rises again to adult values at puberty)
• Total RBC volume: ~28 mL/kg (women), ~36 mL/kg (men)

Clinical note: Hematocrit measures RBC concentration, not total red cell mass. Immediately after hemorrhage, hematocrit may appear normal despite blood loss.

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3. Plasma

• Electrolytes
• Plasma proteins
• Carbohydrates
• Lipids

• Pink plasma = hemolysis (free Hgb in plasma)
• Brown-green = elevated bilirubin
• Cloudy = cryoglobulinemias

Plasma Proteins (normal: ~7.0 g/dL)

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4. Red Blood Cells (Erythrocytes)

Structure

• Biconcave disc shape - maximizes surface area for gas exchange
• No nucleus or organelles in mature form
• Contains hemoglobin (Hgb) - the primary O₂-carrying protein
• Contains carbonic anhydrase II (CA II) - converts CO₂ ⇌ HCO₃⁻ at extraordinarily high speed (>1 million CO₂ molecules/second per enzyme molecule)

Life Span

• 120 days - entire life spent in circulating blood

Function

1. O₂ transport - hemoglobin carries O₂ from lungs to tissues
2. CO₂ transport - carbonic anhydrase converts CO₂ → HCO₃⁻ for transport to lungs (see CO₂ carriage below)
3. pH buffering - hemoglobin acts as a buffer

CO₂ Transport

• HCO₃⁻ exits RBC via Cl⁻/HCO₃⁻ exchanger (band 3 protein) - the chloride shift
• This allows 70% of CO₂ to be carried as plasma HCO₃⁻

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5. White Blood Cells (Leukocytes)

Classification

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6. Platelets (Thrombocytes)

• Anucleate cell fragments derived from megakaryocytes
• Life span: 10 days - entire life spent in circulating blood
• Function: primary hemostasis (platelet plug formation), coagulation support
• Mean platelet volume (MPV) reflects average platelet size

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7. Blood Cell Formation (Hemopoiesis / Hematopoiesis)

Developmental Phases (Ontogeny)

Stem Cell Hierarchy

Hemopoietic Stem Cell (HSC/PPSC)
        │
        ├─► Common Myeloid Progenitor (CMP/CFU-GEMM)
        │         │
        │         ├─► Granulocyte/Monocyte Progenitor (GMP)
        │         │         ├─► Neutrophil progenitor → Neutrophil
        │         │         ├─► Basophil/Mast cell progenitor → Basophil / Mast cell
        │         │         ├─► Eosinophil progenitor → Eosinophil
        │         │         └─► Monocyte progenitor → Monocyte → Macrophage
        │         │
        │         └─► Megakaryocyte/Erythrocyte Progenitor (MEP)
        │                   ├─► Megakaryocyte progenitor → Megakaryocyte → Platelets
        │                   └─► Erythrocyte progenitor (CFU-E) → Erythrocytes
        │
        └─► Common Lymphoid Progenitor (CLP/CFU-L)
                  ├─► Pre-NK cell → NK cell
                  ├─► Pre-T cell → T cell
                  └─► Pre-B cell → B cell → Plasma cell

Erythropoiesis (RBC maturation sequence)

Proerythroblast
    → Basophilic erythroblast (ribosome-rich, basophilic)
        → Polychromatophilic erythroblast (last stage capable of mitosis; ~30 hrs)
            → Orthochromatophilic erythroblast / normoblast
               (nucleus ejected; Howell-Jolly bodies = nuclear remnants; ~48 hrs)
                → Polychromatophilic erythrocyte (reticulocyte)
                    → Mature erythrocyte

• Progressive hemoglobin accumulation (cytoplasm shifts from basophilic → eosinophilic)
• Progressive RNA loss (organelles disappear)
• Nuclear condensation and ejection at orthochromatophilic stage
• Reticulocytes retain some polyribosomes; still synthesize Hgb

Regulation of Erythropoiesis

• Erythropoietin (EPO) - ~34 kDa glycoprotein; acts via tyrosine kinase-associated receptor
  • In adults: produced mainly by fibroblast-like type I interstitial cells of renal cortex/outer medulla
  • In fetal life: produced mainly by liver; liver (Kupffer cells) produce some EPO in adults too
  • Stimulus: hypoxia (low O₂ delivery to kidney)
  • Effect: stimulates proerythroblast production; accelerates RBC progenitor development

Thrombopoiesis (platelet formation)

• Megakaryocytes in bone marrow shed cytoplasmic fragments → platelets
• Regulated by thrombopoietin (TPO)

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8. Key Normal Blood Values (Reference)

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9. Quick Summary Table: Formed Elements

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🩸 Blood - Notes for DMLT (Diploma in Medical Laboratory Technology)

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UNIT 1: BLOOD COLLECTION & SPECIMEN HANDLING

1.1 Venipuncture - Key Points

• Patient should be seated/supine for 15-20 min before blood draw to avoid hemoconcentration
• Tourniquet application: <60 seconds (longer causes hemoconcentration)
• Allow alcohol to dry completely before puncture (wet alcohol causes hemolysis)
• Use correct gauge needle; pulling syringe plunger too fast = hemolysis
• Preferred site: antecubital fossa (reduces hemolysis)
• Mix anticoagulant tubes immediately after filling by gentle inversion

1.2 Blood Collection Tubes - Color Code System

Correct order of draw: Blood culture → Blue → Red/SST → Green → Purple → Grey

1.3 Causes & Effects of Hemolysis

Important: Strong hemolysis can raise K⁺ by 2-3 mEq/L into a critical range.

1.4 Preanalytical Variables Affecting Results

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UNIT 2: BLOOD COMPOSITION

2.1 What is Blood?

• Total volume: ~70 mL/kg (women) | ~80 mL/kg (men)
• Hematocrit = % of blood occupied by RBCs

2.2 Plasma

• Electrolytes (Na⁺, K⁺, Cl⁻, HCO₃⁻)
• Plasma proteins (~7.0 g/dL total)
• Glucose, lipids, hormones, waste products

Plasma Proteins

• Oncotic pressure = ~25 mmHg (prevents fluid leaking out of capillaries)
• Albumin made by liver; half-life ~20 days

Lab note: Serum = plasma minus fibrinogen (after clot formation)

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UNIT 3: FORMED ELEMENTS (BLOOD CELLS)

3.1 Red Blood Cells (Erythrocytes)

1. O₂ transport via hemoglobin (Hgb)
2. CO₂ transport via carbonic anhydrase (CA-II): CO₂ + H₂O ⇌ HCO₃⁻ + H⁺ → Chloride shift
3. Buffering (hemoglobin acts as buffer)

Hemoglobin

• Normal Hgb: 13.5-17.5 g/dL (men) | 12.0-15.5 g/dL (women)
• Structure: 4 globin chains + 4 heme groups (each contains Fe²⁺)
• Types: HbA (α₂β₂ - adult) | HbF (α₂γ₂ - fetal) | HbA₂ (α₂δ₂)

Abnormal RBC Morphology on Peripheral Smear

3.2 White Blood Cells (Leukocytes)

Neutrophil Granules (Important for DMLT)

1. Azurophilic (primary) granules - lysosomes; contain myeloperoxidase (MPO), defensins, cathelicidin, acid hydrolases
2. Specific (secondary) granules - contain lactoferrin, lysozyme, collagenase
3. Gelatinase (tertiary) granules - contain gelatinase/MMP-9

Drumstick appendage on neutrophil nucleus = Barr body (inactive X chromosome; seen in females)

Neutrophil Killing Mechanisms

• Oxygen-dependent (main): NADPH oxidase → superoxide (O₂⁻) → H₂O₂ → hypochlorite (via MPO) = respiratory burst
• Oxygen-independent: Defensins, lysozyme, lactoferrin, cathepsins

CGD (Chronic Granulomatous Disease) = NADPH oxidase mutation → recurrent bacterial infections

Lymphocyte Types

3.3 Platelets (Thrombocytes)

1. Primary hemostasis - adhere to damaged vessel wall → form platelet plug
2. Support coagulation - provide phospholipid surface for coagulation factors

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UNIT 4: BLOOD FILM - PREPARATION & STAINING

4.1 Peripheral Blood Smear Preparation (Wedge Method)

1. Place small drop of blood 1-2 cm from one end of slide
2. Place spreader slide at 30-45° angle in front of the drop
3. Pull back to touch the drop, then push forward smoothly
4. Dry rapidly by waving in air
5. Label with pencil at the thick end

• Thick end - good for scanning, not examining cell morphology
• Body - RBCs overlapping
• Feather edge (thin end) - ideal area for differential count; WBCs well-spread; monolayer of RBCs

Caution on wedge smear: monocytes concentrate at the tip/feather edge; neutrophils crowd the lateral edges

4.2 Staining Methods

Romanowsky Stains (most common for blood films)

Wright's Stain Procedure (Step-by-Step)

1. Prepare and air-dry blood film
2. Fix with absolute methanol for 1-2 minutes
3. Cover with undiluted Wright's stain for 2 minutes
4. Add equal volume of buffer (pH 6.4), mix gently (blow); stain for 4-6 minutes
5. Flush with distilled water/buffer
6. Allow to dry; examine under oil immersion (100×)

Staining Characteristics (What stains what)

4.3 Special Stains Used in DMLT

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UNIT 5: HEMATOPOIESIS (Blood Cell Formation)

5.1 Sites of Hematopoiesis (Developmental Stages)

Extramedullary hematopoiesis = blood cell formation outside bone marrow (spleen/liver) - seen in thalassemia, myelofibrosis

5.2 Sites of Red Bone Marrow in Adults

• Flat bones: sternum, ribs, vertebrae, skull
• Upper ends of long bones: femur, humerus
• Iliac crest (site of bone marrow biopsy/aspiration)

5.3 Stem Cell Hierarchy

Pluripotent Hemopoietic Stem Cell (HSC)
        │
        ├─► Common Myeloid Progenitor (CMP/CFU-GEMM)
        │         ├─► GMP → Neutrophil, Eosinophil, Basophil, Monocyte
        │         └─► MEP → Megakaryocyte (→Platelets) + Erythrocyte
        │
        └─► Common Lymphoid Progenitor (CLP)
                  ├─► T cell (matures in thymus)
                  ├─► B cell (matures in bone marrow → plasma cell)
                  └─► NK cell

• CFU-E = erythrocyte, CFU-G = granulocyte, CFU-M = monocyte, CFU-Meg = megakaryocyte, CFU-GEMM = pluripotent myeloid

5.4 Erythropoiesis - Maturation Sequence

As cells mature: nucleus shrinks → cytoplasm turns from blue to pink → cell gets smaller → nucleus expelled

Regulation of Erythropoiesis

• Erythropoietin (EPO): ~34 kDa glycoprotein
  • Adults: produced by peritubular fibroblasts in renal cortex
  • Fetus: produced by liver
  • Trigger: hypoxia → increases EPO → stimulates RBC progenitors
  • Used clinically in anemia of CKD (recombinant EPO / Darbepoetin)

5.5 Granulopoiesis (Granulocyte Maturation)

Myeloblast → Promyelocyte → Myelocyte → Metamyelocyte → Band (Stab) → Mature Neutrophil

• Myeloblast: large nucleus, no granules
• Promyelocyte: primary (azurophilic) granules appear
• Myelocyte: specific granules appear; last stage capable of division
• Metamyelocyte: indented kidney-shaped nucleus; non-dividing
• Band (Stab) cell: horseshoe-shaped nucleus; immature neutrophil in peripheral blood
• Mature neutrophil: 2-5 lobed nucleus

"Shift to the left" = increase in band cells/immature neutrophils = bacterial infection/stress response

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UNIT 6: HAEMATOLOGICAL INDICES & REFERENCE VALUES

6.1 Complete Blood Count (CBC) - Normal Reference Values

6.2 RBC Indices Interpretation

6.3 Anemia Classification by MCV

6.4 Reticulocyte Count

• Stained with New Methylene Blue (NMB) or Brilliant Cresyl Blue (BCB) - precipitates residual RNA
• Normal: 0.5-2.5%
• High (reticulocytosis) = active RBC production = hemorrhage, hemolysis, treatment response
• Low (reticulocytopenia) = bone marrow failure, aplastic anemia, B12/folate deficiency

6.5 ESR (Erythrocyte Sedimentation Rate)

• Westergren method (gold standard): blood drawn into sodium citrate (4:1); measured at 1 hour
• Normal: men 0-15 mm/hr; women 0-20 mm/hr
• Increased ESR: inflammation, infection, malignancy, pregnancy, anemia
• Decreased ESR: polycythemia, sickle cell disease, spherocytosis

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UNIT 7: BONE MARROW EXAMINATION

7.1 Indications

• Unexplained cytopenia (low RBC, WBC, or platelets)
• Suspected leukemia/lymphoma
• Staging of malignancies
• Diagnosis of aplastic anemia, MDS, myeloma

7.2 Sites for Aspiration/Biopsy

• Posterior superior iliac crest (most common)
• Sternum (aspiration only)
• Anterior iliac crest

7.3 Bone Marrow Cellularity (Normal by Age)

• Normal cellularity at 50 years: vertebrae ~75%, sternum ~60%, iliac crest ~50%, rib ~30%
• Hypercellular marrow = increased hematopoiesis (leukemia, hemolysis, B12 deficiency)
• Hypocellular marrow = aplastic anemia

7.4 Myeloid : Erythroid (M:E) Ratio

• Normal: 3:1 to 4:1
• Increased (>5:1): infection, leukemia, erythroid hypoplasia
• Decreased (<2:1): megaloblastic anemia, hemolytic anemia (erythroid hyperplasia)

7.5 Differential Count from Bone Marrow Aspirate

• 300-500 cells are counted
• Scan under low then high magnification
• Allows assessment of maturation, dysplasia, and lineage proportions

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UNIT 8: KEY DMLT EXAM POINTS (QUICK RECALL)

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