Biological Evidence – Blood (Comprehensive Study Notes)

Mission, Vision & Core Values

• Mission: CHRIST seeks holistic formation of individuals who can contribute effectively to society in an ever-changing world.
• Vision: Excellence and Service.
• Core Values: Faith in God ▪ Moral Uprightness ▪ Love of Fellow Beings ▪ Social Responsibility ▪ Pursuit of Excellence.

Basic Physical & Chemical Properties of Blood

• Normal pH = $7.35\,–\,7.45$ (slightly alkaline).
– Venous blood slightly more acidic (↑CO$_2$).
• Temperature ≈ $38^{\circ}\text{C}$ $(100.4^{\circ}\text{F})$ : always ~$1^{\circ}\text{C}$ higher than core body temp.
• Viscosity: $3\text{–}5\times$ that of water; due to cells + plasma proteins; contributes to normal arterial pressure (≈ $120/80\ \text{mmHg}$).
• Colour:
– Arterial (oxyhaemoglobin) – bright scarlet red.
– Venous (reduced Hb) – dull dark red.
• Volume: ~$8\%$ of body mass (≈ $5\text{–}6\ L$ in 70-kg adult).

Fluid Compartments of a 70-kg Adult

• Total Fluid = $42\ L$ (≈ $60\%$ body weight).
– Intracellular fluid = $28\ L$.
– Extracellular fluid = $14\ L$ → Plasma $3\ L$ + Interstitial $10.5\ L$ + Transcellular $0.5\ L$.

Gross Composition of Whole Blood

• Plasma ≈ $55\%$ (water 90-92 %, proteins 7-8 %, other solutes 1 %).
• Formed elements ≈ $45\%$ (RBC $\sim 45\%$, Buffy coat <1 % containing WBC & platelets).

Plasma Proteins

• Albumins (≈ $60\%$): smallest, liver-made; maintain colloid osmotic pressure, carriers for fatty acids & steroid hormones.
• Globulins (≈ $36\%$):
– $\alpha,\,\beta$ (transport Fe, lipids, fat-soluble vitamins).
– $\gamma$ (immunoglobulins from plasma cells/B-cells).
• Fibrinogen (≈ $4\%$): clot precursor; liver origin.
• Other solutes (1.5 %): electrolytes (Na$^+$, K$^+$, Ca$^{2+}$, Mg$^{2+}$, Cl$^-$, HCO$<em>{3}^-$, HPO$</em>{4}^{2-}$, SO$_{4}^{2-}$), nutrients (glucose, lipids, amino acids), gases, hormones, nitrogenous wastes (urea, uric acid, creatinine).

Formed Elements

Erythrocytes (RBCs)

• Biconcave, anucleate discs; dia. $7\text{–}8\ \mu\text{m}$; surface ↑30 % vs sphere → efficient gas diffusion.
• Lifespan ≈ $120\ \text{days}$; $\sim 4.5\text{–}5.5\ \text{million}\,\mu\text{L}^{-1}$.
• Hemoglobin: adult type $\alpha<em>2\beta</em>2$, carries $\approx 98.5\%$ of $O<em>2$ & 23 % of $CO</em>2$.
• Flexible cytoskeleton (spectrin-actin) allows passage through $3\ \mu\text{m}$ capillaries.
• Production (erythropoiesis) — regulated by renal Erythropoietin (EPO); maturation ~7 d from pro-erythroblast → reticulocyte → RBC.

Leukocytes (WBCs)

• <1 % of blood volume (5,000–10,000 $\mu\text{L}^{-1}$). Have nuclei → major source of genomic DNA in blood.
• Granulocytes:
– Neutrophils (55–70 %): multi-lobed, pale lilac granules; first responders; phagocytose bacteria; respiratory burst producing $O<em>2^-$, $H</em>2O_2$, $ClO^-$; lifespan <24 h.
– Eosinophils (1–4 %): bilobed; red-orange granules; combat parasites & modulate allergies (histaminase).
– Basophils (0.5–1 %): dark blue granules; release histamine, heparin, serotonin → amplify inflammation.
• Agranulocytes:
– Lymphocytes (20–45 %): small (6–9 µm) or large (10–14 µm).
• B cells → plasma cells → antibodies (humoral immunity).
• T helper/cytotoxic → coordinate & kill infected/tumor cells (cell-mediated).
• NK cells → innate cytotoxicity.
– Monocytes (3–8 %): largest (12–20 µm); kidney-shaped nucleus; become macrophages in tissues; antigen presentation; secrete pyrogens (fever).

Thrombocytes (Platelets)

• 150,000–400,000 $\mu\text{L}^{-1}$; anucleate fragments (2–4 µm) from megakaryocytes.
• Survive 5–9 days.
• Functions: primary hemostasis (platelet plug), release serotonin/ADP/TXA$_2$ for vasospasm & coagulation cascade activation.

Hematopoiesis

• All blood cells derive from pluripotent hematopoietic stem cells (HSCs) in red marrow; also in umbilical-cord & peripheral blood (after mobilization).
• Differentiation tree:
– Myeloid lineage: proerythroblast → RBC; megakaryoblast → platelets; myeloblast → granulocytes; monoblast → monocytes.
– Lymphoid lineage: lymphoblast → B, T, NK cells.
• Clinical relevance: HSC transplantation treats leukaemias, lymphomas, marrow failure, immunodeficiencies.

Functions of Blood

• Transport: $O<em>2$, $CO</em>2$, nutrients (glucose, fatty acids, amino acids), hormones, waste (urea, lactic acid).
• Regulation: pH buffering (bicarbonate), temperature homeostasis, fluid–electrolyte balance, osmotic pressure.
• Protection: immunity (WBCs, antibodies), hemostasis (platelets, clotting factors), detoxification (binding toxins).

Complete Blood Count (CBC) Reference Ranges

• Hemoglobin (Hb): Men $14\text{–}17\,\text{g dL}^{-1}$; Women $12\text{–}15\,\text{g dL}^{-1}$.
• Hematocrit (Hct): Men ≈ $40\%$; Women ≈ $31\%$.
• RBC count: $4.0\text{–}6.0\times10^{6}\,\mu\text{L}^{-1}$.
• MCV: $80\text{–}95\,\text{fL}$.
• WBC count: $4.5\text{–}10.0\times10^{3}\,\mu\text{L}^{-1}$.
• Platelets: $140\text{–}450\times10^{3}\,\mu\text{L}^{-1}$.

Comparative Blood Colours in Animals

• Red (Fe-hemoglobin): humans, most vertebrates.
• Blue (Cu-hemocyanin): horseshoe crabs, octopus, lobsters, spiders.
• Green (Fe-chlorocruorin): earthworms, leeches.
• Green (biliverdin recycling): skinks, some marine worms.
• Violet-pink (Fe-hemerythrin): lamp shells.
• Colourless (no respiratory pigment): Antarctic ice fish.

Blood vs. Hemolymph

• Blood: closed vascular system of vertebrates; contains RBCs + Hb; confined to vessels; red.
• Hemolymph: fills haemocoel of most invertebrates; lacks RBCs & Hb; bathes tissues directly; colourless or pigmented variably.

Human Blood vs. Other Animal Blood (Key Distinctions)

• Respiratory pigment in mammals located inside enucleate RBCs; many non-mammals possess nucleated erythrocytes; invertebrates often have extracellular pigments.
• Circulatory system: humans closed & warm-blooded; animals may be open/closed, ectothermic/endothermic.
• Blood group antigens: human ABO & Rh; animals show species-specific sets.

Forensic Serology: Detection & Identification of Blood

Collection & Preservation

• Use sterile swabs, allow to air-dry; package in breathable paper bags; refrigerate/freeze asap; document chain of custody; avoid contamination (PPE).

Presumptive Tests

General Principle
• Rely on heme-catalyzed oxidation–reduction. Heme (Fe$^{2+}/Fe^{3+}$) + $H<em>2O</em>2$ (oxidant) converts a colourless/chemiluminescent substrate → coloured/ light-emitting product. Sensitive to $10^{-5}\text{–}10^{-6}$ dilutions.

Colorimetric Assays
• Phenolphthalin (Kastle–Meyer) test: phenolphthalin + $H<em>2O</em>2$ → pink phenolphthalein in basic pH. Rapid, highly sensitive, but false-positives with strong oxidizers/plant peroxidases.
• Leucomalachite Green (LMG): acid medium; colourless leuco-base → green malachite green.
• Benzidine & derivatives: blue product in acid. Benzidine & ortho-tolidine carcinogenic → replaced by Tetramethylbenzidine (TMB, Hemastix); positive = green/ blue-green. Read immediately (colour shifts brown).

Chemiluminescence / Fluorescence
• Luminol (3-aminophthalhydrazide): emits blue light $\lambda_{max}\approx425\,\text{nm}$ in darkness; good for large-area screening & latent patterns (footprints, fingerprints). Light transient; must photograph quickly; can dilute DNA.
• Fluorescin: oxidized product fluoresces bright yellow-green under 425–485 nm excitation; longer-lasting signal than luminol.

Confirmatory Microcrystal Assays

• Goal: visualize characteristic crystals of heme derivatives under microscope (40×). Less sensitive but highly specific for blood (human or animal).

Takayama (Hemochromogen) Test
• Reagent: pyridine + glucose + $10\%$ NaOH.
• Heat $\approx65^{\circ}\text{C}$ 10 s → pink needle-shaped pyridine ferroprotoporphyrin crystals.

Teichmann (Hematin) Test
• Reagent: KCl/KBr/KI in glacial acetic acid.
• Heat $\approx65^{\circ}\text{C}$ → brown rhombic hematin chloride crystals; more reliable for aged/degraded stains.

Limitations
• Cannot distinguish human vs. non-human blood (need immunological or DNA testing). Not as sensitive as presumptive assays.

Oxidation–Reduction Fundamentals (Context for Tests)

• Oxidation = loss of e$^-$ (often loss of H).
• Reduction = gain of e$^-$.
• Oxidant (e.g., $H<em>2O</em>2$) is reduced; reductant (substrate) is oxidized.
• Heme acts as peroxidase-like catalyst cycling between $Fe^{2+}$ / $Fe^{3+}$.

Ethical, Practical & Real-World Connections

• Forensic use illustrates Social Responsibility & pursuit of truth in legal systems; misuse or misinterpretation can lead to injustice.
• Carcinogenicity of benzidine highlights need for moral uprightness in adopting safer reagents.
• Understanding blood physiology guides medical diagnostics (CBC, anemia, leukemias) and therapies (transfusions, stem-cell transplantation).

Quick Concept Map / High-Yield Facts

• $pH_{blood}=7.4$, $T=38^{\circ}\text{C}$, Viscosity $3\text{–}5\times$ water.
• Plasma: Water 90 % ▪ Proteins 7 % (Albumin>Globulin>Fibrinogen) ▪ Solutes 1 %.
• Formed Elements: RBC 4.5–6 million/µL; WBC 5–10 thousand/µL; Platelets 150–400 thousand/µL.
• Lifespan: RBC 120 d ▪ WBC hours–years ▪ Platelets 7 d.
• Hematopoiesis: HSC → Myeloid vs Lymphoid branches.
• Presumptive → fast/sensitive but not definitive; Confirmatory → crystal formation = definitive for blood presence.