Exercise 4: Blood

Blood

Vital connective tissue composed of plasma and formed elements (RBCs, WBCs, platelets); functions in transport, defense, and homeostasis.

Erythrocyte Sedimentation Rate (ESR)

Non-specific screening test for inflammation; measures how quickly RBCs settle in anticoagulated blood.

ESR Principle

Inflammation increases fibrinogen/globulins, reducing zeta potential, promoting rouleaux formation, and increasing sedimentation rate.

ESR Stages

Rouleaux formation (first 10 min), rapid settling (next 40 min), final sedimentation (last 10 min).

Modified Westergren Method

Tube length 200 mm, 3.8% sodium citrate, more sensitive, male 0–15 mm/hr, female 0–20 mm/hr.

Wintrobe Method

Tube length 100 mm, EDTA blood, less sensitive, male 0–9 mm/hr, female 0–20 mm/hr.

Hematocrit (Microhematocrit Method)

Measures proportion of RBCs in blood; assesses anemia, polycythemia, hemodilution/concentration.

Hematocrit Principle

Centrifuge separates RBCs, buffy coat, plasma; % RBC height/total height is hematocrit.

Normal Hematocrit Values

Adult males 40–55%, adult females 36–48%.

Bleeding Time (Duke’s Method)

In vivo test of primary hemostasis; measures time to stop bleeding after skin puncture.

Bleeding Time Principle

Blot blood every 30 sec; Duke’s method: earlobe/fingertip, 2–4 min; Ivy’s method: forearm incision, 2–9 min with BP cuff.

Clotting Time

Measures secondary hemostasis; time to form a stable clot on non-absorbent surface, normal 2–4 min.

Blood Typing (Forward)

Detects ABO and Rh antigens on RBCs using anti-A, anti-B, anti-D sera; agglutination indicates antigen presence.

Osmotic Fragility Test

Measures RBC membrane stability under hypotonic stress; determines hemolysis onset and completion.

OFT Principle

RBCs in decreasing NaCl absorb water, swell, and lyse; fragile cells lyse sooner.

OFT Results

Initial hemolysis: 0.42–0.44% NaCl; complete: 0.32–0.34% NaCl.

Increased Fragility

Hereditary spherocytosis, autoimmune hemolytic anemia.

Decreased Fragility

Thalassemia, iron deficiency anemia, target cells.

Rouleaux Formation Stage

First 10 minutes; RBCs stack like coins as zeta potential decreases due to increased fibrinogen and globulins.

Mechanism in Rouleaux Formation

Acute-phase proteins bridge between RBCs, reducing repulsion and allowing aggregation.

Rapid Settling (Decantation) Stage

Next 40 minutes; aggregated RBCs are heavier and settle quickly under gravity; most sedimentation occurs here.

Plasma Appearance During Rapid Settling

Plasma begins clearing from the top as RBC mass descends.

Final Sedimentation Stage

Last 10 minutes; RBCs compact at the bottom, plasma becomes fully clear at the top.

Factors Affecting ESR in Each Stage

Plasma protein levels, RBC size/shape, and hematocrit can speed up or slow each phase.

Clinical Relevance of Stages

Abnormally fast progression suggests inflammation, high fibrinogen, or abnormal RBC shapes that promote rouleaux.