Unit 1: Introduction and Haematology

1. Importance of medical diagnostics

Medical diagnostics are the tools and techniques used to gather information about a patient's health. Their importance is fundamental to medicine:

• Disease Detection & Diagnosis: They help identify the cause of symptoms, allowing for a precise diagnosis.
• Monitoring: They allow doctors to track the progression of a disease (e.g., blood sugar in diabetes) and the effectiveness of a treatment.
• Screening: They can detect diseases in asymptomatic individuals, allowing for early intervention (e.g., cancer screening).
• Prognosis: They can help predict the likely course and outcome of a disease.
• Prevention: Identifying risk factors (e.g., high cholesterol) allows for lifestyle changes to prevent disease.

2. Blood composition

Blood is a liquid connective tissue. When centrifuged, it separates into two main parts:

1. Plasma (Approx. 55%-60%): The straw-colored liquid matrix. It is mostly water (90%+) and contains:
   • Proteins: Albumin (maintains osmotic pressure), Fibrinogen (for clotting), Globulins (antibodies).
   • Other solutes: Nutrients (glucose, amino acids), hormones, electrolytes (Na+, K+, Cl-), and waste products (urea).
2. Formed Elements (Approx. 40%-45%): The cellular components.
   • Erythrocytes (Red Blood Cells - RBCs): The vast majority of cells. They are biconcave discs, lack a nucleus when mature, and are filled with haemoglobin to transport oxygen.
   • Leucocytes (White Blood Cells - WBCs): Part of the immune system. They are larger than RBCs and have a nucleus.
   • Platelets (Thrombocytes): Small, anucleated cell fragments that are essential for blood clotting.

3. Preparation of blood smear

A blood smear is a thin layer of blood on a glass slide, used for microscopic analysis (like DLC). A good smear has a "tongue-shaped" appearance with a head, body, and "feathered" tail.

Procedure:

1. Clean Slides: Use two clean, grease-free glass slides.
2. Place Blood Drop: Place a small drop of blood near one end of the first slide (the "smear slide").
3. Spread: Hold the second slide (the "spreader") at a 45° angle. Touch its edge to the smear slide just in front of the blood drop.
4. Capillary Action: Draw the spreader back until it makes contact with the blood, allowing the blood to spread along its edge by capillary action.
5. Push: In one smooth, steady motion, push the spreader slide forward to the other end of the smear slide. This action pulls the blood, creating a thin film.
6. Dry: Allow the smear to air dry completely. Do not blow on it.

4. Differential Leucocyte Count (DLC) using Leishman's stain

Principle: To determine the percentage of each type of white blood cell in a sample. This is crucial for diagnosing infections, allergies, and leukaemias.

Leishman's stain is a Romanowsky stain (like Giemsa). It contains eosin (an acidic, red dye) and methylene blue (a basic, blue/purple dye) in methanol. The methanol acts as a fixative.

Staining Procedure:

1. Place the dried blood smear on a staining rack.
2. Cover the smear with Leishman's stain (undiluted). Let it sit for 1-2 minutes. This fixes the cells.
3. Add double the volume of buffered distilled water (pH 6.8) directly onto the stain. Mix by gently blowing. A metallic green scum should appear.
4. Allow the diluted stain to act for 15-30 minutes.
5. Gently wash the slide with distilled water until the smear has a pinkish tint.
6. Wipe the back of the slide and let it air dry in a vertical position.

Counting Procedure (Practical):

1. Observe the slide under the microscope, starting at low power to find the "feathered edge".
2. Add a drop of immersion oil and switch to the 100x oil immersion lens.
3. Move the slide in a "battlement" (zigzag) pattern, identifying and counting each WBC seen.
4. Count a total of 100 WBCs, tallying each type. The final tally for each type is its percentage.

Identification of Leucocytes:

• Neutrophil (50-70%): Multi-lobed nucleus (2-5 lobes), pale pink/lilac cytoplasm with fine granules. (Increase = bacterial infection).
• Lymphocyte (20-40%): Large, round, dark purple nucleus that takes up most of the cell; thin rim of clear blue cytoplasm. (Increase = viral infection).
• Monocyte (2-8%): Largest WBC; kidney-bean or C-shaped nucleus; abundant grey-blue cytoplasm. (Increase = chronic infection).
• Eosinophil (1-4%): Bi-lobed nucleus; cytoplasm filled with large, bright red/orange granules. (Increase = parasitic infection, allergies).
• Basophil (0.4-1%): Large, S-shaped nucleus (often hidden); cytoplasm filled with large, dark purple/black granules. (Increase = allergic response).

5. Platelet count using haemocytometer

Principle: To manually count the number of platelets in a known volume of diluted blood using a specialized counting chamber (haemocytometer).

Procedure (Practical):

1. Dilution: Draw blood into an RBC pipette to the 0.5 mark. Immediately draw 1% ammonium oxalate (diluting fluid) up to the 101 mark. This gives a 1:200 dilution. (Ammonium oxalate lyses the RBCs, leaving platelets and WBCs).
2. Mix: Mix the contents of the pipette thoroughly for 2-3 minutes.
3. Charge Chamber: Discard the first few drops. Carefully load the diluted blood under the coverslip of a Neubauer (or similar) haemocytometer.
4. Settle: Place the chamber in a moist petri dish (with a wet filter paper) for 15-20 minutes. This allows the platelets to settle.
5. Count: Under 40x magnification, count the platelets in the entire large central square (which contains 25 smaller squares). Platelets appear as small, bright, refractile bodies.
6. Calculation:
   • Let N = number of platelets counted.
   • Volume of large central square = 1 mm (L) x 1 mm (W) x 0.1 mm (Depth) = 0.1 mm³ (or 0.1 µL).
   • Dilution Factor = 200.
   • Platelets/mm³ = (N × Dilution) / Volume = (N × 200) / 0.1 = N × 2000

6. Erythrocyte Sedimentary Rate (ESR)

Principle: A non-specific test that measures the rate (in mm/hr) at which RBCs settle in a vertical tube of anticoagulated blood. In conditions with inflammation, certain proteins (like fibrinogen) increase in the plasma, causing RBCs to clump together (rouleaux formation). These heavier clumps settle faster, resulting in a higher ESR.

Westergren Method (Procedure):

1. Sample: Collect blood in a black-top vacuum tube containing 3.2% sodium citrate. The ratio is 4 parts blood to 1 part anticoagulant.
2. Setup: Place the blood in a Westergren tube, a 300 mm long glass tube with a 2.5 mm bore, graduated from 0 to 200 mm.
3. Wait: Place the tube in a special rack, ensuring it is perfectly vertical and free from vibrations, at room temperature.
4. Read: After exactly 1 hour, measure the distance in millimeters (mm) from the top of the plasma to the top of the sedimented RBC column.
5. Normal Values (approx.): Men < 50 yrs: 0-15 mm/hr; Women < 50 yrs: 0-20 mm/hr.

7. Packed Cell Volume (PCV)

Principle: PCV (or haematocrit) measures the percentage of the total blood volume occupied by red blood cells. It is a primary test for diagnosing anaemia (low PCV) or polycythaemia (high PCV).

Microhaematocrit Method (Procedure):

1. Sample: Collect blood in a capillary tube (heparinised for finger-prick, plain for EDTA blood). Fill it to about 3/4 full.
2. Seal: Seal one end of the tube with clay sealant.
3. Centrifuge: Place the sealed tube in a special microhaematocrit centrifuge (sealed end facing outwards). Centrifuge at high speed (11,000-12,000 rpm) for 4-5 minutes.
4. Read: The blood separates into three layers: packed RBCs at the bottom, a thin "buffy coat" (WBCs and platelets) in the middle, and plasma at the top.
5. Measure: Use a microhaematocrit reader card. Align the bottom of the RBC column with the 0% line and the top of the plasma with the 100% line. Read the value at the top of the RBC layer. This is the PCV.
6. Normal Values (approx.): Males: 40-54%; Females: 36-48%.

Practical Component: Identification of Vials

Blood collection tubes are color-coded based on the additive (anticoagulant or clot activator) they contain.