Leukocyte Count – denotes the number of WBC per 1L of WB (use to follow progress of disease and therapies)

1. Collect blood (EDTA or Heparinized blood)

2. Suck blood to mark “0.5” then wipe the outer part of the pipet
3. Suck Diluting fluid to mark “11”
4. Mix for 3 minutes before charging (shaker/by hand)
5. Charge in the counting chamber (Discard 3-5 drops before charging, because it is cell free)
DO NOT OVERCHARGE!
6. Stand for 1 minute then count ASAP to prevent evaporation
7. Count Cells in the 4 CORNER SQUARES at LPO
8. Calculate:

Erythrocyte Count:
1. Collect blood (EDTA or Heparinized blood)
2. Suck blood in mark “0.5”, and then suck diluting fluid to mark “101”.
3. Mix thoroughly then discard few drops
4. Charge in the counting chamber (DO NOT OVERCHARGE)
5. Stand for 1-2min then count ASAP to prevent evaporation
6. Count cells in the 5 INTERMEDIATE SQUARES at HPO
7. Calculate:

Factors RBC WBC Errors:

Area Correction factor 5 ¼ - Failure to mix properly and presence of bubbles
Depth Correction factor 10 10 - Contaminated diluents
Dilution correction factor 200 20 - Drawing blood too far past the dilution mark

Hematocrit – after centrifugation, RBCs will settle at bottom, the volume of RBC that settled is “PACKED CELL VOLUME”
- Hematocrit is the ratio of volume occupied by RBCs to volume of WB

Microhematocrit: “Adam’s” Red band: with Heparin Spun HcT:

Tube – approx. 75mm long (7-7.5cm) Blue band: w/o Heparin 1st layer – fatty layer
Inner bore – 1.2mm (double anticoagulation is not allowed) 2nd Layer – Plasma
Capacity – 0.05mL of WB 3rd layer – buffy coat
Volume – 2/3 of the tube should be Males: 40-54% Bottom – PRBC
filled (0.05mL) or 5cm of the tube Females: 37-47%
- Start at colored band area Birth: 45-60%
Plug – at least 4mm long (4-6mm)
- Clay then paraffin wax
- Inserted at end with colored band
Labelling – masking tape/micropore
Anticoagulant – heparin
Specimen – EDTA/Capillary blood

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Erythrocyte Sedimentation Rate:
 Non specific for inflammation. Stages of ESR:
 Rate of fall/RBCs settling to the bottom of the tube (in 1 hour) 1.LAG PHASE=
- Formation of ROULEAUX (1st 10 mins)
2. DECANTATION PHASE=
- more RAPID and constant
sedimentation (next 40 mins)
3. END OF SEDIMENTATION=
- SLOW bec. RBCs are already at the
bottom of the tube (last 10 mins.)
Factors that influence Sedimentation:
Intrinsic factors Extrinsic factors
- Number of RBCs/mm3= inversely proportional to ESR - Length of the tube= Longer  Faster ESR
- Size of RBC = directly proportional to ESR - Diameter of the tube= 2mm to 3.75mm
- Shape of RBCs= abnormal/irregular shapes hinder  < 2mm= Slower ESR
rouleaux formation therefore leads to SLOWER ESR.  >3.75mm= Faster ESR
- Plasma protein contents - Temperature= 22-27C
 ALBUMIN & LECITHIN = Slower ESR  <22C= Slower ESR
 GLOBULIN, FIBRINOGEN & CHOLESTEROL=  >27C= Faster ESR
Faster ESR - Inclination of the tube (faster)=A tilt of 3 degrees =30%
- Plasma viscosity= inversely proportional to ESR error
 MORE VISCOUS= Slower ESR - Wet Glasswares= will cause hemolysis  faster ESR
 LESS VISCOUS= Faster ESR - Bubbles= less blood- Faster ESR
- Higher conc. Of AC= Slower ESR (RBC shrinks)
- Delay= must be done within 1-2 hrs. (>2 hrs= slow ESR)

Westergren – ICSH recommended

Rgt: 3.8% Na Citrate (1:4) Male:
<50y/o = 0-15mm
Length: 30cm (300mm +/- 0.5mm) >50y/o= 0-20mm
External bore: 2.5mm (+/-0.5mm) Female:
Internal bore: 2.65mm (+/- 0.15mm) <50y/o = 0-20mm
Grad up to 200 >50y/o = 0-30mm
Kids = 0-10mm

INCREASED ESR NORMAL ESR (slow)

- Inflammatory conditions - Polycythemia
- Acute & Chronic Infection - Spherocytes
- Rheumatic fever - Sickle cells
- Rheumatoid Arthritis - Sickle cell anemia
- Myocardial infarction - Hereditary Spherocytosis
- Leukemia - Acute allergies
- Nephrosis, severe anemia - Peptic ulcer
- Tuberculosis, Hepatitis - Undulant fever
- Multiple Myeloma - Typhoid fever
- Waldenstrom’s macroglobulinemia - Pertussis
- Internal hemorrhage Malarial paroxysm
- Subacute bacterial endocarditis
- Menstruation
- Pregnancy- after 3rd month
- Ulcerative colitis