2. PRINCIPLE AND APPARATUS-
• PRINCIPLE-SINCE NORMAL RBC COUNT RUNS IN MILLIONS, THE COUNT IS
MADE POSSIBLE BY DILUTING BLOOD SAMPLE BEFORE COUNTING AND
SUBSEQUENTLY MULTIPLING THE COUNT BY THE DILUTION FACTOR.
• APPARATUS- IMPROVED NEUBAUER’S CHAMBER, COMPOUND MICROSCOPE,
COVER SLIP, PRICKING APPARATUS, RBC PIPETTE, RBC DILUTING FLUID
(HAYEM’S FLUID) WHICH CONTAINS-
SODIUM SULPHATE (Na2SO4)- 2.5 gm TO PREVENT AGGREGATION OF RBCs
KNOWN AS ROULEAUX FORMATION.
3. APPARATUS
SODIUM CHLORIDE (NaCl)- 0.5 gm. IT HELPS MAINTAIN ISOTONICITY OF THE
FLUID THEREFORE RBCs REMAIN SUSPENDED IN THE SOLUTION.
MERCURIC CHLORIDE (HgCl2)- 0.25 gm. PRESERVATIVE, ANTIBACTERIAL AND
ANTIFUNGAL.
DISTILLED WATER- 100 ml (SOLVENT).
THE FINAL DILUTING FLUID FORMED IS ISOTONIC.
5. PROCEDURE
• CLEAN THE APPARATUS AND TAKE ADEQUATE AMOUNT OF RBC DILUTING
FLUID IN A WATCH GLASS.
• PRICK THE FINGER TAKING THE NECESSARY PRECAUTIONS AND OBTAIN
OPTIMAL BLOOD DROP SIZE.
• WIPE OFF THE FIRST DROP OF BLOOD. HOLD THE PIPETTE, GENTLY DIPPING IT’S
TIP IN THE DROP, SUCK THE BLOOD UPTO 0.5 MARK TAKING CARE THAT THERE
SHOULD BE NO AIR BUBBLES.
• EXCESS BLOOD SHOULD BE DISCARDED FROM PIPETTE BY TAPPING THE TIP
AGAINST THE PALM RATHER THEN STRIKING IT AGAINST HARD SURFACE, BY
BLOWING IT OUT OR BY USING COTTON OR FILTER PAPER. HOWEVER DO WIPE
OFF EXTRA BLOOD STICKING TO THE TIP.
7. PROCEDURE
• IMMIDIATELY AFTER THIS, HAYEM’S FLUID SHOULD BE SUCKED IN UPTO 101
MARK JUST ABOVE THE BULB. THE PIPETTE IS THE HELD HORIZONTAL BETWEEN
THE PALMS AND ROLLED GENTLY FOR ABOUT A MINUTE TO ENSURE THE
THOROUGH MIXING OF BLOOD AND FLUID GIVING A SUSPENSION OF RBC IN
BULB OF THE PIPETTE (1/200).
• COVER SLIP IS PLACED ON NEUBAUER’S CHAMBER OVER RULED AREA ON
BOTH THE SIDES.
• FOCUS RBC SQUARES OF NEUBAUER’S CHAMBER IN 10X AND THEN REMOVE
THE CHAMBER WITHOUT DISTURBING THE FOCUS.
9. PROCEDURE
• DISCARD FIRST FEW DROPS FROM PIPETTE AS THEY CONTAIN UNMIXED FLUID.
• CHARGING THE CHAMBER-
A SMALL DROP OF BLOOD MIXED WITH FLUID IS ALLOWED TO FORM AT THE TIP
OF THE PIPETTE AND THIS DROP IS GENTLY BROUGHT INTO CONTACT WITH THE
EDGE OF THE COVER SLIP AT AN ANGLE OF 45 DEGREES. THE FLUID IS DRAWN
IN THE PIPETTE BY CAPILLARY ACTION. BOTH SIDES OF NEUBAUER’S CHAMBER
ARE CHARGED.
10. PROCEDURE
• AN IDEALLY CHARGED CHAMBER IS ONE WHICH HAS BEEN CHARGED WITH A
SINGLE ADEQUATE SIZE DROP WHICH JUST FILLS THE CHAMBER WITHOUT
LEAVING ANY AIR BUBBLES. THIS IS LEARNT BY PRACTICE.
• IF THE FLUID OVERFLOWS INTO GUTTERS, IT IS CALLED OVER CHARGING. IF IT IS
INSUFFICIENT TO FILL THE CHAMBER OR HAVING AIR BUBBLES, IT IS SAID TO BE
UNDER CHARGED. IN CASE OF INADEQUADE CHARGING, CLEAN THE
CHAMBER WITH SOAP AND WATER AND CHARGE THE CHAMBER AGAIN.
• ALLOW SOME TIME FOR CELLS TO SETTLE DOWN IN THE COUNTING CHAMBER
SO THAT ALL THE CELLS ARE PRESENT IN THE SAME PLANE AND THEN BRING IT
UNDER 10X MICROSCOPE.
11. PROCEDURE
• USING ONLY FINE ADJUSTMENT, THE CELLS ALONG THE RULED AREA ARE BROUGHT
INTO FOCUS. UNIFORM DISTRIBUTION OF CELLS IN THE CHAMBER MUST BE ENSURED.
(A DIFFERENCE OF >20 RBCs AMONG MEDIUM SIZED RBC SQUARE INDICATES
UNEVEN DISTRIBUTION)
• THEN SWITCH OVER TO 40X AND COUNT RBCs IN 4 MEDIUM SIZED CORNER
SQUARES AND 1 MEDIUM SIZED CENTRAL SQUARE (1/5th
mm SIDE) i.e IN 80 SMALL
SQUARES (1/20 mm SIDE)
• RULES FOR COUNTING THE CELLS-
ANY CELL LYING ON UPPER OR LEFT BORDER OF THE SQUARE SHOULD BE
COUNTED IN THAT SQUARE AND LOWER AND RIGHT BORDER CELLS MUST NOT
BE COUNTED OR VICE VERSA. THIS RULE IS FOLLOWED TO AVOID COUNTING A CELL
TWICE. MIDDLE LINE IS CONSIDERED TO BE BORDER OF CELLS WITH TRIPLE LINES.
13. PROCEDURE
• COUNT THE RBCs in 4 MEDIUM SIZED CORNER SQUARES AND 1 MEDIUM
CENTRAL SQUARE. ENTER YOUR OBSERVATIONS IN THE CORRESPONDING
SQUARES.
• CALCULATIONS-
DILUTION FACTOR- 0.5 PART OF BLOOD AND 100.5 PART OF HAYEM’S FLUID
ARE PRESENT IN THE PIPETTE. AS BLOOD MIXES UP WITH THE DILUTING FLUID
ONLY IN BULB OF THE PIPETTE, THEREFORE ONE PART OF THE RBC FLUID WHICH
REMAIN IN THE STEM OF THE PIPETTE DOES NOT MIX WITH BLOOD. THUS 0.5
PART OF BLOOD MIXES WITH 99.5 PARTS OF DILUTING FLUID IN THE BULB OF THE
PIPETTE TO FORM 100 (0.5 + 99.5) PARTS OF SOLUTION.
14. PROCEDURE
• FINAL VOLUME ACHIEVED (100 PARTS) = 200 (DILUTION)
ORIGINAL VOLUME OF BLOOD TAKEN (0.5 PARTS)
CALCULATION OF VOLUME OF FLUID EXAMINED-
AREA OF MEDIUM SIZED RBC SQUARES= 5* 1/5* 1/5* = 1/5 sq.mm
DEPTH OF THE CHAMBER = 1/10 mm
HENCE, VOLUME OF FLUID OVER 5 RBC SQUARES = 1/5 sqmm * 1/10 sqmm =
1/50 microL.
15. PROCEDURE
• CALCULATION OF TOTAL RBC COUNT-
LET ‘N’ BE THE TOTAL NUMBER OF RBCs i.e IN1/50 microL OF DILUTED BLOOD.
THEREFORE NUMBER OF RBCs IN 1 MICROLITER OF UNDILUTED BLOOD
= N* 50* DILUTION FACTOR (200) = N* 10,000.
SOURCES OF ERROR- INSPITE OF UTMOST PRECAUTIONS, THE ERROR IN THE
COUNT IS +/- 20% BECAUSE OF THE FOLLOWING FACTORS-
PIPETTE ERROR, STATISTICAL ERROR (CAN BE AVOIDED BY
COUNTING MORE No. OF CELLS (E= 1/UNDERROOT OF n), CHAMBER ERROR, FIELD
ERROR.
16. PRECAUTIONS-
• PRICK SHOULD BE BOLD ENOUGH TO GIVE FREE FLOWING BLOOD.
• BOTH HEMOCYTOMETER AND COVER SLIP MUST BE DRY AND FREE OF GREASE.
• USE ONLY DRY PIPETTE.
• NEVER USE A BROKEN COVER SLIP.
• BEFORE CHARGING THE CHAMBER, THE FLUID IN THE STEM OF THE PIPETTE HAS
TO BE DISCARDED.
• THE COVER SLIP SHOULD BE PLACED SYMMETRICALLY SO AS TO COVER THE
RULED AREA COMPLETELY.
• THERE SHOULD BE NO UNDER OR OVER CHARGING OF THE CHAMBER
(COUNT WILL BE LOW IN BOTH CASES).
17. PRECAUTIONS-
• AFTER CHARGING THE CHAMBER, ALLOW SOME TIME FOR CELLS TO SETTLE
DOWN BUT THE COUNT SHOULD BE MADE BEFORE THE FLUID IN THE CHAMBER
STARTS TO DRY UP.
• WHILE COUNTING CELLS, THE STAGE OF THE MICROSCOPE SHOULD NOT BE
TILTED.
• COUNTING OF CELL SHOULD BE DONE WITHIN 30 MIN AFTER WHICH
DISCOLORATION OF CELLS BEGIN.
18. AUTOMATED METHODS
• THERE ARE 2 AUTOMATED METHODS-
IMPEDENCE COUNTING
LIGHT SCATTERING TECHNOLOGY
IMPEDENCE COUNTING-
RBCs ARE POOR CONDUCTORS OF ELECTRICITY WHERE AS CERTAIN DILUENTS
ARE GOOD CONDUCTORS. BLOOD IS HIGHLY DILUTED IN A BUFFERED
ELECTROLYTE SOLUTION. AN EXT VACUUM INITIATES MOVEMENT OF MERCURY
SIPHON WHICH CAUSES A MAJOR VOLUME OF SAMPLE TO FLOW THROUGH AN
19. AUTOMATED METHODS
- APERTURE TUBE OF SPECIFIC DIMENSION. BY MEANS OF A CONSTANT SOURCE
OF ELECTRICITY, A DIRECT CURRENT IS MAINTAINED B/W TWO ELECTRODES,
ONE IN THE SAMPLE BEAKER/ CHAMBER SURROUNDING THE APERTURE TUBE
AND THE OTHER INSIDE THE APERTURE TUBE.
WHEN A BLOOD CELL IS CARRIED THROUGH THE APERTURE, IT DISPLACES SOME
OF THE CONDUCTING FLUID AND INCREASES THE ELECTRICAL RESISTANCE. THIS
PRODUCES A CORRESPONDING CHANGE IN POTENTIAL BETWEEEN
ELECTRODES WHICH LASTS AS LONG AS THE RED CELLS PASS THROUGH THE
APERTURE.
THE HEIGHT OF THE PULSE PRODUCED INDICATES THE VOLUME OF THE CELLS
PASSING THROUGH. THE PULSES CAN BE DISPLAYED ON AN OSCILLOGRAPH
SCREEN. THE HEIGHT OF THE PULSES IS USED TO DETERMINE THE VOLUME OF THE
RED CELLS.
20. LIGHT SCATTERING TECHNOLOGY
RED CELLS ARE COUNTED BY MEANS OF ELECTRO-OPTICAL DETECTORS. A
DILUTED CELL SUSPENSION FLOWS THROUGH AN APERTURE SO THAT THE CELLS
PASS IN SINGLE FILE, IN FRONT OF THE LIGHT SOURCE. LIGHT IS SCATTERED BY
THE CELLS PASSING THROUGH THE LIGHT BEAM.
SCATTERED LIGHT IS DETECTED BY A PHOTOMULTIPLIER OR A PHOTODIODE
WHICH CONVERTS IT INTO ELECTRICAL IMPULSES WHICH ARE ACCUMULATED
AND COUNTED.
THE AMOUNT OF LIGHT SCATTERED IS PROPORTIONAL TO THE SURFACE AREA
AND THEREFORE THE VOLUME OF THE CELLS, SO THAT THE HEIGHT OF THE
ELECTRICAL PULSES CAN BE USED TO ESTIMATE THE CELL VOLUME.
27. DACIE’S FLUID
• IT CAN ALSO BE USED FOR COUNTING RBCs IN MICROSCOPY BY
HEMOCYTOMETERY.
IT CONSISTS OF-
• TRISODIUM CITRATE (3.13g) WHICH PREVENTS AGGREGGATION OF RBCs AND
PROVIDE CORRECT OSMOTIC PRESSURE.
• FORMALDEHYDE (1 ml) IT IS USED AS PRESERVATIVE.
• DISTILLED WATER (100 ml)- SOLVENT
