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1. DIFFERENTIAL LEUCOCYTE
COUNT (PY-2.11)
BY,
Dr. NEETIKA SHARMA
S.R PHYSIOLOGY
FELLOWSHIP CLINICAL CARDIOLOGY

2. APPARATUS
• 4-5 GLASS SLIDES, PRICKING APPARATUS, COMPOUND MICROSCOPE, CEDAR
WOOD OIL AND LEISHMAN’S STAIN.
• LEISHMAN’S STAIN-
IT BELONGS TO ROMANOWOSKY GROUP OF STAINS WHICH CONTAINS AN
ACIDIC AND BASIC DYE. IT HAS FOLLOWING PROPERTIES-
1) TO MAKE CLEAR DISTINCTION IN SHADES OF STAINING.
2) TO STAIN GRANULES DIFFERENTIALLY.

3. APPARATUS
• 1. EOSIN- IT IS AN ACIDIC DYE (NEGATIVELY CHARGED) AND STAINS BASIC
POSITIVELY CHARGED PARTICLES NAMELY, RBCs AND GRANULES OF
EOSINOPHILS….
• 2. METHYLENE BLUE- IT IS A BASIC DYE (POSITIVELY CHARGED) AND STAINS
NEGATIVELY CHARGED PARTICLES LIKE CYTOPLASM,
NUCLEI OF WBCs AND GRANULES OF BASOPHILS.
• 3. ACETONE FREE OF METHYL ALCOHOL- IT IS A FIXATIVE i.e –
a) IT PRESERVES THE CELLS IN WHATEVER CHEMICAL AND METABOLIC
STATES
THEY ARE AT THE TIME OF STAINING.

4. APPARATUS
• b ) THE BLOOD SMEAR ALSO GETS FIXED TO THE SLIDE (PRECIPITATION OF
PROTEIN IN IT, BY ALCOHOL) SO THAT IT MAY NOT BE WASHED OFF.
METHYL ALCOHOL SHOULD BE ACETONE FREE SINCE ACETONE PRODUCES THE
SHRINKAGE OF CELLS AND MAY EVEN PRODUCE CRENATION (INDENTED EDGE)
AND LYSIS OF THE CELLS. IT SHOULD ALSO BE COMPLETELY WATER FREE SINCE
WATER MAY AFFECT THE FILM BY CAUSING ROULEAUX FORMATION.

5. PROCEDURE
• A. PREPARATION OF BLOOD SMEAR
1. TAKE 4-5 CLEAN GREASE FREE GLASS SLIDES WITH SMOOTH EDGES, 3-4
TO
BE COVERED WITH BLOOD FILM AND 1 TO BE USED AS SPREADER.
2. LAY 3-4 GLASS SLIDES ON A TABLE, PRICK THE FINGER, TOUCH THE
BLEEDING POINT LIGHTLY IN THE CENTRE LINE ABOUT 1 OR 2 CM FROM
ONE END OF THE SLIDES TO OBTAIN A SMALL DROP OF BLOOD ON
EACH SIDE.

6. PROCEDURE
• 3. PLACE THE NARROW EDGE OF THE SPREADER ON THE SURFACE OF THE
SLIDE JUST IN FRONT OF BLOOD DROP AT AN ANGLE OF 45 DEGREE. DRAW
THE SPREADER BACKWARD SO AS TO TOUCH THE BLOOD DROP AND HOLD
THERE TILL THE BLOOD RUNS ALONG THE WHOLE WIDTH OF THE SPREADER
AT THE LINE OF JUNCTION.
• 4. THE SPREADER IS THEN MOVED SLOWLY AND SMOOTHLY TO THE OTHER END
OF THE SLIDE MAINTAINING AN ANGLE OF 45 DEGREE.

8. PROCEDURE
• A PROPERLY MADE BLOOD FILM WILL BE FAIRLY UNIFORM WITHOUT UNEVEN
STREAKS OR SPOTS/VACOULES IN IT AND NEITHER TOO THICK OR TOO THIN.
• ALLOW THE FILM TO DRY IN AIR. REPEAT THE PROCEDURE WITH THE OTHER
SLIDES.
ROULEAUX FORMATION IS USUALLY SEEN IN VARYING DEGREES IN THE WET
PREPARATION IN OF WHOLE BLOOD.

9. PROCEDURE
• CRITERIA OF A GOOD BLOOD SMEAR-
1. IT SHOULD COVER ALMOST ENTIRE WIDTH AND IS ABOUT 3-4 CM OR 3/4th
THE
LENGTH OF THE SLIDE.
2. IT IS TONGUE SHAPED (BROAD BASE) WITH NO TAILS AT THE END.
3. IT IS OF UNIFORM THICKNESS i.e IT IS NEITHER TOO THICK NOR TOO THIN. (A
THICK SMEAR WHEN PLACED ON WHITE BACKGROUND, APPEARS RED.)
4. THERE ARE NO LONGITUDINAL OR CROSS STRIATIONS OR AIR GAPS IN THE
BLOOD SMEAR.

10. 11

11. PROCEDURE
• B. FIXING AND STAINING OF BLOOD SMEAR-
i) FIXING THE BLOOD SMEAR
1) PLACE THE BLOOD SMEARS ACROSS TWO OF THE PARALLEL SUPPORTS ON
THE GLASS RODS, SO THAT THE SLIDES MAY BE HORIZONTAL.
2) POUR 8-12 DROPS OF LEISHMAN’S STAIN ON EACH SLIDE, JUST ENOUGH
TO
COVER BLOOD SMEAR. NOTE THE TIME. LEAVE IT FOR ABOUT 2 MIN. IT IS
CALLED FIXATION TIME.

12. PROCEDURE
• 1. NOTE THE NUMBER OF LEISHMAN’S STAIN DROPS POURED.
• 2. IN HOT WEATHER IT IS TO BE OBSERVED THAT THE STAIN SHOULD NOT
BECOME THICK DUE TO EVAPORATION OF ALCOHOL. IF IT OCCURS, THE
STAIN PRECIPITATE ON THE FILM. THIS CAN BE AVOIDED BY ADDING MORE
STAIN TO THE ONE ALREADY ON THE SLIDE.

13. PROCEDURE
• ii) STAINING BLOOD SMEAR
1) AT THE END OF 2 MINUTES, ADD AN EQUAL NUMBER OF DROPS OF
BUFFERED WATER (pH 6.8) OVER THE STAIN. TAKE CARE THAT THE WATER IS
NOT SPILLED OVER.
DOUBLE THE NUMBER OF DROPS OF DISTILLED WATER MAY BE USED IN CASE
OF NON AVAILABILITY OF BUFFERED WATER.

14. PROCEDURE
• 2) MIX THE STAIN AND WATER EVENLY BY GENTLY BLOWING AIR
INTERMITTENTLY WITH THE HELP OF A DROPPER.
• 3) IF THE DILUTION OF THE STAIN IS CORRECT, THE FLUID WILL BE COVERED BY
THIN GREENISH SCUM (i.e DIRTY SUBSTANCE). LEAVE THE STAIN TO ACT FOR
10 MIN. IT IS CALLED STAINING TIME.
• 4) AT THE END OF 10 MIN, POUR OFF THE STAIN, HOLD THE SLIDE IN A SLANTING
POSITION BELOW THE TAP AND THE WATER IS ALLOWED TO FLOW THROUGH
THE SMEAR.
• 5) WASH THE SLIDE UPTO 2 MIN WITH TAP WATER GENTLY AND THOROUGHLY
TILL THE FILM GETS A PINKISH TINGE. CONT..

17. PROCEDURE
• i) NONE OF THE GREENISH SCUM SETTLES ON THE SURFACE OF THE BLOOD
FILM.
• ii ) WATER FILM SHOULD NOT STRIKE THE BLOOD SMEAR DIRECTLY OR ELSE IT
MAY BE WASHED OFF.
• 5) WIPE CLEAN THE BACK OF THE SLIDE AND SET IT UPRIGHT TO DRY.

18. PROCEDURE
• C) ASSESSMENT OF THE QUALITY OF THE STAINED SMEAR-
CHECK THE STAINING AND THICKNESS OF THE BLOOD SMEAR BY EXAMINIG
IT FIRST UNDER LOW POWER OBJECTIVE AND THEN UNDER HIGH POWER
OBJECTIVE. A WELL STAINED BLOOD SMEAR HAS FOLLOWING
CHARACTERSTICS:
1) THE SMEAR IS SINGLE CELL THICK, WITH NOT MUCH OVERLAPPING OF THE
CELLS AND THE CELLS ARE UNIFORMLY DISTRIBUTED.
2) AT LEAST 1 WBC IS SEEN/HIGH POWER FIELD

19. PBS AT 10X, 40X, OIL IMMERSION 100X

20. PROCEDURE
• 3) THE RBCs ARE STAINED LIGHT PINK.
• 4) IN AN UNDERSTAINED SMEAR, RBCs APPEAR VERY PALE AND WBCs LOOK
ALMOST COLORLESS.
• 5) IN AN OVERSTAINED SMEAR, RBCs LOOK BLUISH BLACK AND WBCs WILL
TAKE UP MORE METHYLENE BLUE. HENCE WBCs LOOK TOTALLY PURPLE.

21. UNDER AND OVERSTAINED SLIDE

22. PROCEDURE
IF SMEAR IS UNDERSTAINED, ADD THE STAIN AND REPEAT THE PROCESS FOR A
FEW MINUTES. IF IT IS OVERSTAINED THEN WASH THE SLIDE FOR A FEW MINUTES.
D) EXAMINATION OF THE STAINED SMEAR UNDER OIL IMMERSION
THE IDENTIFICATION OF DIFFERENT TYPES OF BLOOD CELLS IS DONE UNDER OIL
IMMERSION. HOW?
1. PLACE A DROP OF CEDAR WOOD OIL ON THE BLOOD FILM. SWING THE OIL
IMMERSION OBJECTIVE INTO POSITION. CAREFULLY LOWER THE OBJECTIVE
UNTIL IT JUST TOUCHES THE OIL DROP. NOW SLIGHTLY RAISE THE OBJECTIVE
AND FOCUS THE CELLS USING FINE ADJUSTMENT ONLY.

24. PROCEDURE
• 2. STUDY THE DISTRIBUTION AND CHARACTERSTICS OF DIFFERENT TYPES OF
CELLS FROM DIFFERENT PARTS OF THE SMEAR.
i) THE MOST ABUNDANT CELL TYPE FOUND ANYWHERE IN THE SMEAR IS RBCs.
THESE ARE SEEN AS LIGHT PINK, NON NUCLEATED DISCS OF UNIFORM SIZE
AND SHAPE.
ii ) AT THE HEAD END OF THE SMEAR THE RBCs ARE CROWDED AND
SUPERIMPOSED AND THE WBCs ARE POORLY STAINED.
iii ) AT THE EXTREME TAIL END, THE CELLS ARE WIDE APART AND WBCs ARE
DISTORTED.

25. PROCEDURE
• iv) AT THE UPPER AND THE LOWER EDGES OF THE SMEAR, THE WBCs ARE
FOUND IN PLENTY BUT ARE POORLY STAINED AND ABNORMALLY RICH IN
GRANULOCYTES.
IRREGULARITY IN THE DISTRIBUTION OF WBCs IS THE RULE; NEUTROPHILS AND
MONOCYTES PREDOMINATE AT THE MARGINS AND THE TAIL, AND
LYMPHOCYTES IN THE MIDDLE OF THE FILM.

27. PROCEDURE
• E) IDENTIFICATION OF DIFFERENT TYPES OF LEUCOCYTES-
THE IDENTIFICATION AND COUNTING OF DIFFERENT TYPES OF LEUCOCYTES
ARE DONE UNDER OIL IMMERSION OBJECTIVE. THE WBCs CAN BE
DIFFERENTIATED FROM RBCs BY THE PRESENCE OF NUCLEUS AND LARGE SIZE.
WHILE IDENTIFYING THE WBCs, FOLLOWING FACTORS NEEDS TO BE KEPT IN
MIND-
1) SIZE OF THE CELLS (COMPARE WITH THE SURROUNDING RBCs WHICH ARE OF
UNIFORM SIZE, 7.2 µm)
2) FEATURES OF NUCLEUS- COLOR, No. OF LOBES.

28. PROCEDURE
• 3) FEATURES OF CYTOPLASMIC GRANULES ( PINK OR BLUE, FINE OR COARSE).
• 4) NUCLEAR : CYTOPLASM RATIO.
• F) COUNTING OF DIFFERENT TYPES OF LEUCOCYTES-
• A MINIMUM OF 100 WBCs ARE IDENTIFIED IN A SYSTEMIC MANNER AND
COUNTING IS MADE USING THE TALLY-BAR METHOD. IDEALLY ALL THE CELLS
SHOULD BE COUNTED IN A SINGLE STRIP RUNNING THE LENGTH OF THE SMEAR,
PROCEEDING FROM BASE TO APEX. USE THE STAGE TO TRAVERSE THE FULL
LENGTH OF THE SMEAR. MOVE THE SLIDE ALONG VERTICALLY BY A DISTANCE
EQUIVALENT TO 2 mm AND AGAIN TRAVERSE THE FULL LENGTH OF THE FILM.

29. BATTLEMENT

30. PROCEDURE
• MOVE THE SLIDE ALONG VERTICALLY BY A DISTANCE EQUIVALENT TO 2 mm
AND AGAIN TRAVERSE THE FULL LENGTH OF THE FILM, THIS TIME IN THE
OPPOSITE DIRECTION. THIS METHOD ENSURES THAT CELLS ARE NOT COUNTED
MORE THEN ONCE.
• IN PRACTICE, A 200 CELL COUNT IS RECOMMENDED AS A ROUTINE
PROCEDURE.
• KEEP PUTTING A VERTICAL BAR AGAINST A CELL IDENTIFIED, THE 5th
BAR
SHOULD BE MARKED OBLIQUELY SO AS TO CUT ALL PREVIOUS 4 VERTICAL
BARS. THIS WILL LEAVE YOU WITH A BUNDLE OF FIVE FOR EASY CALCULATION
AT THE END.

39. ABSOLUTE COUNT

40. PRECAUTIONS
• THE SLIDES SHOULD BE CLEAN AND GREASE FREE, THEREFORE, THESE SHOULD
BE CLEANED THOROUGHLY WITH SOAP AND WATER IMMIDIATELY BEFORE USE.
• THE GLASS SPREADER SHOULD BE CAREFULLY SELECTED. IT SHOULD HAVE A
SMOOTH AND CLEAN EDGE. IF A ROUGH EDGED SPREADER IS USED, MANY
OF THE LEUCOCYTES ACCUMULATE AT THE EDGES AND IN THE TAIL OF THE
SMEAR. ALSO, A GROSS QUALITATIVE IRREGULARITY IN DISTRIBUTION OF CELLS
OCCUR.

41. PRECAUTIONS
• MAKE ABOUT 4-5 BLOOD FILMS SO THAT AT LEAST 1 MAY BE SATISFACTORY.
• MARK THE SMEARED SURFACE WITH GLASS MARKING PENCIL.
• DO NOT HEAT DRY OR BLOT DRY THE SMEAR.
• STORED BLOOD SHOULD NOT BE USED TO MAKE THE SMEAR.
• SMEAR SHOULD’T BE STAINED AFTER 2 HOURS.
• ASSESS THE QUALITY OF THE SMEAR, BOTH GROSSLY AND MICROSCOPICALLY
BEFORE STAINING IT.
• ONLY A PROPER WELL STAINED SLIDE SHOULD BE EXAMINED UNDER OIL
IMMERSION LENS.

42. MANUAL QUESTIONS

43. ANSWER-1
1. ROULEAUX FORMATION IS USUALLY SEEN IN VARYING DEGREES IN THE WET
PREPARATION IN WHOLE BLOOD.

44. ANSWER-2
2. SLIDE IS CLEANED WITH SOAP AND WATER.
ADDITIONAL INFORMATION-

45. ANSWER-3
• 3. MAKE ABOUT 4-5 BLOOD FILMS SO THAT AT LEAST 1 MAY BE SATISFACTORY.

46. ANSWER-4 AND 8
• RBCS, PLATELETS, GRANULOCYTES, AGRANULOCYTES. BASOPHIL MAY BE
RARE.

47. ANSWER-5
5. INITIAL 2 MIN IS FIXATION PERIOD. ALCOHOL PRECIPITATES THE PLASMA
PROTEINS WHICH ACTS AS GLUE AND ATTACH/FIX THE BLOOD CELLS ON THE
GLASS SLIDE AND IT MAINTAINS MORPHOLOGY AND CHEMICAL
CHARACTERSTICS AS NEAR THE LIVING STATE AS POSSIBLE. THE CELLS ARE NOT
STAINED DURING THIS TIME BECAUSE THE STAIN PARTICLES CANNOT ENTER THE
CELLS IN THEIR UNIONIZED STATE. THEIR IONIZATION OCCURS ONLY WHEN
WATER IS ADDED TO THE SALTS IN THE UNDILUTED STAIN. (IF DILUTED STAIN IS
ADDED TO BEGIN WITH, THE BLOOD SMEAR ITSELF WOULD BE WASHED AWAY.
DILUTED LEISHMAN’S STAIN CAN BE USED IF THE BLOOD FILM IS FIRST FIXED IN
ABSOLUTE ALCOHOL).

48. ANSWER-6
• 6. BUFFERED WATER HAS pH OF 6.8. IT IS A PHOSPHATE BUFFER. AT THIS pH,
THERE IS OPTIMAL IONIZATION OF THE STAIN PARTICLES SO THAT THEY CAN
PENETRATE THE CELL BETTER.
• DISTILLED WATER IN DOUBLE THE AMOUNT CAN BE USED IN CASE BUFFERED
WATER IS NOT THERE BUT TAP WATER IS USUALLY NOT RECOMMENDED.

49. ANSWER-7
• 7. METHYL ALCOHOL SHOULD BE ACETONE FREE SINCE ACETONE PRODUCES
THE SHRINKAGE OF CELLS AND MAY EVEN PRODUCE CRENATION (INDENTED
EDGE) AND LYSIS OF THE CELLS. IT SHOULD ALSO BE COMPLETELY WATER FREE
SINCE WATER MAY AFFECT THE FILM BY CAUSING ROULEAUX FORMATION.

50. ANSWER-9

51. ANSWER-10

52. CONT..

53. CONT..

54. CONT..

55. CONT..

56. CONT..

57. LYMPHOCYTES

58. CONT..

59. ANSWER-11

60. CONT……

61. CONT..

62. CONT…

63. DISCUSSION
ADDITIONAL RELEVANT POINTS

69. THANKS
BEST/WARM WISHES
HAVE ENJOYABLE LEARNING
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