1. Blood smear
examination

2. Preparation of blood smear
There are three types of blood smears:
The cover glass smear.
The wedge smear .
The spun smear.
The are two additional types of blood smear used for specific purposes
Buffy coat smear for WBCs < 1.0×109/L
Thick blood smears for blood parasites .

3. WEDGE BLOOD SMEAR
Specimen : EDTA blood within 2 to 3 hours & collected to the mark on tube.
Not's : May change RBCs morphology such as crenated cells if :
Excessive amount of anticoagulant to specimen
Old blood: long standing.
Warm environment (room temperature) may hasten changes.

4. Procedure
placing a drop of blood from mixed sample on a clean glass slide.
Spreader slide using another clean glass slide at degree angle.
Control thickness of the smear by changing the angle of spreader slide
Allow the blood film to air-dry completely before staining. (Do not blow to dry, The moisture from your breath will cause RBC artifact).

6. large angle
low HCT
small angle
high HCT

8. CHARACTERISTICS OF A GOOD SMEAR
Thick at one end, thinning out to a smooth rounded feather edge.
Should occupy 2/3 of the total slide area.
Should not touch any edge of the slide.
Should be margin free, except for point of application.

9. tail body head

10. Common causes of a poor blood smear
Drop of blood too large or too small.
Spreader slide pushed across the slide in a jerky manner.
Failure to keep the entire edge of the spreader slide against the slide while making the smear.
Failure to keep the spreader slide at a 30° angle with the slide.
Failure to push the spreader slide completely across the slide.
Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide
Holes in film: Slide contaminated with fat or grease
Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

11. Slide fixation and
staining

12. Romanowsky staining Leishman's stain : a polychromatic stain
Methanol : fixes cells to slide
Methylene blue stains RNA, DNA
Blue-grey color
Eosin stains hemoglobin, eosin granules
Orange-red color
PH value of phosphate buffer is very important

13. Procedure Thin smear are air dried. Flood the smear with stain.
Stain for 1-5 min. Experience will indicate the optimum time.
Add an equal amount of buffer solution and mix the stain by blowing an eddy in the fluid.
Leave the mixture on the slide for min.
Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain.
Stand slide on end, and let dry in air.

14. Prolonged buffering or washing Old stain
CAUSES & CORRECTION
Too Acid Stain:
Insufficient staining time
Prolonged buffering or washing
Old stain
Correction:
Lengthen staining time
Check stain and buffer pH
Shorten buffering or wash time

15. Alkaline pH of stain components
Too Alkaline Stain:
Thick blood smear
Prolonged staining
Insufficient washing
Alkaline pH of stain components
Correction :
Check pH
Shorten stain time
Prolong buffering time

16. Manual differential

17. Principle White Blood Cells Perform the differential count.
Check for even distribution and estimate the number present.
Perform the differential count.
Examine for morphologic abnormalities.

18. Check the WBC distribution over the smear.
Observations Under 10X
Check to see if there are good counting areas available free of ragged edges and cell clumps.
Check the WBC distribution over the smear.
Check that the slide is properly stained.
Check for the presence of large platelets, platelet clumps, and fibrin strands.

19. Observing direction:
Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction
*avoid repeat or miss some cells

20. WBC estimation Under 40X Using the × 40 high dry with no oil.
Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs.
Count 10 fields, take the total number of white cells and divide by 10.
To do a WBC estimate by taking the average number of white cells and multiplying by 2000.

21. Reference values vary depending on age
14 yr
Total WBC x 103 /µL
Cell Type
50-65
Neutrophils %
30-40
Lymphocyte %
0-10
Monocyte %
0-4
Eosinophil %
0-1
Basophil %

22. Morphology of WBC

23. Normal blood smear

25. Stab neurophile Diameter:12-16 Cytoplasm : pink
Granules: primary, secondary
Nucleus: dark purple blue
Dense chromatin

27. Segmented neurophile Diameter: 12-16 Cytoplasm : pink
Granules: primary, secondary
Nucleus: dark purple blue
Dense chromatin
2-5 lobes

28. SEGMENTED NEUTROPHIL

29. Basophils Diameter: 14-16 Cytoplasm : full of granules
Granules: large refractile, orange-red
Nucleus: blue
Dense chromatin
2 lobes like a pair of glass

30. EOSINOPHIL

31. Eosinophils Diameter: 14-16 Cytoplasm : pink
Granules: dark blue –black obscure nucleus
Nucleus: blue

32. BASOPHIL

33. Lymphocyte Diameter: small 7-9 large 12- 16 Cytoplasm: medium blue
Granules: small agranular large a few
Nucleus: dark blue \round dense chromatin

34. LYMPHOCYTE

35. Monocytes Diameter: 14-20 Cytoplasm : grey blue
Granules: dust-like lilac color granules
Nucleus: blue
large irregularly shaped and folded

36. MONOCYTE

37. LEFT-SHIFT AND RIGHT-SHIFT OF NEUTROPHIL:
Left-shift: non-segmented neutrophil > 5%
(Increased bands Means acute infection, usually bacterial).
Right-shift: hypersegmented neutrophil >3% (Increased hypersegmented neutrophile )

38. Leukocytosis, a WBC above 10,000, is usually due to an increase in one of the five types of white blood cells
Neutrophilic leukocytosis neutrophilia
Lymphocytic leukocytosis lymphocytosis
Eosinophilic leukocytosis eosinophilia
Monocytic leukocytosis monocytosis
Basophilic leukocytosis basophilia