1. Introduction To Medical Technology
- Lab 19 -
Blood Smear Examination
Making Blood Smear

2. I- 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. Specimen Note Wedge Blood Smear
EDTA blood within 2 to 3 hours & collected to the mark on tube.
Note
May change RBCs morphology such as Speculated (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.)

5. Steps For Making Blood Film

6. Characteristics of a good smear
Procedural Notes
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.
As soon as the drop of blood is placed on the glass slide, the smear should be made without delay. Any delay results in an abnormal distribution of the white blood cells, with many of the large white cells accumulating at the thin edge of the smear.

7. The Thickness Of The Spread
If the hematocrit is increased, the angle of the spreader slide should be decreased.
If the hematocrit is decreased, the angle of the spreader slide should be increased

8. 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.

9. Examples Of Unacceptable Smears

10. Biologic Causes Of A Poor Smear
Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear.
Lipemia - holes will appear in the smear. There is nothing you can do to correct this.
Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this.

11. Although this is the easiest and most popular methods for producing a blood smear, it does not produce a quality smear as a result of.
The WBCs are unevenly distributed and RBC distortion is seen at the edges.
Smaller WBCs such as lymphocytes tend to reside in the middle of the feathered edge.
Large cells such as monocytes, immature cells and abnormal cells can be found in the outer limits of this area.
Spun smears produce the most uniform distribution of blood cells.

12. Slide Fixation And Staining
Leishman's Stain

13. II- Fixing the films
To preserve the morphology of the cells, films must be fixed as soon as possible after they have dried.
Methyl alcohol (methanol) is the choice, although ethyl alcohol ("absolute alcohol") can be used.
To fix the films, place them in a covered staining jar or tray containing the alcohol for 2-3 minutes. In humid climates it might be necessary to replace the methanol 2-3 times per day; the old portions can be used for storing clean slides.

14. Notes
It is important to prevent contact with water before fixation is complete. The presence of water during methanol fixation produces refractile body artifacts (water spots) in the erythrocytes.
These water spots persist through staining of the smear and cover items of interest in the smear. Further, they are distracting to the person evaluating the smear. In some cases, the water spots may interfere with diagnosis.
To prevent the alcohol from becoming contaminated by absorbed water, it must be stored in a bottle with a tightly fitting stopper and not left exposed to the atmosphere, especially in humid climates.

15. III. Staining the film
Romanowsky Stains are universally employed for staining blood films and are generally very satisfactory.
There are a number of different combinations of these dyes, which vary, in their staining characteristics.
May-Grunwald-Giemsa is a good method for routine work.
Giemsa stain is thought to produce more delicate staining characteristics.»Wright's stain is a simpler method».
Leishman's is also a simple method, which is especially suitable when a stained blood film is required urgently or the routine stain is not available (e.g. at night).
Field's stain is a rapid stain used primarily on thin films for malarial parasites.

16. Principle The main components of a Romanowsky stain are:
A cationic or basic dye (methylene blue or its oxidation products such as azure B), which binds to anionic sites and gives a blue-grey color to nucleic acids (DNA or RNA), nucleoproteins, granules of basophils and weakly to granules of neutrophils
An anionic or acidic dye such as eosin Y or eosin B, which binds to cationic sites on proteins and gives an orange-red color to hemoglobin and eosinophil granules.
pH value of phosphate buffer is very important.

17. Staining Procedure (Leishman’s Stain)
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 center of the slide to prevent a residue of precipitated stain.
Stand slide on end, and let dry in air.

18. Staining Characteristics Of A Correctly Stained Normal Film
Nuclei Dark Purple
Cytoplasm
Erythrocytes Reddish-Brown
Neutrophils Orange-pink
Lymphocytes Blue; some small lymphocytes deep blue
Monocytes Grey-blue
Basophils Blue
Granules
Neutrophils Fine purple
Eosinophils Red-orange
Basophils Purple-black
Monocytes Fine reddish (azurophil)
Platelets Purple

19. Band Neutrophil
Segmented Neutrophil
Eosinophil
basophil
lymphocyte
Monocyte
platelets
Red Blood Cells

20. Observations Under ×10 Observations Under × 100 Procedures
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.
Check the RBCs distribution & shape.
Observations Under × 100
100 WBCs are counted using the zigzag method. This method of counting is done by going back and forth lengthwise or sidewise.

21. *avoid repeat or miss some cells
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

22. Normal Peripheral Blood Smear

23. Thank You