Blood Smear Examination Lab 19 Making Blood Smear

Blood Smear Examination - Lab 19 - Making Blood Smear

I- Preparation Of Blood Smear ◊ There are three types of blood smears: 1. The cover glass smear. 2. The wedge smear. 3. The spun smear. ◊ The are two additional types of blood smear used for specific purposes 1. Buffy coat smear for WBCs < 1. 0× 109/L 2. Thick blood smears for blood parasites.

Wedge Blood Smear ◊ Specimen EDTA blood within 2 to 3 hours & collected to the mark on tube. ◊ Note May change RBCs morphology such as Speculated (crenated) cells if : 1. Excessive amount of anticoagulant to specimen. 2. Old blood - long standing. 3. Warm environment (room temperature) may hasten changes.

Procedure § placing a drop of blood from mixed sample on a clean glass slide. § Spreader slide using another clean glass slide at 30 -40 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. )

Steps For Making Blood Film

Procedural Notes 1. Characteristics of a good smear 1. Thick at one end, thinning out to a smooth rounded feather edge. 2. Should occupy 2/3 of the total slide area. 3. Should not touch any edge of the slide. 4. Should be margin free, except for point of application. 2. 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.

3. The Thickness Of The Spread 1. If the hematocrit is increased, the angle of the spreader slide should be decreased. 2. If the hematocrit is decreased, the angle of the spreader slide should be increased

4. Common Causes Of A Poor Blood Smear 1. Drop of blood too large or too small. 2. Spreader slide pushed across the slide in a jerky manner. 3. Failure to keep the entire edge of the spreader slide against the slide while making the smear. 4. Failure to keep the spreader slide at a 30° angle with the slide. 5. Failure to push the spreader slide completely across the slide. 6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide 7. Holes in film: Slide contaminated with fat or grease 8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

Examples Of Unacceptable Smears

5. Biologic Causes Of A Poor Smear 1. Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear. 2. Lipemia - holes will appear in the smear. There is nothing you can do to correct this. 3. Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this.

6. 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. A. B. C. D. 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.

Slide Fixation And Staining Leishman's Stain

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.

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.

III. Staining the film Romanowsky Stains are universally employed for staining blood films and are generally very satisfactory. Ø There a number of different combinations of these dyes, which vary, in their staining characteristics. 1. May-Grunwald-Giemsa is a good method for routine work. 2. Giemsa stain is thought to produce more delicate staining characteristics. «Wright's stain is a simpler method «. 3. 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). 4. Field's stain is a rapid stain used primarily on thin films for malarial parasites.

Principle Ø 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. § p. H value of phosphate buffer is very important.

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 10 -15 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.

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

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

Procedures v Observations Under × 10 1. Check to see if there are good counting areas available free of ragged edges and cell clumps. 2. Check the WBC distribution over the smear. 3. Check that the slide is properly stained. 4. Check for the presence of large platelets, platelet clumps, and fibrin strands. 5. Check the RBCs distribution & shape. v Observations Under × 100 ü 100 WBCs are counted using the zigzag method. This method of counting is done by going back and forth lengthwise or sidewise.

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

Normal Peripheral Blood Smear