Methods of
Hematology

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3-8. SLIDE STAINING

a. Cover the slide completely with Wright's stain and allow it to remain on the smear for about 2 minutes to fix the blood cells. The stain should cover the slide but should not be allowed to overflow the edges; the stain must be replenished should it begin to evaporate.

b. Add an equal volume of Wright's stain buffer directly to the stain and blow the mixture gently to assure maximum mixing. Allow it to remain for about 4 minutes.

NOTE: The times recommended for staining and buffering are approximate and

should be adjusted with each fresh batch of stain to give the most satisfactory

results.

c. Using tap water, float off the mixture of stain and diluent from the slide to avoid the deposition of metallic scum on the smear. The scum appears after the addition of the buffer to the stain. Wash the slide thoroughly under cold, slowly-running water.

d. Air dry the smear and wipe the excess stain from the under surface of the slide.

e. Discussion.

(1) A properly prepared blood smear is margin-free; has no lines, ridges, or holes; is placed centrally on the slide; has an adequate thin area; and has a uniform distribution of leukocytes.

(2) It is preferable that blood smears not be made from blood containing anticoagulants since the leukocytes change their staining characteristics, develop vacuoles, engulf oxalate crystals, and show nuclear deformities. However, satisfactory slides are made with blood anticoagulated with EDTA.

(3) Avoid the following errors:

(a) Thick films made from an excess amount of blood placed on the slide.

(b) Delay in transferring the blood to the slide.

(c) A spreader slide that has damaged or unpolished ends.

(d) The use of dirty, dusty, greasy, or scratched slides.

(4) If slides cannot be stained immediately, they should be dried and then fixed in methyl alcohol for 30 minutes.

(5) In cases of marked leukopenia, smears can be prepared from the white cell layer ("huffy coat") obtained by centrifuging the blood slowly in a Wintrobe hematocrit tube at 500-800 rpm for 5 minutes.

(6) It is important that the blood film be completely dried before staining; otherwise the wet areas will wash off the slide.

(7) Protect blood slides from insects such as flies, cockroaches, etc. They can "clean" raw blood slides very rapidly.

(8) Protect slides from areas of high humidity. Excessive moisture tends to hemolyze red blood cells.

(9) Slides should be stained as soon as possible after preparation. White cells tend to become distorted and disintegrate very rapidly, thus causing considerable difficulty in identification.

(10) Very little actual staining takes place during the fixation stage. Most of the staining actually occurs during the buffering stage.

(11) During the buffering stage, it is important that only amounts of buffer equal to the stain be added, otherwise there is a tendency to over-dilute, causing the smear to stain weakly.

(12) After the staining is complete, do not blot the smear but air-dry it. To speed up the drying process, the smear can be placed in the heat of the substage light. It is important that the slide not be heated too intensely or too long since overheating tends to darken the staining reaction.

(13) A good quality smear should macroscopically pinkish-gray in hue. It should not be blue, green, or red. Microscopically, the red blood cells should be pink to orange and the white blood cells bluish if they display their true staining color.

(14) If the RBCs are bluish or green, this indicates that the stain is too alkaline. With an alkaline stain, the WBCs stain heavily and generally display fair distinguishing characteristics. However, the heavy stain masks any abnormalities of the RBCs. Heavy staining can be caused by:

(a) Blood smears which are too thick.

(b) Over-staining (prolonged buffer action).

(c) Evaporation of the methanol in the stain.

(d) Stain or diluent which is alkaline.

(e) Alkaline fumes.

(15) If the red blood cells are bright red, the stain is too acid. In this condition they stain well but the white blood cells (except eosinophilic granules) stain very poorly if at all. Thus, the stain is of no value for differential studies. ''Tendency toward acid staining is caused by:

(a) Incomplete drying before staining.

(b) Insufficient staining (insufficient buffer action).

(c) Overdilution of the stain with buffer.

(d) Prolonged washing of the slide after staining.

(e) Stain or buffer which is acid.

(f) Acid tunes.

(16) The staining reactions of blood are as follows:

Type of blood cell

or component

Good stain

Acid stain

Alkaline stain

Erythrocytes

All neclei

Eosinophilic granules

Pink to orange

Purple-blue

Granules red

Bright red

Pale blue

Brilliant red, distinct

Blue or green

Dark blue

Deep gray or blue

Neutrophilic Granules

Lymphocyte Cytoplasm

Violet-pink

Blue

Pale

Pale blue

Dark, prominent

(17) The technician should strive for a staining reaction that is neither too alkaline nor too acid. Such a stain gives good distinguishing features for all the cells of the blood system.

(18) If the staining reaction is excessively alkaline, this can be corrected by decreasing time of staining or neutralizing the stock stain solution with 1 percent acetic acid or 1 percent hydrochloric acid. Add the acid a drop at a time. Check the results after the addition of each drop of acid with trial slides.

(19) If the staining reaction is excessively acid, this may be corrected by increasing the time of staining or neutralizing the stock stain solution with 1 percent potassium bicarbonate or a weak solution of ammonia water. Add the ammonia water or potassium bicarbonate one drop at a time. Check the results on trial slides after the addition of each drop of the neutralizer.

(20) Staining reactions can also be varied by adjusting the proportions of disodium phosphate and potassium acid phosphate used in preparing the buffer. For example, increasing the proportion of disodium phosphate will make the buffer more alkaline; reducing it will make the buffer more acid.

(21) A poorly stained smear can sometimes be saved by washing rapidly with 95 percent alcohol, washing quickly in water, then restraining.


Curriculum design: David L. Heiserman
Publisher: SweetHaven Publishing Services

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