2. PS most important, valuable, frequently asked Ix in Hemat
Labs.
It provides information on:
RBC morphology:- Diagnose anemia.
WBC disorders and DLC.
Platelet number and morphology:- Diagnose bleeding disorder.
Cross check CBC parameters.
Detect hemoparasites.
INTRODUCTION
3. Blood cells contain cellular structures with different reaction (pH), some
are acidic while others are basic.
Aniline dyes are of two types:
Basic dye – Methylene blue.
Acidic dye – Eosin.
All stains with combination of acidic and basic dye are called
ROMANOWSKY STAINS.
STAINS
4. First prep by ROMANOWSKY in 1891.
Methylene blue on oxidation produces coloured compounds – AZURES
have ability to combine with EOSIN.
Oxidation – maturation / chemical treatment.
Azures - different shades of staining the smears.
RBC – pink.
Granules of Eosinophils – orange.
Granules of Basophil – bluish black.
Granules of neutrophils – lilac.
Nuclei and structures – stained by basic dye – basophilic.
Nuclei and structures – stained by acidic dye – eosinophilic.
ROMANOWSKY
STAINS
5. Dissolve 1 g of Leishman powder in 500ml of acetone free methyl alcohol
(fixative) “{acetone destroys cells}” in conical flask.
Warm it at 50 c – 15 min.
Ripening – sunlight exposure – 2 days.
LEISHMAN STAIN
6. Dissolve 1 g of Giemsa stain powder in 66 ml of glycerol at 60 C for 2 hrs.
After cooling add 66ml of acetone free methyl alcohol – mix.
37 C – one week – ripening.
OTHER STAINS:
WRIGHT STAIN.
JENNER STAIN.
JENNER – GIEMSA STAIN.
FIELD STAIN.
GIEMSA STAIN
7. Drop of blood – clean grease free glass slide – 1 cm away from one end.
Place smooth edge of spreader over blood drop and spread the blood along
its edge.
Place slide at 30-40 degree angle – make smear – smooth forward
movement.
Ideal smear – 2.5-3.5cm – length.
Dry at room temperature.
Label.
PERIPHERAL SMEAR
8.
9. Length : 2/3 to 3/4 of slide length.
Shape : tongue.
Head, body, tail.
SOURCES OF ERROR:
Grease – blank oval areas.
Spreader edge not smooth – tail of smear ragged (TAILING OF SMEAR).
Error in neutrophil count.
WELL MADE
PERIPHERAL SMEAR
10.
11. Fix with in 4 hours.
Fix – acetone free methyl alcohol.
SMEAR FIXATION
12. Place smear on staining rack.
Pour undiluted stain on slide with dropper.
Stain has to cover entire upper surface.
Wait 2 min – methyl alcohol fixes the smear.
Pour double the amount buffer water.
Gently mix and wait 8 to 10 min.
Look for greenish metallic scum to appear.
Displace the stain – pouring running water from one side od slide – stain scum
drained off – no more stain left behind.
DO NOT DISCARD STAIN FROM SLIDE BY TILTING IT – RESULTS IN
STAIN PRECIPITATES.
Keep slide in slanting position.
Examine under microscope.
SMEAR STAINING
13. Buffered pH is important.
Acidic pH – cells pink – WBC light stained.
Basic pH – cells blue – WBC bluish black.
If overstained – wash with Leishman stain / methanol – few seconds.
If understained – restain – add Leishman stain and buffer.
SOURCE OF ERROR
14. Methanol fixed PS – pour diluted stain ( 1 in 10 ).
Wait 20 – 3- min.
Wash slide.
Dry it.
THICK FILM – MALARIA.
GIEMSA STAIN
15. RBC: size, shape, colour.
WBC: number and distribution – 100 cells count.
PLATELET: number and distribution.
HEMOPARASITES: malaria, microfilaria.
PS EXAMINATION
