PBS using Leishman Stain

1. Peripheral smear

2. Preparations
• Specimen-direct capillary blood or
anticoagulated blood
• Apparatus-clean glass slides,lancet,
spreader slide,microscope&microscope
oil
• Stain-leishman stain,wright or gimsa
• Diluent-distilled water or buffer solution
with Ph 6.8

3. Composition of leishman’s stain
• Polychromed methylene blue
• Eosin azure leishman powder
• Acetone free absolute alcohol
• 1.5gms leishman powder in 1 liter
acetone free absolute alcohol

4. Procedure
MAKING A SMEAR
• One drop of blood on one end of slide
• Spreader slide is placed at 45°on the drop
and moved along the slide
• It is moved smoothly and once such that
the blood film is thin
• Care should be taken to prevent the
formation of air bubbles
• Air dry the smear
• Make identification mark on one edge

5. Staining a smear
• Place the smear on the staining rack
• Pour leishman stain to cover the smear
completely allow to fix for 2-3 mins
• Add water twice the amount of
leishman’s stain allow to fix for 7-10 mins
• Appearance of golden scum or sheen on
the surface of stain
• Wash the stain off the slide with running
water
• Wipe the back of slide and air dry the
slide

6. Examination of the smear
• The stained slide is placed on the
microscope and is focused under low
power
• Place a drop of oil on the slide and
observe under oil immersion objective
• Observe RBC,platelet series
• WBC series is observed and a differential
count is done
• Record the findings

7. Precautions
• Objective must be clean
• Objective must be cleaned after every use
• Good smear is tongue shaped occupying
2/3rd of the slide
• Divided into head,body and tail
• Must be focused at the junction of body
and tail

8. Diagnostic value
RBC’s SERIES
• Size-microcytic/normocytic/macrocytic/
anisocytosis
• Shape-poikilocytosis,sickle cells,target
cells,tear drop cells,burr cells
spherocytosis,ovalocytosis
• Color-hypochromic/normochromic
• Immature cells-
normoblasts,polychromatophillic cells

9. WBC’s SERIES
DIFFERENTIAL COUNT
• Neutrophills-40--70%
• Eosinophills-2--4 %
• Basophills-0--1 %
• Monocytes-2--4 %
• Lymphocytes-25—40 %

10. Immature cells
• Myeloid series-
myeloblasts,promyelocyte,myelocyte,met
amyelocyte,band forms
• Lymphoid series-lymhoblasts

11. Thank you

12. PLATELET SERIES
• General score-6--10platelets/oil
immersion field
PARASITES
• Intracellular-malarial parasite
• Extracellular-microfilarial