Blood Smear preparation.pdf

Smear preparation and types

1. BLOOD SMEAR PREPRATION

2. ✿To study morphology of rbc
✿To study morphology of wbc
✿To study morphology of platlets
✿To study morphology of inclusion bodies
✿To confirm diagnosis of blood cell disorders
✿To examine hemoparasites(malaria, trypanosoma etc..)

3. TYPES OF BLOOD SMEAR
✿Thin blood smear
✿Thick blood smear

4. THIN BLOOD SMEAR

5. THIN BLOOD SMEAR
✿This can be done achieved by anticoagulated blood... either obtained
by venipuncture or finger prick
✿There are 3 methods:
✿1) Slide method
✿2)cover glass/cover slip method
✿3) Spin method

6. 1)SLIDE METHOD

7. PROCEDURE
✿Take a grease free, clean and dry slide
✿ place a drop of blood 1cm away from the end of the slide
✿Keep the spreader slide infront of an <gle og 30-45° to the slide
✿Draw the spreader backwards until it touches the blood
✿Then blood will be flowing along the edge of the spreader slide
✿Keep the <gle of spreader constant and push it front wards
✿Dry it stain it and observe

12. PARTS OF THIN BLOOD SMEAR
✿They have 3 parts
✿1)Head
✿2)Body
✿3)Tail

14. QUALITIES OF A IDEAL BLOOD SMEAR

16. CAUSES OF POOR BLOOD SMAER
✿Drop of blood too large or too small.
✿Spreader slide pushed across the horizontal slide in a jerky manner.
✿ Failure to keep the entire edge of the spreader slide against the
horizontal slide while making the smear.
✿Failure in using appropriate angle for the spreader slide.
✿Failure to push the spreader slide compleyely acrosss the slide
✿Holes in film slide contaminated with fat or grease
✿Cellular degenerative changes delay in fixing inadequate fixing time or
methanol contaminated with water

19. 2)COVER GLASS METHOD

20. PROCEDURE

22. 3)SPIN METHOD

26. STAINING OF THIN BLOOD SMEAR

27. METHOD
✿Leishman's staining
✿ Wright's stain

28. LEISHMAN'S STAINING

29. WRIGHT'S STAIN: DIP METHOD

30. WRIGHT'S STAIN: RACK METHOD

31. THICK SMEAR

32. ✿It is used for detection of blood parasites
✿For differential count
✿In case of severe leucopenia

33. METHOD 1:BLOOD SMEAR PREP
✿Thick blood smear are prepared by taking 4 drops of blood on a slide
✿ Joining the corners of the drop with a needle or with the cornerof
another slide
✿Spread the drop in one direction to make a thick film, size of a icon
diamter
✿The thickness of the film should be such that it allows a mews print
to be tead through dry prep

34. METHOD 1: DEHAEMOGLOBULIZATION
✿Dry the smear, dip in a copling jar containing 10drops pf glacial acetic
acid and 50ml normal saline
✿Repeat process till dehemoglobulisation (slide is colour less)is
complete
✿Drain it completely and wash with normal saline
✿Stain smear with dil Gemsa or Leishman stain

35. METHOD 2
✿prepare smear
✿Without drying immediatly put in 95% ethyl alcohol or 85% isopropyl
alochol for 15 min
✿When fixation is complete take out and pour few drops of glacial
acetic acid
✿Wait for few sec
✿When dehemoglobulisation is complete througly wash with water

36. METHOD 2: STAINING
✿Fields stain A
✿Fields stain B
✿Two contaniner of clean water
✿Air dry, dip into stainA fpr 5 sec drain
✿Wash for abt 5 sec on watet drain off
✿dip in to stain B for 5 sec drain off
✿Wash with water gently, dry it and observe

37. EXAMINATION OF UNSTAINED SMEAR
✿2-3 drops of blood is taken in a slide and coverslip us put on the rim is
then smear with vaseline to prevent drying up of the blood
✿Slid is examined under low power, if microfilaria is present shows
wriggling movement
✿Also used to identify trypanosomes
✿Advantages
✿Simple and inexpensive
✿If trypanosomes is detected disease is diagnosed

39. BUFFY COAT PREPRATION
✿It is well known that a typical or primitive blood cell circulate in a
small no in the peripheral blood in health
✿A typical mononuclear cells, metamyelocyte and megakaryocytes may
be found even premonocyte, premonoblast, nucleated rbc may also bee
seen but in small number
✿BC prep is well suitable for the study of morphology of plateltes and
white cells in case oof thrombocytopenia, leucopenia
✿Buffy coat prep are also used to detect the abnormal circulating
elements such as cancer cells, lymphosarcoma cells, fragments of
megakaryocytes
✿Buffy coat films can be used for the detection of bacyeria fungi or
parasite within neutrophil, monocytes or circulating macrophage.

40. THE BUFFY COAT IS THE FRACTION OF AN ANTICOAGULATED BLOOD
SAMPLE THAT CONTAINS MOST OF THE WHITE BLOOD CELLS AND
PLATELETS

41. PROCEDURE
✿Venous blood/anticoagulated blood is taken in a wintrobes
haematocrit tube/ Capillary tube and centrifuged in 300rpm for 15min.
✿After centrifugation remove thr supernatant serum carefully by fine
pipette and with same pipette deposit the platelet anf underlying
leuvocute layer on to slides
✿Emulsify buffy coat with patients plasma and then spread a film
✿Allow to dry in air and then fix and stain asusual
✿As an alternative to centrifugation the blood may be allowed to
sediment with the helpof sedimentation enhancing agents like
fibrinogen, methyl cellulose, Boyum's reagent(leucocye prep)

43. IMPORTANCE OF BUFFY COAT
PREPRATION
✿Buffy coat prep has very much imp bcoz it include only platelet and
wbc
✿In normal case the thickness of buffy coat layer is 0.1mm. The
variation in the thickness of buffy coat layer is the imp infication of
variation in the wbc and platelets
✿Buffy coat prep helps in the identification of cancer cells,
lymphosarcoma cells, fragments of megakaryocytes and
erythrophagocytic cells.
✿It reflect the conditions of thrombopenia, thrombocytosis,
leucocytosis and leucopenia, buffy coat layer is greenish in colour

44. ✿In normal case plasma is straw yellow colour, yellow green, orange
colour of plasma indicates increased bilirubin
✿Milky or cloudy plasma layer inficates nephrosis and lipaemia

45. THANK YOU??????

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