PERIPHERAL SMEAR STAINING

PERIPHERAL SMEAR STAINING

1. What stains are routinely used for staining peripheral smear

   • Romanowsky stains are routinely used.

   • These stains contain the combinations of acidic and basic dyes that produces subtle distinction between staining of cell and staining of granules differentially

   • Granules in neutrophils are stained by Azure complexes, eosinophilic granules by acidic components of dye and basophilic granules which contain acid heparin has affinity for basic component of dye

2. What are the different types of Romanowsky  stains

   • Leishman’s stain

   • Wright’s stain

   • Giemsa’s stain

   • Jenner’s stain

   • Field’s stain

   • May Grunwald Giemsa

3. Which stain is used for identification of intracellular parasites and viral inclusions?

   • Giemsa stain

4. Which stain has very quick staining method ?

   • Field’s stain

5. Enumerate the components of standard Romanowsky stain?

   • Azure B (Trimethyl thionine)

   • Eosin Y (Tetra bromofluorescine)

   • Azure A

   • Azure C

   • Methylviolet

6. Which Romanowsky stain is commonly used for routine staining

   • Leishman’s  stain

7. What is the composition of Leishman’s stain

   • Methylene blue – stains the nucleus

   • Eosin  – stains the cytoplasm

   • Acetone free methyl alcohol – acts as fixative

8. How will you prepare  Leishman’s stain

   • Dissolve 0.2 gm of Leishman’s powder in 100 ml of acetone free Methyl alcohol

   • Mixture is warmed to 50°C for 10 – 15 minutes

   • Then the mixture is filtered.

   • Dye is ripened by placing the filtrate in sunlight for 3-4 days or by placing it in incubator at 37°C for 24 hours

9. Name the types of peripheral blood smear

   • Thin smear

   • Thick smear

10. What are techniques for peripheral smear preparation

    • Slide technique

    • Cover slip technique

    • Automated slide making and staining

11. What is the procedure of preparing peripheral smear ?

    • Drop of  blood is placed about 1-2cms from one end at the central line of slide

    • Place a glass slide or spreader at an angle of 45° to the slide in front of the drop and then move it back so that it comes in contact with blood drop

    • Immediately spread out the drop along the line of contact with spreader

    • Care should be taken to see that the blood drop is pulled along the slide instead pushd ahead of slide

12. What should be the essential feature in a spreader for making a good blood smear

    • The spreader slide should always have a smooth edge that results smooth smear formation without irregularity on tail

13. What should be the angle at which spreader should be kept in contact with slide

    • 45°

14. Why is it necessary to maintain the angle of the spreader

    • If the angle is lesser than 45°, smear obtained will be very thinner

    • If the angle is more than 45°, smears obtained will be thinner

15. What is the procedure of Leishman’s stain?

    • Cover the blood smear with Leishman’s stain and keep it for 2 minutes. Fixation of the smear by methyl alcohol occurs in these 2 minutes

    • The stain should not be allowed to dry

    • Dilute with equal quantity of buffer solution (PH.6.8) or distilled water. Gently blow over the stain for mixing

    • Keep it for 8 minutes for the staining to take place.

    • Then the slide is washed with distilled water

    • Clean the back of the slide and leave it to air dry

16. Which buffer is used in the staining of peripheral smear  by Leishman’s stain

    • Sorensen’s   phosphate buffer

    • It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate.

17. What is the use of buffer for diluting the stain 

    • Romanowsky stain diluted with phosphate buffer of PH 6.8  imparts a reddish hue to red cells and differential staining of granules of granulocytes

18. What is the effect of pH on staining with Romanowsky stain

    • pH of 6.8 is recommended for staining

    • If there is increased pH (alkaline) then the Azure components are accentuated at the expense of Eosin

    • If there is acidic pH then the Eosin components are accentuated at the expense of Azure B

19. Why stained  blood film show scattered granules and  indistinct nucleus 

    •  Due to  Use of chlorinated tap water instead of buffer or fresh distilled water in staining procedure

20. What are characteristics of a good smear

    • Good smear is tongue shaped with smooth tail

    • Has both thin and thick areas

    • No lines or holes

    • Should occupy 2/3 of the total slide

    • Should not touch any edge of slide

    • Should be Margin free except point of application

21. Name the parts of a thin peripheral blood smear

    • Head

    • Body

    • Tail

22. Which part of the smear should be examined for leukocyte count

    • Junction of the body and tail

23. What is the tailing of the smear

    • If the edge of the spreader is not smooth ,  the smear shows streaks which contain large number of neutrophils and platelets

    • In rest of the smear neutrophils are diminished

24. What is the action of Acetone free methyl alcohol in smear interpretation

    • It  fixes the peripheral smear to glass slide and preserves the morphology and chemical status of the cells

25. Why Acetone free methyl alcohol should be used

    • Because Acetone causes lysis of RBC’s

26. What are the acidic and basic components of Romanowsky stain

    • Acidic stain- Eosin Y – stains basic components like cytoplasmic  granules  and RBCs  in a pink colour

    • Basic stain – Methylene blue – stains acidic components like  basophilic granules in the cytoplasm, nuclei of  all leukocytes  in a blue violet colour

27. What can be the cause if the smear  shows excessive blue colour?

    • The possible errors are

      • Over alkalinity of stain

      • Insufficient washing with the buffer

      • Overly thick smear

28. What can be the cause if the smear  shows excessive red colour?

    • The possible errors are

      • Too short staining time

      • Acidic PH of the buffer

      • Excessive washing.

      • Acidic PH of the distilled water if the distilled water is used instead of buffer

29. How does a well stained blood film look like when stained with Romanowsky’s method of staining?

    • Blood film will show pinkish tint

    • Microscopically –

      • RBC’s are pinkish orange

      • The nuclei of leukocytes will be purplish blue

      • Neutrophilic granules appear pinkish violet

      • Eosinophilic granules appear red in colour

      • Basophilic granules appear dark blue in colour

30. Common causes of poor blood smear while making smear

    • Drop of blood too large or two small

    • Failure to keep the entire edge of the spreader against slide on making smear

    • Failure to keep the spreader slide at a 45º angle with slide

    • Slide contaminated with fat grease or air bubbles

    • Edge of spreader dirty, or chipped

    • Failure to push the spreader slide completely across the slide

31. Which stain is used for detection of malaria in surveys

    • Field’s stain as it is rapid stain

32. Which stain is used for identification of intracellular parasites and viral inclusions

    • Giemsa stain

33. Which stain has very quick staining method 

    • Field’s stain

34.  What is method of preparing thick smear?

    • 4 drops of blood are placed at 4 corners

    • Using the corner of a clean slide, spread the drop of blood in a  circle the size of a diameter 1-2 cm

    • Allow the smear to dry thoroughly

    • Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during staining

    • Place the film in vertical position in the distilled water for 5 – 10 minutes for dehemoglobinization

    • Smear is dried and stained by Leishman’s stain

35. What should be the optimum thickness of the thick smear

    • Optimum thickness is when the news print is just readable through it

36. What are the uses of thick films

    • For demonstration of malaria and microfilarial parasite

37. What is the best time for collection of blood to demonstrate malarial parasite

    • Blood should be collected at or just after the height of febrile paroxysms

38. What stages of malarial parasites can be seen in peripheral blood smear

    • All stages of P.vivax, P.ovale, and P.malariae can be seen in smear. In P.falciparum infection early trophozoites (ring forms) and gametes are only seen, as the growing trophozoites and schizonts remain in capillaries of internal organs

39. What is the optimum time for collection of blood to demonstrate microfilaria

    • As the parasite shows nocturnal periodicity, sample should be collected at mid night or by keeping the patient in dark room for atleast 2 hours

40. Why thick blood smear used for parasites detection

    • A thick peripheral blood smear provides large volume of blood to be examined so that parasites can be scanned in shorter time

41. What are the limitations of Thick smear

    • Species identification of malarial parasites may be difficult

    • A thin film should always be examined if a definitive identification based on morphology is required

    • Smears must be prepared from anticoagulated blood within one hour after venipuncture, as the morphology of parasitic forms and the erythrocytes become atypical after that time due to direct action of the anticoagulant

42. Which parasites can be seen in Peripheral blood film

    • Malarial parasite

    • Microfilaria

    • Leishmania

    • Babesia

    • Trypanosoma

43. What is the method of preparing and staining with Wright’s stain

    • Preparation of stain

      • Wright’s stain powder – 0.2gms

      • Acetone free methyl alcohol – 100ml

      • Both are mixed and solution is kept for few days at 37°C before using it. When the stain is ripened, scum is formed on surface

    • Method – same as Leishman’s stain

44. What is the method of preparing and staining with Giemsa stain

    • Preparation of stain

      • Giemsa powder – 0.6gms

      • Glycerol – 50 ml

      • Acetone free methyl alcohol – 50ml

      • 0.6 g of Giemsa powder is added to the 50ml of glycerol and the mixture is warmed at 50°C for 15 minutes. Then 50 ml of methanol is is added to mixture. Stain is filtered and is used at the dilution of 1:10

    • Method –

      • Blood film is fixed with methyl alcohol for 3 to 5 minutes and dried

      • Stain (1:10 dilution) is poured on smear and is left for 20 to 25 minutes

      • Wash smear with neutral water and dry

45. What is the method of preparing and staining with Field’s stain

    • Preparation of stain

      • Stain A  (Polychrome methylene blue)

        • Methylene blue – 0.26g

        • Azure B- 0.1g

        • Disodium hydrogen phosphate – 2.5g

        • Potassium dihydrogen phosphate – 1.25g

        • Water – 100 ml

      • Stain B ( Eosin)

        • Eosin (Water soluble yellow Eosin) – 0.26g

        • Disodium hydrogen phosphate – 2.5g

        • Potassium dihydrogen phosphate – 1.25g

        • Water – 100ml

      • Preparation of stains

        • Phosphates are dissolved in freshly boiled water

        • Azure B is grounded in mortar and added to phosphate solution so that it will be dissolved well

        • Dyes are added in Phosphate solutions and mixed well and filtered

      • Method

        • Keep the smear for 1 second in Stain A

        • Rinse gently in clean water for 5 – 10 seconds until the slide becomes free from stain

        • Place the smear in solution B for 1 second

        • Rinse for 2 to 3 seconds in clean water

        • Place vertically in the rack and allow it to dry

46. What is the method of preparing and staining with May-Grunwald’s- Giemsa  stain

    • Preparation of stain

    • May-Grunwald’s stain

      • May-Grunwald powder – 0.3g

      • Acetone-free methyl alcohol – 100ml

      • Dissolve the powder in 100 ml of methyl alcohol by warming at 50ºC for 10 minutes by shaking. Filter the solution after 24 hours 

    • Method

      • Smear is fixed in methanol

      • Stain the film with dilute dye solution (1:10) for 5 minutes

      • Then stain with dilute Giemsa’s stain (1:10) for 10 to 15 minutes

      • Wash with buffered water and dry in the air.

References

• Sabitri Sanyal. Preparation manual for undergraduates: Clinical pathology

• P. Chakraborthy and Gargi Chakraborthy. Practical Pathology

• Dr.Sundaram Challa and Dr. Radha Sagar. Post graduate Pathology practicals: FAQs

• Dr. Tejindar singh and Dr. K. Uma Chaturvedi. Practical Pathology. 3rd edition

By- 

Edited by 

Dr.V.Shanthi. Professor and HOD, Pathology, Narayana Medical College, Nellore.