1. Lecture 3
PBS
Reticulocyte

2. PERIPHERAL BLOOD SMEAR EXAMINATION
Preparation: -
Clean grease free slides should be used
Place a drop of fresh finger prick blood or EDTA blood (from which it could be prepared as early as possible to avoid distortion of leucocytes) on one slide.

3. Place a glass slide with a smooth edge (spreader) at an angle of 45 degree to the slide and move it back to make contact with the drop of blood.
Ensure that the drop of blood is pulled by the spreader but it is not pushed ahead of the spreader.

6. Preparation: -
A well prepared film is neither too thick nor too thin and has no holes due to grease on slides.
The film should be dried immediately by waving in the air.
If not immediately stained can be fixed acetone free methyl alcohol.

8. Staining : - Leishman's stain is commonly used.
Other stains which can be used are Giemsa, May Grunwald Giemsa and Wright stain.

9. Leishman stain Composition
Leishman powder (eosin-methylene blue powder) 0.5gm
Acetone free methyl alcohol 100ml

10. Ripening
The powder is transferred to a conical flask to which methyl alcohol is added.
Mixture is warmed to 50degree centigrate for 10 to 15 mins.
It is then filtered.
The dye is ripened by keeping the filtrate in sunlight for 3-4 days or in an incubator at 37 degree C for 7 days.

11. Why acetone free methyl alcohol?
Acetone if present, will wash away the nuclear stain, which will result in poor nuclear staining.

12. Staining : -
Place slide horizontally in staining or on 2 glass rods over a sink.
Fix by pouring Leishman stain (undiluted) for 2 min with help of a dropper. The stain should cover the whole film. (This is done to fix the smear).
Add twice the quantity of buffer water (4 to 5 drops of stain 10 drops of buffered water). Mix well by blowing. Allow the stain to act for 8 min. exact optimum time for staining may vary with stain used and should be determined by trial with each batch.
Wash off the stain thoroughly to avoid any stain deposit. Tip the water and dry.

13. Buffered Water Pot dihydrogen phosphate - 0.514 gm
Disod.hydrogen phosphate gm
Dist water ml
The pH of buffered water is about 6.8
If pH is acidic, RBC's will become pink and leucocyte may understain. Very alkaline pH may lead to bluish staining of RBCs and overstaining of leucocytes.

14. Romanowsky stain
Stains which are made up of combinations of acid and basic dyes are known as romanowsky stains.

15. Examination of PS:-
It is a very important investigation in haematology and can offer volumes of information.
A PBS examination should ideally assess RBCs, WBCs, Platelets, immature or abnormal cells and parasite if any.

16. Red Cells :-
Note size (normocytic, microcytic, macrocytic) and variation in size (anisocytosis)
Shape variation in shape (poikilocytes)

17. White Cells:-
Note number which may be normal, high or low. In normal PBS only mature forms are seen.
Proportion :Do a differential count. Minimum of 100 cells should be counted.
Normal Differential Count (DLC)
Neutrophils %
Lymphocytes %
Monocytes %
Eosinophils %
Basophils %

18. Platelets : - Note the number of platelets normal, high or low.
If film is made from finger prick platelet clumps are supposed to be present.

19. Neutrophil Nucleus: Multilobed (2-5lobes)
Cytoplasm: Contains granules which are fine and amphophilic

20. Eosinophil Nucleus: Bilobed or trilobed
Cytoplasm: Contains granules which are coarse and eosinophilic.

22. Lymphocyte Nucleus:Rounded or slightly indented
Cytoplasm:Scanty and agranular

24. Monocyte Nucleus : Large, kidney shaped or convoluted
Cytoplasm: Abundant and agranular

25. Monocyte

26. Basophil Nucleus: Lobed but often obscured by granules
Cytoplasm: Basophilic granules

27. Basophil

28. Neutrophilia Infections: Acute and chronic bacterial infection.
Some viral infections.
Some fungal infections.
Some parasitic infections.

29. Lymphocytosis Chronic infective diseases like T B Viral disease.
Chronic lymphocytic leukaemia

30. Eosinophilia : - Allergic states like bronchial asthma
Parasitic infestation like hook work

31. Monocytosis : -
Parasitic disease like malaria and kala azar.

32. Basophilia : -
CML

33. RBC Indices
MCV: It is the volume of an average red cell or the average cell volume of all the red cells.
Corresponds with the red cell diameter on blood smear/ Average size of red cells

34. ABSOLUTE VALUES MCV (Mean Cell Volume)=80-100 fl
MCV = PCV x 10_______ Femtolitre
RBC Count in millions
MCV  in Macrocytic anaemia and
 in Microcytic hypochromic anaemia

35. MCH
MCH: It is the amount (weight ) of haemoglobin in average red cell or the average content of haemoglobin per cell in all the red cells.

36. MCH(Mean Cell Haemoglobin) = 29  2 pg
MCH = Hb in gm/dl x 10 pg
RBC counts in millions

37. MCHC
MCHC: It is the percentage of haemoglobin in the average red cell.

38. MCHC (Mean Corpuscular Cell Haemoglobin Conc) = 32 - 36%
MCHC = Hb in gm/dl x 100
PCV
MCH & MCHC are decreased in microcytic hypochromic anaemia

39. RETICULOCYTE COUNT
Reticulocyte are juvenile red cells containing remnants of ribosomes and RNA. Ribosomes have the property of reacting with certain basic dyes such as brilliant cresyl blue or new methylene blue to form blue precipitate of granules or filaments. The reaction takes place only when the reticulocytes are still alive (without fixing the cells) i.e in vitally stained unfixed preparations.

40. Method
Diluting and staining fluid: 1 gm brilliant cresyl blue in 100 ml of normal saline.
Take 2 -3 drops of brilliant cresyl blue solution in a glass tube and add equal drops of patients EDTA or double oxalated blood.
Mix thoroughly and incubate at 37oC for min. after mixing, film is made on glass slide as usual and after drying examined under oil immersion.

41. Counting
For greater accuracy 1000 cells are counted making note of number of reticulocytes among these.

42. Normal Range
% for adult & Children
2 - 6 % for infant or cord blood
Interpretation - Number of reticulocytes in peripheral blood is a fairly accurate reflection of bone marrow erythropoietic activity. They decrease in aplastic anaemia and increase in haemolytic anaemia and haemorrhage of any cause.