1. Introduction In Medical Technology
Lecture 11
Making Blood smear

2. Preparation of blood smear
There are three types of blood smears:
The cover glass smear.
The wedge smear .
The spun smear.
The are two additional types of blood smear used for specific purposes
Buffy coat smear for WBCs < 1.0×109/L
Thick blood smears for blood parasites .

3. Wedge blood smear
Specimen : EDTA blood within 2 to 3 hours & collected to the mark on tube.
Not's : May change RBCs morphology such as Spiculated (crenated) cells if :
Excessive amount of anticoagulant to specimen
Old blood - long standing.
Warm environment (room temperature) may hasten changes.

4. Procedure
Placing a drop of blood from mixed sample on a clean glass slide.
Spreader slide using another clean glass slide at degree angle.
Control thickness of the smear by changing the angle of spreader slide
Allow the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)

5. STEPS FOR BLOOD FILM

7. The thickness of the spread
If the hematocrit is increased, the angle of the spreader slide should be decreased.
If the hematocrit is decreased, the angle of the spreader slide should be increased.

8. Characteristics of a Good Smear
Thick at one end, thinning out to a smooth rounded feather edge.
Should occupy 2/3 of the total slide area.
Should not touch any edge of the slide.
Should be margin free, except for point of application.
Note: As soon as the drop of blood is placed on the glass slide, the smear should be made without delay. Any delay results in an abnormal distribution of the white blood cells, with many of the large white cells accumulating at the thin edge of the smear.

9. Common causes of a poor blood smear
Drop of blood too large or too small.
Spreader slide pushed across the slide in a jerky manner.
Failure to keep the entire edge of the spreader slide against the slide while making the smear.
Failure to keep the spreader slide at a 30° angle with the slide.
Failure to push the spreader slide completely across the slide.
Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide
Holes in film: Slide contaminated with fat or grease
Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

10. Examples of unacceptable smears
A: Blood film with jagged tail made from a spreader with a chipped end.
B: Film which is too thick
C: Film which is too long, too wide, uneven thickness and made on a greasy slide.
D: A well-made blood film.

11. Examples of unacceptable smears

12. Biologic causes of a poor smear
Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear.
Lipemia - holes will appear in the smear. There is nothing you can do to correct this.
Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this
Notes:
The WBCs are unevenly distributed and RBC distortion is seen at the edges Smaller WBCs such as lymphocytes tend to reside in the middle of the feathered edge.
Large cells such as monocytes, immature cells and abnormal cells can be found in the outer limits of this area.
Spun smears produce the most uniform distribution of blood cells.

13. Slide Fixation & Staining
LEISHMAN'S STAIN

14. Fixing the films
To preserve the morphology of the cells, films must be fixed as soon as possible after they have dried.
It is important to prevent contact with water before fixation is complete.
Methyl alcohol (methanol) is the choice, although ethyl alcohol ("absolute alcohol") can be used.
Methylated spirit (95% ethanol) must not be used as it contains water.
To fix the films, place them in a covered staining jar or tray containing the alcohol for 2-3 minutes. In humid climates it might be necessary to replace the methanol 2-3 times per day; the old portions can be used for storing clean slides.

15. Staining the film
Romanowsky staining: Romanowsky stains are universally employed for staining blood films and are generally very satisfactory.
There are a number of different combinations of these dyes, which vary, in their staining characteristics.
May-Grunwald-Giemsa is a good method for routine work. Giemsa stain is thought to produce more delicate staining characteristics.
Wright's stain is a simpler method.
Leishman's is also a simple method, which is especially suitable when a stained blood film is required urgently or the routine stain is not available (e.g. at night).
Field's stain is a rapid stain used primarily on thin films for malarial parasites.

16. Principle The main components of a Romanowsky stain are:
A cationic or basic dye (methylene blue or its oxidation products such as azure B), which binds to anionic sites and gives a blue-grey color to nucleic acids (DNA or RNA), nucleoproteins, granules of basophils and weakly to granules of neutrophils
An anionic or acidic dye such as eosin Y or eosin B, which binds to cationic sites on proteins and gives an orange-red color to hemoglobin and eosinophil granules.
pH value of phosphate buffer is very important.

18. Staining procedure (Leishman’s stain)
Thin smear are air dried.
Flood the smear with stain.
Stain for 1-5 min. Experience will indicate the optimum time.
Add an equal amount of buffer solution and mix the stain by blowing an eddy in the fluid.
Leave the mixture on the slide for min.
Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain.
Stand slide on end, and let dry in air.

19. too acidic suitable too basic

20. Causes & correction Too Alkaline Stain: Too Acid Stain:
insufficient staining time
prolonged buffering or washing
old stain
Correction:
lengthen staining time
check stain and buffer pH
shorten buffering or wash time
Too Alkaline Stain:
thick blood smear
prolonged staining
insufficient washing
alkaline pH of stain components
 Correction :
check pH
shorten stain time
prolong buffering time

21. Performing A Manual differential And assessing RBC Morphology

22. Purposes Red Blood Cells, White Blood Cells. Platelets.
Check for even distribution and estimate the number present (also, look for any gross abnormalities present on the smear).
Perform the differential count.
Platelets.
Estimate number present.
 Examine for morphologic abnormalities.
Red Blood Cells,
Size and shape.
Relative hemoglobin content.
Polychromatophilia.
Inclusions.
Rouleaux formation or agglutination

23. Procedures Observations Under ×10
Check to see if there are good counting areas available free of ragged edges and cell clumps.
Check the WBC distribution over the smear.
Check that the slide is properly stained.
Check for the presence of large platelets, platelet clumps, and fibrin strands.

24. Observations Under ×40 : WBC estimation
Using the × 40 high dry with no oil.
Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs.
Count 10 fields, take the total number of white cells and divide by 10.
To do a WBC estimate by taking the average number of white cells and multiplying by 2000.

25. Observations Under × 100: Platelet Estimates
Use the oil immersion lens estimate the number of platelets per field.
Look at 5-6 fields and take an average.
Multiply the average by 20,000.
Note any macroplatelets.
Platelets per oil immersion field (OIF)
<8 platelets/OIF = decreased
8 to 20 platelets/OIF = adequate
>20 platelets/OIF = increased

26. PLATELETS

27. Observing and Recording Nucleated Red Blood Cells (nRBCs)
If 10 or more nucleated RBC's (NRBC) are seen, correct the White Count using this formula:
Example : If WBC = 5000 and 10 NRBCs have been counted
Then 5,000× 100/110 =
The corrected white count is

28. Manual Differential Counts
These counts are done in the same area as WBC and platelet estimates with the red cells barely touching.
This takes place under × 100 (oil) using the zigzag method.
Count 100 WBCs including all cell lines from immature to mature.
Reporting results
Absolute number of cells/µl = % of cell type in differential x white cell count

29. Observing direction:
Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction
* avoid repeat or miss some cells

30. Normal peripheral blood smear

31. Leukocytosis
Leukocytosis, a WBC above 10,000 is usually due to an increase in one of the five types of white blood cells and is given the name of the cell that shows the primary increase.
Neutrophilia : Neutrophilic Leucocytosis
Lymphocytosis : Lymphocytic leukocytosis
Eosinophilia : Eosinophilic leukocytosis
Monocytosis : Monocytic leukocytosis
Basophilia: Basophilic leukocytosis

33. Stab (Band) neutrophil
Diameter:12-16
Cytoplasm : pink
Granules: primary secondary
Nucleus: dark purple blue
dense chromatin

34. Segmented neutrophil Diameter: 12-16 Cytoplasm : pink
Granules: primary, secondary
Nucleus: dark purple blue dense chromatin, 2-5 lobes

35. Segmented neutrophil
Neutrophils are so named because they are not well stained by either eosin, a red acidic stain, or by methylene blue, a basic or alkaline stain.
Neutrophils are also known as "segs", "PMNs" or "polys" (polymorphonuclear).
They are the body's primary defense against bacterial infection.

36. Increased neutrophils count (neutrophilia)
Acute bacterial infection.
Granulocytic leukemia.
Decreased neutrophil count (neutropenia)
Typhoid fever
Brucellosis
Viral diseases, including hepatitis, influenza, rubella, and mumps.

37. LEFT-SHIFT AND RIGHT-SHIFT OF NEUTROPHIL
Normally, most of the neutrophils circulating in the bloodstream are in a mature form, with the nucleus of the cell being divided or segmented. Because of the segmented appearance of the nucleus, neutrophils are sometimes referred to as "segs.”
The nucleus of less mature neutrophils is not segmented, but has a band or rod-like shape. Less mature neutrophils - those that have recently been released from the bone marrow into the bloodstream - are known as "bands" or "stabs".
Left-shift: non-segmented neutrophil > 5%
Right-shift: hypersegmented neutrophil >3%

38. Shift to right  Increased hypersegmented neutrophile.
Band neutrophil
Shift to left  Increased bands mean acute infection, usually bacterial.
Shift to right  Increased hypersegmented neutrophile.

39. Eosinophil Diameter: 14-16 Cytoplasm : full of granules
Granules: large refractile, orange-red
Nucleus: blue dense chromatin 2 lobes like a pair of glass

40. The most common reasons for an increase in the eosinophil count are
Allergic reactions such as hay fever, asthma, or drug hypersensitivity.
Parasitic infection
Eosinophilic leukemia

41. Basophil Diameter: 14-16 Cytoplasm : pink
Granules: dark blue –black obscure nucleus
Nucleus: blue

42. Basophils The purpose of basophils is not completely understood.
Basophile counts are used to analyze allergic reactions.
An alteration in bone marrow function such as leukemia may cause an increase in basophils.

43. Lymphocyte Diameter: small 7-9 large 12-16 Cytoplasm: medium blue
Granules:
small agranular
large a few primary granules
Nucleus: dark blue \round dense chromatin

44. Lymphocytes
Lymphocytes are the primary components of the body's immune system. They are the source of serum immunoglobulins and of cellular immune response.
Two types of lymphocytes:
1. B lymphocyte : Humoral immunity
2. T lymphocyte : Cellular immunity

45. Lymphocytes increase (lymphocytosis) in:
1. Many viral infections
2. Tuberculosis.
3. Typhoid fever
4. Lymphocytic leukemia.
A decreased lymphocyte (lymphopenia) count of less than 500 places a patient at very high risk of infection, particularly viral infections.

46. Monocyte Diameter: 14-20 Cytoplasm : grey blue
Granules: dust-like lilac color granules
Nucleus: blue
large irregularly shaped and folded

47. Diseases that cause a monocytosis include:
• Tuberculosis
• Brucellosis
• Malaria
• Monocytic leukemia

48. Notes 1. Do not count cells that are disintegrating
Eosinophil with no cytoplasmic membrane and with scattered granules
Pyknotic cell (nucleus extremely condensed and degenerated, lobes condensed into small, round clumps with no filaments interconnecting).
smudge cells
Basket cells

49. 2- Abnormal differentials 200 Cell diff: Three or more basophils seen.
WBC > 15.0 (>20.0 for babies under 1 month and labor unit)
Three or more basophils seen.
If more than five immature WBC's are seen (or any blasts) let someone else diff slide and average results.
Correct WBC for NRBC's if you seen ten or more NRBCs/100 WBC.
Always indicate number of cells counted on diff. 
If any cell type is extremely elevated (such as bands, monos, or eos > 20) indicate that you are aware of the abnormality by circling or checking on the card next to the results.

50. 3- Morphologic Changes Due To Area Of Smear
Thin area- Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear.
Thick area - Rouleaux, which is normal in such areas. Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a "stack of coins" fashion..

52. 4. A well-made and well-stained smear is essential to the accuracy of the differential count. The knowledge and ability of the cell morphologist is critical to high-quality results.
5. Before reporting significant abnormalities such as blasts, malaria or other significant finding on a patient’s differential, ask a more experienced tech to review the smear for confirmation. In clinical settings where a pathologist or hematologist is present, the smear is set aside for Pathologist Review.
6. Never hesitate to ask questions concerning morphology or the identification of cells. The differential is one of the most difficult laboratory tests to learn. In fact, learning about cells and their morphology is a process that continues for as long as you perform differentials.

53. Thanks