1. Examination of Peripheral Blood Smear
Dr S Homathy

2. Complete blood count
The most common test used in clinical medicine
Determine type and severity of blood cell
abnormalities
Nowadays, CBC is fully automated and highly reproducible.
Correct interpretation of automated CBC can reduce rate of unnecessary blood smear examination
Provide useful information for provisional diagnosis of RBC and WBC diseases

3. A well Made and well Stained Smear can provide:
Estimates of cell count
Proportions of the different types of WBC
Morphology

4. 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 .

5. Peripheral Blood Smear
Objective
1. Specimen Collection
2. Peripheral Smear Preparation
3. Staining of Peripheral Blood Smear
4. Peripheral Smear Examination

6. Specimen Collection Venipuncture
should be collected on an EDTA (Disodium or Tripotassium ethylene diamine tetra-acetic acid) Tube
EDTA liquid form preferred over the powdered form
Chelates calcium

7. Specimen Collection Advantages
Many smears can be done in just a single draw
Immediate preparation of the smear is not necessary
Prevents platelet clumping on the glass slide

8. Specimen Collection Disadvantages: PLATELET SATELLITOSIS
causes pseudothrombocytopenia and pseudoleukocytosis
Cause: Platelet specific auto antibodies that reacts best at room temperature

9. Specimen Collection
Platelet satellitosis

10. Peripheral Smear Preparation
Wedge technique
Coverslip technique
Automated Slide Making
and Staining

11. Peripheral Smear Preparation
Wedge technique
Push Type wedge preparation
Pull Type wedge prepartion
Easiest to master
Most convenient and most commonly used technique

12. Material needed
Glass slide 3 in X 1in
Beveled/chamfered edges

13. Peripheral Smear Preparation

14. Peripheral Smear Preparation
Procedures:
Drop 2-3 mm blood at one end of the slide
Diff safe can be used
a. Easy dropping
b. Uniform drop

15. Precaution: Too large drop = too thick smear Too small drop = too thin smear

16. The pusher slide be held securely with the dominant hand in a 30-45 deg angle.
- quick, swift and smooth gliding motion to the other side of the slide creating a wedge smear

17. 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

18. Peripheral Smear Preparation

21. Peripheral Smear Preparation
Precautions:
Ensure that the whole drop of blood is picked up and spread
Too slow a slide push will accentuate poor leukocyte distribution, larger cells are pushed at the end of the slide
Maintain an even gentle pressure on the slide
Keep the same angle all the way to the end of the smear.

22. Peripheral Smear Preparation
Precautions:
Angle correction:
1. In case of Polycythemia:
high Hct
angle should be lowered
- ensure that the smear made is not to thick
2. Too low Hct:
Angle should be raised

23. Feature of a Well Made Wedge Smear
Smear is 2/3 or 3/4 the entire slide
Smear is finger shaped, very slightly rounded at the feathery edge:
widest area of examination
Lateral edges of the smear visible
Should not touch any edge of the slide.

24. Should be margin free, except for point of application
Smear is smooth without irregularities, holes or streaks
When held up in light:
feathery edge should show rainbow appearance
Entire whole drop of blood is picked up and spread

26. 26

27. used for Bone marrow aspirate smears Advantage: Disadvantage:
Cover Slip Technique
rarely used
used for Bone marrow aspirate smears
Advantage:
excellent leukocyte distribution
Disadvantage:
labeling, transport, staining and storage is a problem

28. 22 x 27mm clean coverslip
More routinely used for bone marrow aspirate
Technique:
1. A drop of marrow aspirate is placed on top of 1 coverslip
2. Another coverslip is placed over the other allowing the aspirate to spread.
3. One is pulled over the other to create 1 thin smears
4. Mounted on a 3x1 inch glass slide

29. Precautions:
Very light pressure should be applied between the index finger and the thumb
Crush preparation technique
Too much pressure causes rupture of the cells making morphologic examination impossible
Too little pressure prevents the bone spicules from spreading satisfactorily on the slide

30. tail body head

31. Thin area:
Spherocytes which are really "spheroidocytes" or flattened red cells.
True spherocytes will be found in other (Good) areas of smear.

32. 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.

33. 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.

34. 6.Irregular spread with ridges and long tail:
Edge of spreader dirty or chipped; dusty slide
7.Holes in film:
Slide contaminated with fat or grease
8.Cellular degenerative changes:
delay in fixing, inadequate fixing time or methanol contaminated with water

35. 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

36. Automatic Slide Making and Staining
SYSMEX 1000i

37. Peripheral Smear Preparation
Drying of Smears
Fan
Heating pans
No breath blowing of smears – may produce crenated RBCs or develop water artifact (drying artifact)

38. Slide fixation and staining

39. Romanowsky staining Leishman's stain : a polychromatic stain
Methanol : fixes cells to slide
methylene blue stains RNA,DNA
blue-grey color
Eosin stains hemoglobin, eosin granules
orange-red color
pH value of phosphate buffer is very important

40. Pure Wright stain or Wright Giemsa stain
Blood smears and bone marrow aspirate

41. Procedure 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.

42. Features of a well-stained PBS
Macroscopically: color should be pink to purple
Microscopically:
RCS: orange to salmon pink
WBC: nuclei is purple to blue
cytoplasm is pink to tan
granules is lilac to violet
Eosinophil: granules orange
Basophil: granules dark blue to black

43. Optimal Assessment Area:
RBCs are uniformly and singly distributed
Few RBC are touching or overlapping
Normal biconcave appearance
200 to 250 RBC per 100x OIO

44. Trouble shooting Macroscopic
Overall bluer color: increased blood proteins (multiple myeloma, rouleaux formation)
Grainy appearance: RBC agglutination (cold hemagglutinin diseases)
Holes: increased lipid
Blue specks at the feathery edge: Increased WBC and Platelet counts

45. Microscopic: 10x Objective
Assess overall quality of the smear i.e feathery edge, quality of the color, distributin of the cells and the lateral edges can be checked for WBC distribution
Snow-plow effect: more than 4x/cells per field on the feathery edge: Reject
Fibrin strands: Reject
Rouleaux formation, large blast cell assessment

46. Too acidic Suitable Too basic

47. Too Acid Stain:RBC too pale, WBC barely visible
insufficient staining time
prolonged buffering or washing
old stain
Correction:
lengthen staining time
check stain and buffer pH
shorten buffering or wash time

48. Too Alkaline Stain:RBC gray, WBC too dark, Eosinophil granules are gray
thick blood smear
prolonged staining
insufficient washing
alkaline pH of stain components
heparinized sample
Correction :
check pH
shorten stain time
prolong buffering time

49. Problem encountered during staining
Water artifact:
moth eaten RBC,
heavily demarcated central pallor on the RBC surface,
crenation,
refractory shiny blotches on the RBC 49

50. What contributes to the problem:
humidity in the air as you air dry the slides.
Water absorbed from the humid air into the alcohol based stain
Solution:
Drying the slide as quickly as possible.
Fix with pure anhydrous methanol before staining.
Use of 20% v/v methanol

51. AUTOMATED SLIDE STAINERS
It takes about 5-10 minutes to stain a batch of smears
Slides are just automatically dipped in the stain in the buffer and a series of rinses
Disadvantages:
Staining process has begun, no STAT slides can be added in the batch
Aqueous solutions of stains are stable only after 3-6 hours

52. QUICK STAINS
Fast, convenient and takes about 1 minute to be accomplished
Modified Wrights-Giemsa Stain, buffer is aged distilled water
Cost effective
Disadvantage:
Quality of stains especially on color acceptance
For small laboratories and for physician’s clinic only

53. Manual differential

54. Principle White Blood Cells
Check for even distribution and estimate the number present (also, look for any gross abnormalities present on the smear).
Perform the differential count.
Examine for morphologic abnormalities.

55. Red Blood Cells, Examine for:
Size and shape ( Anisocytosis,Poikilocytosis
Relative hemoglobin content.
Polychromatophilia.
Inclusions.
 Rouleaux formation or agglutination
Platelets.
Estimate number present.
 Examine for morphologic abnormalities.

56. Observations Under 10X
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.

57. WBC estimation Under 40X 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.

58. Platelet estimation Under 100X
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.

59. Platelets per oil immersion field (OIF)
<8 platelets/OIF = decreased
8 to 20 platelets/OIF = adequate
>20 platelets/OIF = increased

60. 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

61. If 10 or more nucleated RBC's (NRBC) are seen, correct the
White Count using this formula:
Corrected WBC Count =
WBC x 100/( NRBC + 100)
Example : If WBC = 5000 and 10 NRBCs have been counted
Then 5,000× 100/110 =
The corrected white count is

62. Determine a quantitative scale

63. Left-shift: non-segmented neutrophil > 5%
Increased bands Means acute infection, usually bacterial

64. Right-shift: hypersegmented neutrophil
Increased hypersegmented neutrophile

65. Leukocytosis, a WBC above 10,000, is usually due to an increase in one of the five types of white blood cells
Neutrophilic leukocytosis neutrophilia
Lymphocytic leukocytosis - lymphocytosis
Eosinophilic leukocytosis - eosinophilia
Monocytic leukocytosis monocytosis
Basophilic leukocytosis basophilia

66. Morphology of WBC
Normal blood smear

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

70. Eosinophil One eosinophil - mature. Normal blood - 100X.
Orange colour granules.
Bi-lobed nucleus

71. Basophil
One mature basophil.
Blackish granules overlying the nucleus

72. Normal lymphocytes Lymphocytes are the smallest WBC.
They have large condensed nucleus, with a scanty bluish cytoplasm.

73. Normal monocyte Monocytes are the largest WBC.
The nucleus is slightly indented .
The cytoplasm is abundant, sky blue in colour.
Some have vacuoles in the cytoplasm.

74. Red cells
Normal red cells or erythrocytes show only slight variation in size and shape.
The blood film should be examined in the area where the red cells are touching but not often overlapping.

75. In this area many red cells have an area of central pallor which may be up to a third of the diameter of the cell.
This is consequent on the shape of a normal red cell, which resembles a disc that is thinner in the centre.

76. Summarizing RBC Parameters
RBC Count )RBC x 1012/L)
Hb (g/dl)
Hct (5 or L/L)
Mean Cell Volume (MCV. Fl)
Mean Cell Hb (MCH, pg)
Mean Cell Hb Concentration (MCHC. %, g/dl)

77. RBC distribution Morphology Size Shape Inclusions Young rbcs Color
Arrangement

78. Platelets Normal platelets are also apparent.
They are small anuclear fragments between the red cells containing small purple-staining granules.

80. Platelet aggregates Platelet aggregates may be composed of
apparently intact platelets,
degranulated pale grey platelets
or a mixture of both, as in this example.
If the platelet count is low it is essential to examine the blood film carefully for platelet aggregates

81. Platelet satellitism
Platelet satellitism describes the phenomenon of adherence of platelets to white cells.
It is an in vitro phenomenon of no clinical significance.
However it is important that it is detected since the platelet count will be factitiously low.

82. Staining of Peripheral Blood Smear
HEMA-TEK STAINER

83. Component of automated CBC
Blood count basic parameters:
Hb, Hct,RBC, WBC, platlet.
Red cell indices: MCV, MCH, MCHC, RDW
WBC differentials
Cytogram or Scattergram
Reticulocyte count

84. Red cell parameters
Direct Measurement
Erythrocyte Concentration (RBC) x 106/ml
Mean Corpuscular Volume (MCV) Femtolitre (fl)
Hemoglobin (Hb) Gram/decilitre (g/dl)
Indirect Measurement
Hematocrit (Hct) = RBC x MCV/10 %
Mean Corpuscular Hemoglobin (MCH) = HB x 10 / RBC (pg)
Mean Corpuscular Hemoglobin Concentration (MCHC) = Hb/Hct x 100 (g/dl)
Red Cell Distribution Width (RDW) %

85. Reticulocyte count Reticulocyte =
non-nucleated RBC with polyribosomal RNA
as stained by supravital stain (new methylene blue or brilliant cresyl blue)
Polychromasia underestimates Reticulocytes
Three methods of reticulocyte Enumeration
Manual count on slide per 1,000RBC
Automated CBC with reticulocyte counter (Coulter VCS, Cell-Dyne 4000, Technicon-H3)
Flow cytometry with fluorescent dyes

86. RBC disorders
Hypochromic microcytic anemia
Iron deficiency anemia
Thalassemia and hemoglobinopathy
Macrocytic anemia
Megaloblastic anemia
Non-megaloblastic macrocytic anemia

87. Hemolytic anemia
Immune hemolytic anemia: AIHA, DHTR
Microangiopathic hemolytic anemia (MAHA)
Red cell enzymopathies: G-6-PD deficiency
RBC membrane defects: spherocytosis,
ovalocytosis, elliptocytosis, stomatocytosis
RBC inclusion bodies and parasites

97. WBC disorders
Leukopenia
with absolute neutropenia: bone marrow failure, agranulocytosis
with atypical lymphocytes: viral infection, chronic lymphoproliferative disorders
with immature myeloid cells: acute leukemia, MDS or myelopthisis

98. Leukocytosis
Reactive leukocytosis: leukemoid reaction
Acute leukemia: AML vs. ALL
Chronic myeloproliferative disorders
Chronic lymphoproliferative disorders
Leukoerythroblastosis

99. Platelet disorders
Quantitative disorders
Isolated thrombocytopenia: Immune vs. non-immune
Thrombocytopenia associated with other hematologic abnormalities
Thrombocytosis
Qualitative disorders
Giant platelets (megathrombocytes)
Platelet inclusion or granule abnormality
Bizarre in shape and size
Megakaryocytes or megakaryoblasts