Examination of the Peripheral Blood smear
A blood smear test is a blood test used to check for abnormal blood cells.
The three chief blood cells that the test centers on are red cells, white cells, and platelets.
Preparation of blood smear
- There are three types of blood smears:
1. The cover glass smear.
2. The wedge smears.
3. The spun smear.
- They are two additional types of blood smear used for specific purposes
1. Buffy coat smear for WBCs < 1.0×109/L
2. Thick blood smears for blood parasites.
Wedge blood smear
- Specimen: EDTA blood within 2 to 3 hours & collected to the mark on the tube.
- Not's: The morphology of RBC may be changed like Spiculated (serrated) cells if the following situations occur:
1. An excessive amount of anticoagulant to specimen
2. Old blood - long-standing.
3. A warm environment (room temperature) may hasten changes.
Procedure
- Placing a drop of blood from a mixed sample on a clean glass slide.
- Spreader slide: using second clean glass slide at a 30-40-degree angle.
- Control the thickness of the smear by changing the angle of the spreader slide.
- Let the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from your breath will cause an RBC artifact.)
Characteristics of a Good Smear
1. One end is thicker and thins to a smooth round feather edge.
2. Should account for 2/3 of the total slide area.
3. Do not touch any edges of the slide.
4. Should be margin free, except for the point of application.
Examination of Blood smear
Macroscopic
1. Mostly blue color: increased blood proteins (multiple myeloma, rouleaux formation)
2. Grainy appearance: RBC agglutination (cold hemagglutinin diseases)
3. Holes: increased lipid
4. Blue specks appear at the feathery edge: white blood cell and platelet count increase.
Microscopic
10x Objective
1. Assess the overall quality of the smear i.e. examine feather edge, color quality, cell distribution, and white blood cell distribution on the side edge
2. Snow-plow effect: over 4x/cells per field on the feathery edge: Reject
3. Fibrin strands: Reject
4. Rouleaux formation, large blast cell assessment.
40x Objective
1. A correct area where to start counting is determined
2. WBC estimate: internal quality control
100x Objective
1. Highest magnification
2. WBC differential counting
Optimal Assessment Area:
1. RBCs are uniformly and singly distributed
2. Few RBC are touching or overlapping
3. Normal biconcave appearance
4. 200 to 250 RBC per 100x OIO
Morphologic changes due to area of the smear
Thin area- Spherocytes which are true "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of the smear.
Thick area - Rouleaux, which is normal in this area. Confirm by examining thin areas.
If it is a real rouleaux, two or three RBCs will be glued together in a " stack of coins " manner.
Common causes of a poor blood smear
1. The drop of blood so large or so small.
2. The spreader slide pushed across the slide in a jerky manner.
3. Fail to keep the whole edge of the spreader slide against the slide while making the smear.
4. Failure to maintain a 30° angle between the spreader rail and the rail.
5. When the spreader slide cannot be pushed completely across the slide.
6. Non-uniform spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide
7. Holes in the film: when the slide contaminated with grease
8. Cellular degenerative changes: delay in fixing, insufficient fixation time, or methanol contamination by water
Biologic causes of a poor smear
1. Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then re-smear.
2. Lipemia - holes will be seen in the smear. You cannot take any action to correct this problem.
3. Rouleaux - RBC’s will become a coin-like stack. cannot take any action to correct this problem.
Performing A Manual Differential And assessing RBC Morphology
Principle
White Blood Cells.
1. Check for even distribution and estimate the number present (also check for obvious abnormalities on the smear).
2. Perform the differential count.
3. Examine for morphologic abnormalities.
Red Blood Cells, Examine for:
1. Size and shape.
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination
Platelets.
1. Estimate number present.
2. Examine for morphologic abnormalities.
Procedures
Observations Under ×10
1. Check if there is a good counting area, there are no jagged edges and cell clumps.
2. Check the WBC distribution over the smear.
3. Check that the slide is properly stained.
4. Examine the existence of large platelets, platelet clumps, and fibrin strands.
Observing direction
Notice one field and record the number of WBC according to the different type then move to another field in the snake-liked direction (avoid repeat or miss some cells).
Observations Under× 40x: WBC Estimates
- Use ×40 high dry, no oil.
- Choose a portion of the peripheral smear where there is only a slight overlapping of the RBCs.
- Calculate 10 fields, take the total number of white blood cells, and divide by 10.
- WBC estimation is performed by multiplying the average number of white blood cells by 2000.
Observations Under × 100: Platelet Estimates
1. Use an oil immersion lens to estimate the number of platelets in each field of view.
2. View at 5-6 fields and take an average.
3. Multiply the average by 20,000.
4. Note any macroplatelets.
Platelets per oil immersion field (OIF)
1) <8 platelets/OIF = decreased
2) 8 to 20 platelets/OIF = adequate
3) >20 platelets/OIF = increased
Evaluate WBC Morphology
o Pay attention to the presence of abnormal white blood cell morphology
o Hypersegmented poly's (5 or more lobes)
o Vacuolation of neutrophils
o Toxic granulation of neutrophils
o Dohle bodies
o Atypical Lymphocytes
o Smudge cells
Manual Differential Counts
- These counts have performed in the same area as the WBC and platelet estimates, with almost no red blood cell contact.
- Use the zigzag method to perform below ×100 (oil).
- Count 100 WBCs, from immature to mature in all cell lines.
- Report the result: Absolute cell count / µl = percentage of cell type in differential x white cell count.
Observing and Recording Nucleated Red Blood Cells (nRBCs)
- If you see 10 or more nucleated RBCs (NRBC), use the following formula to correct the white count:
Right WBC Count = WBC x 100/ (NRBC + 100)
Example: If WBC = 5000 and 10 NRBCs have been calculated
Then 5,000× 100/110 = 4545.50
The corrected white count is 4545.50.
Tips on Diff's
Do not count cells that are disintegrating
• smudge cells
• eosinophil without cytoplasmic membrane and with scattered granules
• Pyknotic cell (extremely condensed and degenerated nuclei, with lobes condensed into small, round clumps with no filaments interconnecting).
• Basket cells
Abnormal differentials
1. 200 Cell diff:
a. WBC> 15.0 (Infants under 1 month and labor units> 20.0)
b. Three or more basophils seen.
3. If you see more than five immature WBCs (or any blasts), ask others to compare the slides and average the results.
4. Correct WBC for NRBC's if you see ten or more NRBCs/100 WBC.
4. Always indicate the number of cells counted on diff.
5. If any cell type is hugely elevated (for example, bands, monocytes, or eosinophils> 20), show that you are aware of the abnormality by circling or checking on the card next to the results.
Recording RBC Morphology
1. Scan area using ×100 (oil immersion).
2. Observe 10 fields.
3. Red cells are examined for size, shape, hemoglobin content, and the presence or absence of inclusions.
4. Abnormal morphology: Red cell morphology is assessed according to See the following sample grading system. Remember that the red blood cell morphology must be scanned in a good counting area.
Two questions should be asked
1. Is the morphology seen in every field?
2. Is the morphology pathologic, isn't it artificially induced?
Red Blood Cell Morphology
The size of normal red blood cells should be the same as the nuclear size of normal lymphocytes.
|
NO.
of Field/ Oil imm. |
Grade
Degree of abnormality |
|
1-6 per oil
imm. field |
1+ |
|
7-10 per OIF |
2+ |
|
11-20 per
OIF |
3+ |
|
> 20 per
OIF |
4+ |
Reporting results
- When describing the morphology of red blood cells, use large cells and small cells as much as possible, rather than simply anisocytosis alone.
- Whenever possible, use a specific cell morphology instead of simply reporting poikilocytosis.
- When red cells are normocytic, normochromic, report them as NORMAL. When you notice abnormal patterns, do not indicate normal on the report form.
- EXAMPLE: 7-10 microcytic RBCs/OIF is reported out as 2+ microcytosis or Moderate microcytosis.
determine a quantitative scale
|
|
Grade
Degree of abnormalities |
|
1 to 5
cells/10 fields |
Slight |
|
6 to 15
cells/10 fields |
Moderate |
|
> 15
cells/10 fields |
Marked |
Grading inclusions
|
|
Grading
inclusions |
|
Rare |
0 to 1/high power
field |
|
Few |
1 to 2/high
power field |
|
Moderate |
2 to 4/high
power field |
|
Many |
> 5/high
power field |
Morphology of WBC in peripheral blood
Normal peripheral blood smear
Stab neutrophil
- Diameter:12-16
- Cytoplasm: pink
- Granules: primary and secondary
- Nucleus: dark purple-blue and dense chromatin
Band neutrophil
Segmented neutrophil
- Diameter: 12-16
- Cytoplasm: pink
- Granules: primary and secondary
- Nucleus: dark purple-blue, dense chromatin, 2-5 lobes
Eosinophil
- Diameter: 14-16
- Cytoplasm: full of granules
- Granules: large refractile, orange-red
- Nucleus: blue, dense chromatin, 2 lobes like a pair of glass
Basophil
- Diameter: 14-16
- Cytoplasm: pink
- Granules: dark blue-black obscure nucleus
- Nucleus: blue
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
Monocyte
- Diameter: 14-20
- Cytoplasm: grey-blue
- Granules: dust-like lilac color granules
- Nucleus: blue, large irregularly shaped and folded
Abnormal changes of WBC morphology
Left-shift and right-shift of neutrophil:
- Left-shift: non-segmented neutrophil > 5%
- Right-shift: hypersegmented neutrophil >3%
- Toxic Granulation
- Auer Bodies (Auer Rod)
- Hypersegmentation
- Anisocytosis of neutrophil
- vacuolization
- Degeneration of nucleus
- Dohle body
Platelet estimate
— Choose an area where RBC barely touch
— No. of platelet in 10 OIO fields is counted multiplied by 20,000
— Anemia or Erythrocytosis
— Average No. of Plts/field x total RBC count
200 RBCs/field
(200 is the average number of RBC/field)
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