In Medical Technology
Making Blood smear
Preparation of blood smear
There are three types of blood smears:
1. The cover glass smear.
2. The wedge smear .
3. The spun smear.
The are two additional types of blood smear used for
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 tube.
Not's : May change RBCs morphology such as Spiculated
(crenated) cells if :
1. Excessive amount of anticoagulant to specimen
2. Old blood - long standing.
3. Warm environment (room temperature) may hasten
1. Placing a drop of blood from mixed sample on a clean
2. Spreader slide using another clean glass slide at 30-40
3. Control thickness of the smear by changing the angle of
4. Allow the blood film to air-dry completely before
staining. (Do not blow to dry. The moisture from your
breath will cause RBC artifact.)
STEPS FOR BLOOD FILM
The thickness of the spread
1. If the hematocrit is increased, the angle of the
spreader slide should be decreased.
2. If the hematocrit is decreased, the angle of the
spreader slide should be increased.
Characteristics of a Good Smear
1. Thick at one end, thinning out to a smooth rounded
2. Should occupy 2/3 of the total slide area.
3. Should not touch any edge of the slide.
4. 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.
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
Failure to push the spreader slide completely across the
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.
Examples of unacceptable smears
A: Blood film with jagged tail made from a spreader with achipped
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.
Examples of unacceptable smears
Biologic causes of a poor smear
1. Cold agglutinin - RBCs will clump together. Warm the
blood at 37° C for 5 minutes, and then remake the smear.
2. Lipemia - holes will appear in the smear. There is nothing
you can do to correct this.
3. Rouleaux - RBC’s will form into stacks resembling coins.
There is nothing you can do to correct this
1. 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.
2. Large cells such as monocytes, immature cells and abnormal cells can be
found in the outer limits of this area.
3. Spun smears produce the most uniform distribution of blood cells.
SLIDE FIXATION & STAINING
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
Methyl alcohol (methanol) is the choice, although ethyl
alcohol ("absolute alcohol") can be used.
Methylated spirit (95% ethanol) must not be used as it
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.
Staining the film
Romanowsky staining: Romanowsky stains are universally
employed for staining blood films and are generally very
There are a number of different combinations of these dyes,
which vary, in their staining characteristics.
1. May-Grunwald-Giemsa is a good method for routine work.
Giemsa stain is thought to produce more delicate staining
2. Wright's stain is a simpler method.
3. 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).
4. Field's stain is a rapid stain used primarily on thin films for
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
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.
Staining procedure (Leishman’s stain)
Thin smear are air dried.
Flood the smear with stain.
Stain for 1-5 min. Experience will indicate the
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 10-15 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.
too acidic suitable too basic
Causes & correction
Too Acid Stain: Too Alkaline Stain:
1. insufficient staining 1. thick blood smear
time 2. prolonged staining
2. prolonged buffering or
3. insufficient washing
4. alkaline pH of stain
3. old stain
1) lengthen staining time
1) check pH
2) check stain and buffer
2) shorten stain time
3) prolong buffering time
3) shorten buffering or
Performing A Manual differential
And assessing RBC Morphology
White Blood Cells. Red Blood Cells,
1. Check for even distribution and 1. Size and shape.
estimate the number present 2. Relative hemoglobin
(also, look for any gross content.
abnormalities present on the
smear). 3. Polychromatophilia.
2. Perform the differential count. 4. Inclusions.
5. Rouleaux formation or
1. Estimate number present.
2. Examine for morphologic
Observations Under ×10
1. Check to see if there are good counting areas
available free of ragged edges and cell
2. Check the WBC distribution over the smear.
3. Check that the slide is properly stained.
4. Check for the presence of large platelets,
platelet clumps, and fibrin strands.
Observations Under ×40 : WBC estimation
1. Using the × 40 high dry with no oil.
2. Choose a portion of the peripheral smear where
there is only slight overlapping of the RBCs.
3. Count 10 fields, take the total number of white
cells and divide by 10.
4. To do a WBC estimate by taking the average
number of white cells and multiplying by 2000.
Observations Under × 100: Platelet Estimates
1. Use the oil immersion lens estimate the
number of platelets per field.
2. Look 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
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
Then 5,000× 100/110 = 4545.50
The corrected white count is 4545.50.
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
Count 100 WBCs including all cell lines from
immature to mature.
Absolute number of cells/µl = % of cell type in
differential x white cell count
Observe one field and record the number of WBC according
to the different type then turn to another field in the snake-
* avoid repeat or miss some cells
Normal peripheral blood smear
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
Stab (Band) neutrophil
Cytoplasm : pink
Nucleus: dark purple
Cytoplasm : pink
Nucleus: dark purple
blue dense chromatin,
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
They are the body's primary defense against bacterial
Increased neutrophils count (neutrophilia)
1. Acute bacterial infection.
2. Granulocytic leukemia.
Decreased neutrophil count (neutropenia)
1. Typhoid fever
3. Viral diseases, including hepatitis, influenza,
rubella, and mumps.
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%
Segmented neutrophile Band neutrophil
Shift to left Increased bands mean acute infection, usually bacterial.
Shift to right Increased hypersegmented neutrophile.
Cytoplasm : full of granules
Granules: large refractile, orange-
Nucleus: blue dense chromatin 2
lobes like a pair of glass
The most common reasons for an increase in the
eosinophil count are
1. Allergic reactions such as hay fever, asthma, or drug
2. Parasitic infection
3. Eosinophilic leukemia
Cytoplasm : pink
Granules: dark blue –black
The purpose of basophils is not completely
Basophile counts are used to analyze allergic
An alteration in bone marrow function such as
leukemia may cause an increase in basophils.
Cytoplasm: medium blue
large a few primary
Nucleus: dark blue \round
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
Lymphocytes increase (lymphocytosis) in:
1. Many viral infections
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.
Cytoplasm : grey blue
Granules: dust-like lilac
large irregularly shaped
Diseases that cause a monocytosis include:
• Monocytic leukemia
1. Do not count cells that are disintegrating
Eosinophil with no cytoplasmic membrane and with
Pyknotic cell (nucleus extremely condensed and
degenerated, lobes condensed into small, round clumps
with no filaments interconnecting).
2- Abnormal differentials
1. 200 Cell diff:
A. WBC > 15.0 (>20.0 for babies under 1 month and labor unit)
B. Three or more basophils seen.
2. If more than five immature WBC's are seen (or any
blasts) let someone else diff slide and average results.
3. Correct WBC for NRBC's if you seen ten or more
4. Always indicate number of cells counted on diff.
5. 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
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
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"
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
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.