BLOOD SMEAR

EXAMINATION
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 specific purposes
4. Buffy coat smear for WBCs < 1.0×109/L
-

5. 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 :
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,
1. Excessive amount of anticoagulant to
specimen
2. Old blood - long standing.
3. Warm environment (room temperature) may
hasten changes.
PROCEDURE
• placing a drop of blood from mixed sample on a
clean glass slide.
• Spreader slide using another clean glass slide at
30-40 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.)
high HCT

small angle
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low HCT

large angle
CHARACTERISTICS OF A GOOD SMEAR

1. [Thick at one end, thinning out to a smooth

Y
rounded feather edge.
2. C S
Should occupy 2/3 of the total slide area.
-

3. 1
Should not touch any edge of the slide.
-

4. Should be margin free, except for point of

application.
tail body head
MORPHOLOGIC CHANGES DUE TO
AREA OF SMEAR
• Thin area-CSpherocytes which are really
&
"spheroidocytes" or flattened red cells.
C
True spherocytes will be found in other
(Good) areas of smear.
C

?
• 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..
C
COMMON CAUSES OF A POOR BLOOD
SMEAR
1. Drop of blood too large or too small.-
2. Spreader slide pushed across the slide in a jerky manner.-
3. Failure to keep the entire edge of the spreader slide against the
slide while making the smear.
4. Failure to keep the spreader slide at a 30° angle with the slide.-
5. Failure to push the spreader slide completely across the slide.-
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.
BIOLOGIC CAUSES OF A POOR SMEAR

1. CCold agglutinin - RBCs will clump

together. Warm the blood at 37° C for 5
&
minutes, and then remake the smear.
2.
3Lipemia - holes will appear in the
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smear. There is nothing you can do to
correct this.
3. Rouleaux -CRBC’s will form into stacks
resembling coins. There is nothing you
can do to correct thisS
SLIDE FIXATION &
STAINING
LEISHMAN'S STAIN
PRINCIPLE LIKE ROMANOWSKY PRINCIPLE

C Leishman's stain((
: a polychromatic stain&
• Methanol : fixes cells to slide
-

• Cmethylene blue stains RNA,DNAC

-

=== blue-grey color

• & Eosin stains hemoglobin,
( eosin granules
=== orange-red color
• &pH value of phosphate buffer is very important
&
STAINING 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 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:
1. insufficient staining time -
2. prolonged buffering or washing -
3. old stain -

• Correction:
4) lengthen staining time -
5) check stain and buffer pH
6) shorten buffering or wash time -
• Too Alkaline Stain:
1. thick blood smear -
2. prolonged staining-
3. insufficient washing ~
4. alkaline pH of stain components
• Correction :
5) check pH -
6) shorten stain time -
7) prolong buffering time -
 PERFORMING A MANUAL
DIFFERENTIAL AND ASSESSING RBC
MORPHOLOGY
PRINCIPLE

• White Blood Cells.

1. Check for even distribution and
estimate the number present (also, look
for any gross abnormalities present on
the smear).
2. Perform the differential count.
3. Examine for morphologic
abnormalities.
PRINCIPLE

• Red Blood Cells, Examine for:

1. Size and shape.
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination
PRINCIPLE

• Platelets.
1. Estimate number present.
2. Examine for morphologic
abnormalities.
PROCEDURES
• Observations Under ×10
1. Check to see if there are good counting
areas available free of ragged edges and cell
clumps.
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.
 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
OBSERVATIONS UNDER× 40X : WBC
ESTIMATES
• 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.
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)
5) <8 platelets/OIF = decreased
6) 8 to 20 platelets/OIF = adequate
7) >20 platelets/OIF = increased
EVALUATE WBC MORPHOLOGY
• Note if any abnormal white cell morphology is present
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 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
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:
Corrected WBC Count =
WBC x 100/( NRBC + 100)
Example : If WBC = 5000 and 10 NRBCs have
been counted
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 with no cytoplasmic membrane and
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with scattered granules
• Pyknotic cell (nucleus extremely condensed and
degenerated, lobes condensed into small, round
clumps with no filaments interconnecting).
• Basket cells
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.
• 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
NRBCs/100 WBC.
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 the
results.
 RECORDING RBC MORPHOLOGY
1. Scan area using ×100 (oil immersion).

2. Observe 10 fields.

3. Red cells are observed 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. Note that red cell morphology must be
scanned in a good counting area.

Two questions should be asked

5. Is the morphology seen in every field?

6. Is the morphology pathologic and not artificially induced?

Table 1 & 2 represents a system derived to determine a quantitative scale.

RED BLOOD CELL MORPHOLOGY
• A normal red blood cell should be approximately the
same size as a normal lymphocyte nucleus or 2 normal
sized red blood cells should fit side by side across a
normal sized poly (not a hypersegmented poly).

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
• Where possible use macrocytic and microcytic,
rather than simply anisocytosis alone, when
describing red cell morphology.
• Use specific cell morphology when possible, rather
than simply reporting poikilocytosis.
• When red cells are normocytic, normochromic,
report out as NORMAL. When abnormal
morphology has been noted, DO NOT indicate
normal on the report form.
• EXAMPLE: 7-10 microcytic RBC's/OIF is reported
out as: 2+ microcytosis or Moderate microcytosis.
DETERMINE A QUANTITATIVE SCALE

1
GRADING INCLUSIONS

2
MORPHOLOGY OF WBC
IN PERIPHERAL BLOOD
Normal
NORMAL PERIPHERAL BLOOD SMEAR
STAB NEUTROPHIL

● Diameter:12-16
● Cytoplasm : pink
● Granules: primary
secondary
● Nucleus: dark purple blue
● dense chromatin
BAND NEUTROPHIL
SEGMENTED NEUTROPHIL

● Diameter: 12-16
● Cytoplasm : pink
● Granules: primary
secondary
● Nucleus: dark purple blue
dense chromatin
2-5 lobes
SEGMENTED NEUTROPHIL
EOSINOPHIL

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

● Diameter: 14-16
● Cytoplasm : pink
● Granules: dark blue –black
obscure nucleus
● Nucleus: blue
BASOPHIL
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
LYMPHOCYTE
MONOCYTE

● Diameter: 14-20
● Cytoplasm : grey blue
● Granules: dust-like lilac
color granules
● Nucleus: blue
large irregularly shaped and
folded
MONOCYTE
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
Blood Grouping&
Typing
Principle
 Landsteiner’s law
• If an antigen is present on a patient’s red blood
cells , the corresponding antibody will not be
present in the patient’s plasma under normal
conditions. Loading…
 Rh system
• Rh factor

• If it is present Rh D-
Positive

• If it is absent Rh D-
Negative
 Methods

• Slide or Tile

• Tube
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• Microplate

• Gel system
Loading…
GROUP
A

GROUP B

GROUP
AB
 • Advantages
1. Preliminary typing tests
2. Use during camps
• Disadvantages
3. Less sensitive
4. Drying of reaction giving rise to false
positive tests
Frequently Asked
Questions
Importance of
• Blood transfusion: Blood grouping and
Blood grouping cross matching are always done prior to
blood transfusion to any person.
• Haemolytic disease of newborn: There is a
role of Rh and ABO blood grouping in HDN.
• Paternity disputes: ABO and Rh blood
grouping are used as a routine test in such
cases. It is possible to disprove parentage
but impossible to prove parenthood.
• Medicolegal use: In criminal cases, whether
a stain is blood or not, and its blood group
can be detected.
• Susceptibility to various diseases:
Persons with blood group O are more
susceptible to peptic ulcer while persons with
blood group A are more susceptible to
gastric cancer
 Discussion…
Other grouping system:
• Lewis
• MNSs
• Kell
• Duffy
• Kidd
• Lutheran
•P
• Ii
 Cross Matching
• Major cross match

Mixing donor’s red cells with recipient’s

serum

• Minor cross match

Patient’s cell with donor’s plasma

 Bombay blood group

• Blood group which lacks

-A
-B
-H antigens.
• Anti-A, anti-B, anti-A,B, and anti-H present in
the serum.
• Can only be transfused with blood from
another Bombay
 Transfusion reactions
• Hemolytic transfusion reaction

• Non hemolytic transfusion reactions

-Febrile illness
-Utricaria
-Anaphylaxis
-Circulatory overload
-Pulmonary edema
-GVHD
 Donor criteria
• Age:
18-60yrs

• Weight:
Minimum 45 kg

• Haemoglobin:
Minimum 12.5gm/dl
• Normal vitals

Types of blood grouping

• Forward typing
The unknown test cells are antigen typed
against potent and specific anti-A & anti-B

• Reverse typing
The unknown serum is tested against known
group A & group B cells
• Universal donor
O Blood group

• Universal recipient
AB Blood group
 Screening
• Malaria
• Syphilis
• Hepatitis B
• Hepatitis C
• HIV 1&2
 Blood components
• Packed red cells
• Fresh frozen plasma
• Platelet rich plasma
• Cryoprecipitate
• Leukocyte poor red cell concentrate
• Platelet concentrate
• Granulocyte concentrate
• Single donor plasma
• Cryo poor plasma
 Anticoagulants
• Acid Citrate Dextrose

• Citrate Phosphate Dextrose

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• Citrate Phosphate Dextrose Adenine-1

• Additive solution: ADSOL/SAG-M

 Plasma Derivatives
• Albumin
• Plasma protein fraction
• Factor VIII concentrate
• Fibrinogen
• Immunoglobulins
• Other coagulation factors
Thank you
Hemoglobin estimation
 Methods for Hb estimation
• Colorimetric method - based on colour
• Physical method – based on specific gravity
• Chemical method – based on iron content of
haemoglobin
• Gasometric method – based on oxygen
combining capacity of haemoglobin
 Colorimetric method
• The colour of a solution is directly proportional to the amount
of a coloured substance in the solution
BEER’S LAW
• Optical density of the coloured solution is directly proportional
to the concentration of the coloured material in the solution.
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• The amount of hemoglobin determines the optical density
Methods based on above law:
• Alkaline haematin method -
•~Photoelectric method(cyanmethemoglobin,oxyhemoglobin)
• Acid haematin method - Sahli’s method -
• Cell counter or autoanalyser v
 Sahli’s method
Principle
• Haemoglobin is converted to acid hematin by
using N/10 HCL.
• Brown colour of acid hematin is matched
against the brown colour of the comparator.
HAEMOMETER

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 Equipments
• Sahli haemoglobinometer -
• Sahli pipette (marked at 0.02ml) ~
• Stirrer
• Dropping pipette
• N/10 hydrochloric acid -
• Distilled water
 Technique
• Take N/10 HCL upto lowest mark on Hb tube.
• Add 20µl blood in Sahli pipette
• Add blood sample to the acid solution ,mix
with a glass stirrer and wait for 10 mins.
• Then add distilled water drop wise ,with
continuous mixing until colour matches with
the colour of glass plate.
• Note the reading in gm/dl.
 Advantages
• Simple bedside test
• Easy to perform
• Quick
• Reagents and apparatus are cheap
• Does not require technical expertise
 Disadvantages

•The colour of the

comparator fades
• Visual error in
matching
•Acid hematin colour
 starts fading after 10
Errors

• Air bubble in the pipette

• Blood outside the tip of the pipette.
• Blood column should not exceed the
mark
• HCl should not be less
• Wait for a minimum of 10 min after
mixing acid and blood.
• Normal values (Depend on the age and sex of
the individual)
l Reference range

Birth
1 year
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12.6 +/- 1.5 gm/dl
2-6 years 12.5 +/- 1.5 gm/dl
6- 12 years 13.5 +/- 2.0 gm/dl
Adult female 12 – 15 gm/dl
Adult male 13 - 17 gm/dl
 Significance of Hb value
• Low Hb values- Anemia

• High Hb values- Polycythemia

Primary - Polycythemia rubra vera

Secondary– 1. Kidney tumours

2. High altitude
3. Hypoxia-COPD
 4.Congenital heart

Cyanmethaemoglobin method

• More accurate method for the

estimation of Hb.

• Reagents:
• Drabkin’s solution
Dihydrogen potassium
phosphate-
 Principle
• Hb is oxidized to
methemoglobin by
potassium
ferricyanide.
• MethHb in turn is
converted to a stable
cyanmethemoglobin
by Potassium
cyanide.
• Colour of this
 Procedure
• Take 5 ml of Drabkin
(Cyanmethaemoglobin) reagent in a
test tube
• Pipette 20µl of blood using a Sahlis
pipette/micropipette, add to the
Drabkin reagent and mix thoroughly
• Allow to stand for 5 min
• Take reading of test and standard in a
spectrophotometer or photoelectric T
510

colorimeter at a wavelength of 540 nm

using Drabkin (Cyanmethaemoglobin)
reagent in the blank tube to set optical
 Calculations:
• Hb concentration in test (g %) =
OD of test X Hb Concentration of STD X 251*
OD of STD 100

* Dilution factor
ADVANTAGES:
•No visual error
•All HB except
sulfhaemoglobin are
converted
•Cyanmethaemoglobin
 is Frequently
a stableasked
compound
questions
1.List different methods of estimation of
hemoglobin
2. State the method used for Hb estimation in a
blood donation camp – Copper sulphate
method.
3.State the principle of cyanmethaemoglobin
method
4.List the contents of Drabkin reagent
5. Explain the advantages and disadvantages of
cyanmethaemoglobin method