3. INTRODUCTION
• Hemoglobin is a CHROMOPROTEIN. (gives red color to the whole
blood)
• Major component of RBC cytoplasm
- 90% of dry weight of mature RBC
- 32% of wet weight of mature RBC
WHAT IF HEMOGLOBIN WAS PRESENT IN THE PLASMA?
1. There would be an increase in viscosity of the blood
leading to increased blood pressure.
2. There would be an increase in osmotic pressure
leading to increased fluid exchange
3. Secretion in urine
4. Rapid destruction by reticuloendothelial system.
4. HEMOGLOBIN
HEME
Iron Protoporphyrin
4 pyrrole rings
derived from acetyl
CoA and glycine
GLOBIN=4
polypeptide chains
2 α chains
- 141 amino acids
- chromosome 16
2 β chains
- 146 amino acids
- chromosome 11
5. SYNTHESIS OF HEMOGLOBIN
• Begins in: PROERYTHROBLAST
STAGE
• First appears in: INTERMEDIATE
NORMOBLAST STAGE
• Continues till: RETICULOCYTE
STAGE
EARLY
NORMOBLAST
INTERMEDIATE
NORMOBLAST
LATE NORMOBLAST
6. PHYSIOLOGICAL
OXYHEMOGLO
BIN
REDUCED
HEMOGLOBIN
DERIVATIVES
CARBAMINOHE
MOGLOBIN
CARBOXYHEM
OGLOBIN
METHHEMOGL
OBIN
SULFHEMOGLO
BIN
CYANMETHEM
OGLOBIN
Derived from physiological by
action of acids, alkalis,
oxidizing or reducing agents
- Loose and
reversible
binding of
oxygen
with iron
atom.
- Each
molecule
of Hb
carries 4
molecules
of oxygen.
- Formed by
release of
oxygen from
hemoglobin.
- Carbon
dioxide is
attached to
the globin
part of Hb.
- Affinity of
Hb for
carbon
dioxide is 20
times that
of oxygen.
- Carbon
monoxide
binds to sites
of oxygen
binding in Hb.
- Affinity for
carbon
monoxide is
200-300 times
that of
oxygen.
- Causes:
1. Drugs
2. Oxidising
agents
- Ferrous iron
converted
to ferric iron
- Gives dusky
appearance
to skin
- Causes:
1. Drugs
(Sulphonam
ides)
2. Chemicals
- Irreversible
- Action of
cyanide on Hb.
7. NORMAL VARIANTS
EMBRYONIC
LIFE FETAL LIFE
AT BIRTH AND
ONWARDS
ABNORMAL
VARIANTS
HBS HBC HBD OTHERS
GOWER 1 HB:
ζ2 ε2
GOWER 2 HB:
α2 ε2
HB PORTLAND:
ζ2 γ2
HBF: α2 γ2
- After 8th week of
development
- γ chain differs from
β in 37 amino acids
- Lesser affinity to 2,3
DPG- so accepts
more oxygen from
maternal blood
- 80% of total Hb at
birth
- Disappears by 5th
month
VARIANTS OF
HEMOGLOBIN
- Different structural forms of hemoglobin that vary in the
structure of the polypeptide chains.
- Identified by hemoglobin electrophoresis
HBA: α2 β2
HBA2: α2 δ2
- Seen in sickle
cell anemia
- Glutamic acid
is replaced by
valine in the
6th position in
β chain
- Seen in
African
Americans
- Glutamate is
replaced by
lysine in the
6th position
- Glutamate is
replaced by
glutamine in
the 12th
position
- HbE
- HbI
- HbJ
- HbH
- Barts’
8. Hb polymerises at low
oxygen tensions
Elongated crystals in
red blood cells
Sickle shape
Difficult to pass
through capillaries
Spiked ends of crystals
rupture red blood cells
Sickle cell anemia
PATHOGENESIS OF SICKLE CELL ANEMIA
9. HEMOGLOBIN LEVELS
AGE GROUP HEMOGLOBIN LEVELS (in
gm/dl)
AT BIRTH 13.6-19.6
2-6 MONTHS 9.5-14.0
6 MONTHS-6 YEARS 11.0-14.0
6 YEARS- 12 YEARS 11.5-15.5
ADULT FEMALE
Pregnant 11.0-14.0
Non Pregnant 12.0-15.0
ADULT MALE 13.0-17.0
WHY ARE HEMOGLOBIN LEVELS LOWER IN FEMALES?
- NOT DUE TO MENSTRUAL LOSSES (20-30 ML)
- Estrogen has inhibitory effect on erythropoietin
- Androgen has stimulatory effect on erythropoietin
WHY ARE HEMOGLOBIN LEVELS HIGHER IN
NEWBORNS?
- Increased RBC count
- Relative hypoxia in infants acts as a potent stimulus
for erythropoietin secretion
10. INDICATIONS OF HEMOGLOBIN ESTIMATION
To detect presence
and severity of
anemia
To screen for
polycythemia
To assess response to
a specific treatment
in anemia
Estimation of red cell
indices
Selection of blood
donors
- Anemia refers to reduced
hemoglobin or reduced
oxygen carrying capacity of
blood.
- Anemia is best assessed by:
1. Hemoglobin estimation
2. Packed cell volume
estimation
- According to severity,
anemia can be classified
into:
1. Mild: >10 gm/dl but less
than lower side normal
range
2. Moderate: 7-10 gm/dl
3. Severe: <7 gm/dl
- Refers to hemoglobin levels
above normal
- Classification:
1. PRIMARY: Total red cell
mass increased,
erythropoietin normal eg:
POLYCYTHEMIA VERA
2. SECONDARY: Total red cell
mass increased,
erythropoietin increased
Eg: HYPOXIA
3. RELATIVE: Plasma volume
reduced,total red cell mass
normal Eg: DEHYDRATION
- Along with Packed Cell
Volume (PCV) and RBC
count
𝑀𝐶𝐻(𝑀𝑒𝑎𝑛 𝑐𝑜𝑟𝑝𝑢𝑠𝑐𝑢𝑙𝑎𝑟
ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛)
=
𝐻𝑏 (𝑔𝑟𝑎𝑚𝑠 𝑝𝑒𝑟 𝑑𝑙) × 10
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑚𝑖𝑙𝑙𝑖𝑜𝑛𝑠 𝑝𝑒𝑟 𝑐𝑢. 𝑚𝑚)
𝑀𝐶𝐻𝐶(𝑀𝑒𝑎𝑛 𝑐𝑜𝑟𝑝𝑢𝑠𝑐𝑢𝑙𝑎𝑟
ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛)
=
𝐻𝑏 (𝑔𝑟𝑎𝑚𝑠 𝑝𝑒𝑟 𝑑𝑙) × 100
𝑃𝐶𝑉 (%)
11. METHODS OF HEMOGLOBIN ESTIMATION
COLORIMETRIC GASOMETRIC CHEMICAL
SPECIFIC
GRAVITY
ELECTRONIC
ANALYSER
Color comparison
is done between
standard and test
sample.
VISUAL
- Tallqvist
- Sahli’s
acid
hematin
method
- WHO
Hemoglob
in color
scale
PHOTO
ELECTRIC
- Cyanmet
hemoglo
bin
- Oxyhem
oglobin
- Alkali
hematin
method
- Measures
oxygen carrying
capacity
- Apparatus: VAN
SLYKE
- 1 gram Hb
contains 1.34 ml
of oxygen
- Only measures
oxyHb and
reduced Hb
- Does not
measure
carboxyHb,
sulfHb or metHb
- Time consuming
- Expensive
- Iron content of
hemoglobin is
estimated.
- 100 grams of
hemoglobin
contains 374 mg
iron.
- Tedious
- Time consuming
- Rapid and
simple method
- Used for
selection of
blood donors
- It is done by
CuSO4 method
- Gives an idea about
cell count and
hemoglobin
concentration
- Hemolysing agent
causes release of
hemoglobin.
- Potassium ferricyanide
and cyanide are added
to this forming
cyanmethemoglobin.
- Hb is analysed by
spectrophotometry,
12. TALLQVIST CHART
• Contains a series of
lithographed colors.
• Corresponds to Hb levels
between 10-100%
• Cheap and simple method.
• Margin of error=20-50%
• Method is currently
obsolete.
Drop of
blood is
obtained
by finger
puncture
Placed on
absorbent
paper
Color
produced
is matched
against
color on
the chart
13. SAHLI’S HEMOGLOBINOMETER METHOD
PRINCIPLE:
Blood containing Hb is
mixed with acid
Acid hematin is
produced
Diluted till the color is
the same as the golden
brown tinted glass rods
in the comparator box.
Hemoglobin level is
read from the scale
(g%)
EQUIPMENT: REAGENTS:
DISTILLED
WATER
0.1 N HCl
OTHERS:
STERILE
LANCET
STERILE
GAUZE
COTTON
SWABS
70%
ALCOHOL
14. PARTS
COMPARATOR BOX - Rectangular plastic box
- Slot in the middle to accommodate the calibrated
hemoglobin tube
- Non fading, standardised, golden brown glass rods fitted on
each side of the slot matching the color.
- Opaque white glass fitted behind the slot to provide uniform
illumination during direct visual color matching.
HEMOGLOBIN TUBE - Square or round glass tube is calibrated in two colors:
1. Yellow color: g Hb% (preferred)
2. Red color: % Hb
- Brush is also provided to clean the tube.
HEMOGLOBIN PIPETTE - Glass capillary pipette
- Single calibration mark: 20 μL
- No bulb because there is no dilution of blood
STIRRER - Thin glass rod with flattened end
- Use: Stirring and mixing the blood and dilute acid
PASTEUR PIPETTE - 8 to 10” glass tube
- Drawn to a long thin nozzle with a rubber teat
- Acts as a dropper
15. PROCEDURE
8-10 drops
of N/10 HCl
are placed in
the Hb tube.
Finger
pricked
under
aseptic
conditions.
First two
drops wiped
away.
When a
large drop
begins to
form, blood
is drawn
using the
pipette till
the 20
cu.mm mark
Blood
sticking to
the tip of
the pipette
is wiped
using a
cotton swab
Tip of the
pipette is
immersed in
the bottom
of the acid
solution and
the blood is
expelled
gently
Pipette is
withdrawn
from the
tube. We
ensure that
no mixing is
done
outside the
tube.
Hb tube is
put back in
the
comparator
and allowed
to stand for
6-8 minutes
Now the
solution
thus formed
is diluted
with
distilled
water till its
color
matches
the color of
the
standard
tinted glass
rods in the
containter.
16.
17. QUESTIONS!
• WHAT IS NORMALITY?
• WHAT IS N/10 HCL? HOW TO PREPARE IT?
• CAN WE USE STRONG ACIDS OR ALKALIS INSTEAD OF N/10 HCL? WHY?
• WHAT IF WE TAKE LESS THAN 8-10 DROPS OF N/10 HCL?
• WHAT IF WE TAKE MORE THAN 8-10 DROPS OF N/10 HCL?
• WHY DO WE DISCARD THE FIRST TWO DROPS?
• WHY DO WE HAVE TO WAIT FOR 6-8 MINUTES?
• CAN WE USE TAP WATER FOR DILUTING INSTEAD OF DISTILLED WATER?
• CAN WE USE N/10 HCL FOR DILUTING INSTEAD OF DISTILLED WATER?
18. WHAT IS NORMALITY?
- No of gram equivalents in 1 LITRE of water.
WHAT IS N/10 HCL? HOW TO PREPARE IT?
- 1 N HCl= 1+35 g=36 g of HCl in 1 litre of water
- So N/10 HCl is 36 g of HCl in 10 litres of water or
3.6 gm HCl in 1 litre of water.
- So basically diluting a 1 N solution 10 times will
give us N/10 HCl.
CAN WE USE STRONG ACIDS OR ALKALIS INSTEAD OF
N/10 HCL? WHY?
- NO. Because strong acids and alkalis will cause
disruption of hemoglobin.
WHAT IF WE TAKE LESS THAN 8-10 DROPS OF N/10
HCL?
1. Blood may not mix well and may clot.
2. All of the Hb will not be converted to acid hematin.
So this will lead to a false low value.
WHAT IF WE TAKE MORE THAN 8-10 DROPS OF N/10
HCL?
The final color that would develop would be much
lighter than the actual. This would give a false high
value.
CAN WE USE TAP WATER FOR DILUTING INSTEAD OF
DISTILLED WATER?
No. Acid hematin is already not a true solution.
Hence, there is a risk of turbidity. Using tap water
which has salt, would further increase the chance of
turbidity.
CAN WE USE N/10 HCL FOR DILUTING INSTEAD OF
DISTILLED WATER?
Yes. All the Hb would have already been converted to
acid hematin, so adding N/10 HCl would not further
deepen the color.
19. CAN WE USE N/10 HCL FOR DILUTING INSTEAD OF
DISTILLED WATER?
Yes. All the Hb would have already been converted to
acid hematin, so adding N/10 HCl would not further
deepen the color.
WHY DO WE HAVE TO WAIT FOR 6-8 MINUTES?
To give time for the acid hematin to form.
WHY DO WE DISCARD THE FIRST TWO DROPS?
1. To prevent contaminants in the blood that may
come from the skin surface.
2. Initial drops might be diluted with tissue fluid and
can give a false estimation of Hb.
20. ADVANTAGES DISADVANTAGES
- Simple
- Quick
- Accurate
- Inexpensive
- Can be used for mass
surveys
- Acid hematin is not in a
true solution. So there
may be turbidity.
- Only measures
oxyhemoglobin and
reduced hemoglobin.
- CarboxyHb and MetHb
are not estimated.
SOURCES OF ERROR
PERSONAL
- Not taking exact
20 cu.mm
- Not giving
enough time for
acid hematin to
form
- Using old
comparator with
faded glass rods
TECHNICAL
- Color matching is
done visually so it
becomes subjective
PRECAUTIONS
- Donot squeeze finger while collecting blood.
- No blood should be collected that sticks outside
the pipette tip.
- Only recommended time should be allowed for
each step.
- Avoid overdilution because color cannot be
concentrated once it becomes lighter than the
color in the comparator.
- There should be a uniform golden brown color
throughout the solution. Dark color near the
bottom of the tube indicates improper mixing.
- Take 3 readings to reduce personal error.
21. CAUSES OF ALTERED HEMOGLOBIN
LEVELS
INCREASED
Experimental
- >20 cu mm blood
- Blood sticking outside pipette tip
Increased red cell count
PHYSIOLOGICAL
- Females
- Newborns
- High altitude
PATHOLOGICAL
- Polycythemia
- Hypoxia due to cardiac or
pulmonary cause
DECREASED
Experimental
- Sample diluted with tissue fluid
<20 cu mm blood
- Acid hematin not allowed to
develop fully
Decreased red cell count
PHYSIOLOGICAL
Pregnancy (due to hemodilution)
PATHOLOGICAL
Anemia
22. WHO HEMOGLOBIN COLOR SCALE
• Similar to Tallqvist method
• Technical modifications done to
improve accuracy and reliability
• Rapid, simple, inexpensive and
reliable.
• Consists of printed set of
colours with hemoglobin values
between 4-14 g/dl.
• Developed by WHO after
extensive field trials.
USES
Detection and management
of anemia in
underdeveloped countries.
Screening blood donors
Screening children and
females in health programs
Monitoring iron treatment
Decision making about
referral
Point of care device
23. CYANMETHHEMOGLOBIN METHOD
METHOD OF CHOICE
PRINCIPLE:
Blood is mixed with potassium
ferricyanide, potassium cyanide
and a non ionic detergent
RBCs are lysed producing a Hb
solution
Potassium ferricyanide convers
hemoglobin to MetHb
MetHb is converted to
CyanmetHb by potassium
cyanide
Absorbance is read from a
spectrophotometer at 540 nm
EQUIPMENT:
SAHLI’S
PIPETTE
PHOTOELECTRIC
COLORIMETER OR
SPECTRO
PHOTOMETER
5 ML
PIPETTE
REAGENTS:
DRABKIN’S
SOLUTION
CYANMETHB
STANDARD
SOLUTION WITH
KNOWN HB
VALUE
SPECIMEN:
- EDTA
ANTICOAGULAT
ED VENOUS
BLOOD
- BLOOD
OBTAINED
FROM SKIN
PUNCTURE
TEST TUBE
24. PROCEDURE
5 ml of
Drabkin’s
reagent taken
in a test tube.
20 μL of blood
is added to it.
The tube is
stoppered,
mixed by
inverting
several times
and allowed to
stand for 5
minutes.
The sample is
transferred to
a cuvette.
The
absorbance is
read in a
spectrophoto
meter at 540
nm or a photo
electric
colorimeter
using a yellow
green filter
Absorbance of
the known
standard is
also taken.
Hb value is
derived from
the formula
shown or a
previously
prepared
graph or table.
- Diluted cyanmet
standards are available
for preparation of a
calibration graph.
- Standard cyanmetHb
solution can also be
serially diluted with
Drabkin’s solution.
25. COMPOSITION OF DRABKIN’S
SOLUTION
pH= 7.0-7.4
1. Potassium ferricyanide: 200 mg
2. Potassium cyanide: 50 mg
3. Potassium dihydrogen phosphate:
140 mg
4. Non ionic detergent: 1 ml
5. Distilled water: 1000 ml
POINTS TO NOTE:
1. Erroneous results can be seen in:
- Lipemic blood
(HYPERTRIGLYCERIDEMIA)
- Leucocytosis (>25,000/μL)
- Plasma protein abnormality
(MULTIPLE MYELOMA,
WALDENSTORM’S
MACROGLOBULINEMIA)
2. CyanmetHb is stable. So delay in
taking the reading of absorbance will
not affect the results.
3. Measures all forms except sulfHb
26. ADVANTAGES DISADVANTAGES
- All forms of Hb except sulfHb are
converted to cyanmetHb
- No visual error as color matching is
not required
- Stable cyanmetHb
- Absorbance may be measured soon
after dilution.
- Reliable and stable reference standard
is available from WHO for direct
comparison.
- Diluted blood has to stand for a period
of time
- Potassium cyanide is poisonous so it
can NEVER BY PIPETTED BY MOUTH.
- Abnormal plasma proteins or
leukocytosis can cause turbidity when
dilute with Drabkin’s solution.
27. SPECIFIC GRAVITY METHOD:
- SG of CuSO4=1.053, equivalent to Hb
level=12.5 g/dl
- Rapid and simple
- Gives approximate Hb value
- Used for selection of donors in blood
banks.
- Abnormally high values can be seen
in:
1. Leucocytosis (eg: CML)
2. Hypergammaglobulinemia (eg:
Multiple myeloma)
Blood is mixed
with a weak
ammonia
solution.
Absorbance of
the solution is
measured at
540 nm or in a
photoelectric
colorimeter
using a yellow
green filter.
Absorbance of
test sample is
then
compared to
standard.
OXYMETHB METHOD:
- Rapid and simple
- No stable standard solution available
- Color of oxyHb fades quickly
- No Hb derivatives other than oxyHb
are measured.
A drop of
blood is
allowed to
fall on CuSO4
solution of
SG=1.053
from a height
of 1 cm.
Drop of blood
gets covered
with copper
proteinate
and remains
discrete for
15-20
seconds.
If drop sinks,
SG is higher
than CuSO4
and
ACCEPTABLE
FOR
DONATION.
28. AUTOMATED ANALYSER
• Modified cyanmetHb method
• Other chemicals are sodium lauryl sulphate, imidazole, sodium
dodecyl sulphate
• Measurements are made at various wavelengths depending on the
final stable product.
3 PART DIFFERENTIAL ANALYSERS 5 PART DIFFERENTIAL ANALYSERS
- Two chambers:
1. Hb/WBC chamber
2. RBC/Platelet chamber
- It is called so because it categorizes
white blood cells into three main types:
neutrophils, lymphocytes, and
monocytes.
- Provides accurate red cell parameters,
platelet parameters and reticulocyte
parameters.
- Called so because it categorises white
blood cells into five types: neutrophils,
lymphocytes, eosinophils, monocytes
and basophils.
31. PERIPHERAL BLOOD SMEAR
• Specimen for microscopic examination prepared by spreading a drop
of blood across a glass slide followed by staining using Romanowsky’s
stains.
• Uses:
1. Cause of anemia or thrombocytopenia
2. Identifying type of leukemia
3. Diagnosing hemoparasitic infections
4. Monitoring effect of chemotherapy on bone marrow
5. To provide a direction for further investigations that will help in
arriving at the correct diagnosis.
32. PREPARATION OF A BLOOD SMEAR
Small drop of
blood is
placed in the
centre line
about 1 cm
away from
one end of
the glass slide
Spreader slide
is placed at an
angle of 30° in
front of the
drop and then
drawn back to
touch the
drop of blood.
Smear is
made by
smooth,
forward
movement of
the spreader
along the
slide.
Rapidly air
dried
Patient’s
name, lab
number and
date are
written
Fixed
immediately
with absolute
methyl
alcohol for 2-3
minutes.
• Length of slide: 75x25 mm
• Thickness of slide: 1 mm
• Slide should be clean, dry and grease free.
• Blood sample: EDTA anticoagulated venous blood or
capillary (finger prick) blood.
• BEST IF DIRECTLY MADE FROM SKIN PUNCTURE
• Length of smear: 3 cm
33. IDEAL BLOOD SMEAR
• Tongue shaped with a
smooth tail
• Does not cover the entire
area of the slide
• Has both thick and thin areas
with gradual transition
• Does not have any lines or
holes.
34. STAINING OF BLOOD SMEAR
• Blood smears are routinely
stained by one of the
Romanowsky stains.
• Stains included are:
1. May-Grunwald-Giemsa stain
2. Jenner
3. Wright’s
4. Leishman’s
5. Field’s
COMPONENTS OF ROMANOWSKY STAINS
ACIDIC BASIC
- Negatively charged
- AFFINITY FOR BASIC
COMPONENT.
- Binds to cationic sites
- Gives orange red color to
Hb and eosinophilic
granules.
- Eg: Eosin Y
- Positively charged
- AFFINITY FOR ACIDIC
COMPONENT.
- Binds to anionic sites
- Gives blue gray color to
nucleic acids,
nucleoproteins and
basophilic granules.
- Eg: Methylene blue,
Azure B
35. LEISHMAN STAIN
• Compound dye- eosin and methylene blue dissolved in acetone free
methyl alcohol.
EOSIN METHYLENE BLUE ACETONE FREE METHYL ALCOHOL
- Acidic dye
- Stains basic substances
- Gives orange red color to Hb and
eosinophilic granules.
- Basic dye
- Stains acidic granules, leukocyte nuclei,
basophil granules and neutrophilic granules
(weakly)
- Methyl alcohol is a fixative
- Alcohol precipitates plasma proteins which
acts as a glue and fixes the blood cells to
the slide
- Alcohol also preserves the morphology and
chemical status of the cells.
- Acetone is a strong lipid solvent so it will
cause crenation, shrinkage and even lysis of
cells. Hence, ACETONE FREE SOLUTION.
- Water also causes hemolysis and rouleaux
formation. So, FREE FROM WATER also.
36. PROCEDURE
The smear is
air dried and
fixed with
methanol for
2-3 minutes.
The smear is
covered with
Leishman
stain for 2
minutes
Twice the
volume of
buffered
water is
added and left
for 5-7
minutes
The stain is
then washed
away in a
stream of
buffered
water
The back of
the slide is
cleaned and
the slide is
then kept to
dry
The slide is
mounted with
a suitable
mounting
media with a
clean and dry
coverslip
37. QUESTIONS!
• Why do we wait for 2 minutes after applying Leishman’s stain?
• Sometimes, a greenish scum can be seen over the smear. Is it bad?
• Why is buffered water used and not distilled
water?
• Can we use tap water?
38. WHY DO WE WAIT FOR 2 MINS?
1. Alcohol will fix the blood cells on to the glass slide.
2. Alcohol will preserve the shape and chemistry of
the cells to its living state as much as possible.
SOMETIMES, A GREENISH SCUM CAN BE SEEN
FLOATING OVER THE SMEAR. IS IT BAD?
No! It indicates that staining has been done properly.
Indicates ripening of the stain.
WHY IS BUFFERED WATER USED AND NOT DISTILLED
WATER?
- Buffered water is phosphate buffer where pH is
adjust to 6.8
- This pH allows stain to penetrate better.
CAN WE USE TAP WATER?
No. Cells will not be stained properly because of the
unknown pH and salt content.
Impurities in tap water can also show up as artefacts
on the blood film.
39. FEATURES OF A WELL STAINED BLOOD SMEAR
UNDER NAKED EYE UNDER LOW POWER (100X) UNDER HIGH POWER (400X)
- Smear appears translucent and
bluish pink when seen against a
white surface
- Thickness is uniform
throughout.
- Red cells appear as dots,
uniformly spread out in a single
layer.
- WBC cannot be differentiated.
- No stain precipitate is present
on the smear.
- Red cells are stained dull orange
pink and show central pallor
- WBCs lie scattered among the
red blood cells.
1. Neutrophils: Pale pink
cytoplasm and mauve purple
granules
2. Eosinophils: Pale pink
cytoplasm and orange red
granules
3. Basophils: Blue cytoplasm and
dark blue-violet granules.
4. Monocytes: Gray-blue
cytoplasm, fine reddish
granules
5. Small lymphocytes: Dark blue
cytoplasm
6. Platelets: purple
40.
41.
42. UNDERSTAINED
Causes:
1. Insufficient staining
time
2. Prolonged buffering or
washing
3. Old stain
OVERSTAINED
Causes:
1. Thick blood smear
2. Prolonged staining
3. Insufficient washing
4. Alkaline pH of stain
components
WELL STAINED
43. TO READ
• Abnormalities in shape and size of RBC.
• Leucocyte classification
• Causes of increase or decrease in each type of leucocyte
• Function of leucocytes
• Causes of increase or decrease in platelet count.
