PHYSIOLOGICAL VARIATIONS IN Hb
CONCENTRATIONS
- Hemoglobin can change
depends on the status of your
body
INCREASED HEMOGLOBIN
1. Exercise- your body is panting
which increased the oxygen
need. Hemoglobin is increased
when there is an increased RBC
production.
2. Posture- Lying to sitting
increased Hb Concentration
3. Altitude- The higher the altitude
the lesser the oxygen. The body
will produce more RBC as a
coping mechanism leading to
more hemoglobin.
4. Smoking- Kind of hemoglobin
found in smokers:
carboxyhemoglobin; causes
less oxygen to bind in
Hemoglobin which makes the
body to produce more RBC.
DECREASED HEMOGLOBIN
ELDERLY
: Due to Renal insufficiency,
Testosterone deficiency, Diminished
erythropoiesis and Myelodysplasia.
Renal insufficiency - (kidney is
related. Hormones in kidney that
helps in RBC production is the
erythropoietin which managed by jak
2. If there is a problem in the kidney,
the erythropoietin is not properly
produced causing decreased RBC
and hemoglobin).
Testosterone deficiency -
Testosterone promote erythropoiesis
which increased RBC to increased
Hb. In this case, RBC is decreased and
hemoglobin is decreased.)
Diminished erythropoiesis- (Bone
marrow is damaged leading to
decreased RBC and decreased
hemoglobin)
Myelodysplasia- (cell related in
myelo: WBC. There is an increased
production of WBC specifically the
young cells(blast). The WBC will over
run the bone marrow leading to a
decreased RBC in bone marrow.
INDICATIONS FOR Hb ESTIMATION
1. Determine Presence and
severity of anemia
- What is the level of your
hemoglobin if you have
anemia: low.
2. Screening for polycythemia
- Confirmation is via blood
smear: what we saw in the
blood smear is:
1. Poikilocytes
2. Anisocytes
- Decreased RBC, WBC, Platelets
3. Response to specific therapy in
anemia
- In Iron deficiency Anemia, the
drug is Ferrous sulfate. 1 week
take but prior to taking, Hb is
 tested. Ex: 10g/ dl after 1 week,
the Hb became 15g/dL. It is
used if the treatment is
effective or not.
4. Estimation of RBC indices
- MCH and MCHC is used.
5. Selection of blood donors
- Test the donors if have a normal
Hb (12.5g/dl) if <12.5 g/dl –
rejected. If > or equal 12.5/dL-
accepted.
METHODS FOR Hb DETERMINATION
A. GASOMETRIC METHOD- Oldest
1. Van Slyke Method
: Oxygen carrying capacity
measured by Van Slyke
apparatus
: Based on the formula 1gm of
Hb= 1.34 ml of Oxygen
: It does not measure
Carboxyhemoglobin,
Sulfhemoglobin and
Methemoglobin. Only normal
Hb is measured.
: Time consuming and
expensive
: Results are 2% less accurate
than other methods
B. CHEMICAL METHOD
: Iron content of Hb is 1st
estimated
What is the iron form of Hb:
Ferrous form
: Indirectly Hb is derived – 100
grams of Hb contains 374
grams of iron. 1g of Hb = 3.74
; Time consuming method
: Used to calibrate all other
methods of Hb estimation
1. SPECIFIC GRAVITY METHOD
: Rough estimate is made
from S.G of blood
: Also known as Copper
Sulfate Technique
: Used in mass screening, like
selection of donors
: A drop of blood is allowed
to fall in copper sulphate
solution that has a S.G of
1.053 from a height of 1cm.
: 1.053 SG= 12.5 grams/dl
: Once the blood is dropped
into the copper sulfate, it
gets covered with copper
proteinate
: If it bloods sinks(accept)
(>12.5g/dl), if it floats
(reject) (<12.5g/dl)
C. VISUAL METHOD
1. SAHLI’S ACID HEMATIN
METHOD
PRINCIPLE
: Blood is mixed with an acid solution,
so that Hb is converted to brown
colored acid hematin
: Diluted with water till brown color
matches that of brown glass standard
(reference, comparator)
: Hb value is read directly from the
scale
STEPS FOR SAHLI’S ACID HEMATIN
METHOD
 Prepare materials
solvent is
SAHLI’S called 1
HAEMO- NORMAL
GLOBINOMETER solution.

Q; Can we use
N-HCL or any
strong acid or
HAEMO- Has a alkali?
GLOBINOMETER marking of
PIPETTE 20uL= 1 drop A: No, we can
of blood. not use
because it will
cause
destruction of
proteins
HAEMO- Marking of present in Hb
GLOBINOMETER gram %. and Hb value
TUBE Lowest will alter.
marking is 2 STIRRER
(GLASS
ROD)

Standard
COMPARATOR basis or
BOX reference

DISTILLED
WATER

N/10 HCL Q: Define

NORMALITY.
Or what is
normal
solution?

A: When 1 m
equivalent
weight of a
substance is
dissolved in 1
litre of the
 STEPS 5. Draw blood in
1. With the help of dropper, fill Haemoglobinometer
N/10 HCl in
Haemoglobinometer Tube up
to its lowest marking (up to 2
gm%, yellow marking).

Mix contents thoroughly and

put Haemoglobinometer tube
in a comparator box

2. Put Haemoglobinometer Tube

in a Comparator box.

6. Wait for 10 minutes

- During this time
haemoglobin is converted
to Acid-Haematin ( brown
3. Prick your finger color)

4. With the help of

Haemoglobinometer pipette
7. After 10 minutes, add distilled
suck the blood up to 20 uL
water drops till the color of the
marking.
solution is same as to that of the
Comparator Box color strip.
(Mix it with stirrer)
 4. Development of color is slow,
acid hematin is not stable
5. Source of light will influence the
comparison of colors.

D. PHOTOELECTRIC METHOD
When color of the solution is same 1. CYANMETHEMOGLOBIN
Comparator box color strip, hold the METHOD
Tube- lift the stirrer – look the readings
: Most accurate method for
in Hb gram % (Lower meniscus need
estimation of Hb
to be read)
: Recommended by the international
Committee for Standardization in
hematology due to the ff reason
1. Almost all form of hemoglobin
are converted to
cyanmethemoglobin (Except
sulfhemoglobin)
2. Stable and reliable standard is
available
ADVANTAGES OF SAHLI’S ACID
Photoelectric method:
HEMATIN METHOD
Spectrophotometer is used. Beer’s
1. Easy to perform law is used.
2. Quick
CYANMETHEMOGLOBIN METHOD
3. Inexpensive
4. Bedside procedure Principle
5. No technical expertise
- Blood is mixed with Drabkins
DISADVANTAGES OF SAHLI’S ACID solution
HEMATIN METHOD
1. For maximum color, longer time
Drabkins solution- pH 7.0 – 7.4
is required
2. Performed match with the • Potassium ferricyanide-
brown glass standard is not convert Hb to Methemoglobin
possible • Potassium cyanide- converts
3. Carboxy, Methemo and Methemoglobin to
Sulfhemoglobin are not cyanmethemoglobin
converted to acid hematin • Potassium dihydrogen
phosphate
 • Non-ionic detergent
• Distilled water
✓ Erythrocytes are lysed
producing an evenly
distributed Hb solution
✓ Potassium ferricyanide
converts Hb to methemoglobin
✓ All Hbs present in blood are
converted to this form
✓ Absorbance is measured in
spectrophotometer at 540 nm
✓ To obtain amount of unknown
Hb sample, its absorbance is
compared with the standard
cyanmethemoglobin solution.
CYANMETHEMOGLOBIN METHOD-
EQUIPMENT
• Photoelectric colorimeter or
spectrophotometer
• Sahlis pipette at 20 micro litre
• Pipette 5 mL
CYANMETHEMOGLOBIN METHOD
• Take 5 ml of Drabkins solution
and to it add 20 microlitres of
blood
• Stopper the tube, mix by
inverting several ties (Lyse RBC=
Hb) Faster
• Allow to stand for 5 minutes
• Transfer the sample to cuvette
• Read the absorbance in the
spectrophotometer at 540 nm
• Also take the absorbance of
the standard solution.
CYANMETHEMOGLOBIN METHOD
ADVANTAGES
• All forms of Hb except
sulphemoglobin are converted
to
hemiglobincyanide/cyanmeth
emoglobin (HiCN).
Sulphemoglobin is not normally
seen in blood and irreversible
• Visual error is not there as no
color matching is required
• Cyanmethemoglobin solution
is stable and its color does not
fade with time so readings may
not be taken immediately.
• Absorbance may be
measured soon after dilution
• A reliable and stable reference
standard is available from
World Health Organization for
direct comparison.
DISADVANTAGES
• Diluted blood has to stand for a
period of time to ensure
complete Conversion of Hb
• Potassium cyanide is a
poisonous substance and that
is why Drabkin’s solution must
never be pipetted by mouth
• The rate of conversion of blood
containing
carboxyhemoglobin is slowed
• Considerably. Prolonging the
reaction time to 30 min can
overcome this problem
• Abnormal plasma proteins
cause turbidity when blood is
diluted with Drabkin’s solution.
 What happens to WBC when
centrifuge? They sink in the
bottom and they were called
sediments. Supernatant is
clear.
• A high leukocyte count also
causes turbidity on dilution of
blood. Centrifuging the diluted
blood can help overcome the
turbidity.