Hemoglobin is the main component of red blood cells and transports oxygen throughout the body. It is composed of four protein subunits and iron. Various methods can be used to estimate hemoglobin levels, including visual colorimetric methods like Sahli's method, photometric methods like cyanmethemoglobin, and specific gravity testing. The cyanmethemoglobin method is currently recommended, as it converts all forms of hemoglobin except sulfhemoglobin to cyanmethemoglobin, which can then be accurately measured spectrophotometrically. Estimating hemoglobin levels provides important information about oxygen carrying capacity and anemia diagnoses.

1. Hemoglobin Estimation

2.  (RBC) contains two pairs of
polypeptide chains α2 β2 and four
haem groups each having an atom of
ferrous iron(Fe+2).
Hb is the main component of RBCs
and accounts for approximately 34%
of the RBCs by weight.
Iron content of HB is 3.47 mg /100 g.
Main role to transport oxygen.

4. Various forms of Hb
 Oxyhemoglobin (Hb O2)
 Carboxy hemoglobin (Hb CO)
 Sulfhemoglobin (SHb)
 Methaemoglobin -Fe3+ (ferric)

5. Variants of Hb
 Normal: Hb F ,Hb A, Hb A2-delta
Abnormal: HbS, HbC, HbD & HbE
 Embryo are two zeta and two epsilon
chains
 Fetal hemoglobin Haemoglobin F
(α2γ2) .
 adult -Haemoglobin A (α2β2) – Has
two alpha chains and two beta chains,
 Haemoglobin A2 (α2δ2). 97% HBA
adult

6.  Samples used for Hb estimation
 Capillary blood from finger prick.
 Intravenous sample—It should be well
anticoagulated, preferably in EDTA.
Liquid anticoagulants should not be
used at all as these dilute and
decrease Hb concentration.

7. Normal Values of Hb
 Men 13.0 to 16.0 g/dl
 Women 11.0 to 15.0 g/dl
 Infants 16.0 to 19.0 g/dl
Critical Values-
 Less than 7 gm/dl- Severe
Anemia More than 20 gm/dl-
Hperviscosity

8. METHODS FOR ESTIMATION
OF HAEMOGLOBIN
The measurement of concentration of
Hb in the blood is known as
HEMOGLOBINOMETRY.
 Colorimetric method
 Gasometric method.
 Specific gravity method
 Chemical method.

9. Colorimetric method:
 Colorimetric method is based on
colorimetric measurement of the
intensity of color developed on
addition of some substance to the
blood. values are measured by
comparing with known standard.
 Include the following:
 A. Visual Methods
 B. Photoelectric Methods

10.  Visual Methods
◦ Sahli’s method (Acid haematin
method)
◦ Tallquist chart ( color comparison)
obsolete
◦ Hemoglobin Color Scale obsolete
 Photoelectric Method
◦ Cyanmethemoglobin.
◦ Oxyhaemoglobin & Alkaline Hematin

11. Commonly used methods
Sahli’s Method – A Color Based Method
 Visual method.
 Principle :
◦ Hb is converted into acid hematin with the
action of dilute hydrochloric acid (N/10 HCl).

◦ The acid hematin is brown in color and its
intensity is matched with a standard brown
glass comparator in a visual colorimeter
called Sahli’s colorimeter.

12.  Equipment:
◦ Sahli’s Hemaglobinometer consisting of :
 Graduated hemoglobin tube
 Comparator box with a brown glass
standard
◦ Hb Pipette
◦ Stirrer
◦ Dropper (dropping pipette)
 REAGENTS:
 a. N/10 HCl b. Distilled Water
 Sample:
 EDTA anticoagulated venous blood.
 Blood obtained by skin puncture

13. Sahli’s Method
 Procedure
 Fill Sahli’s Hb tube upto mark 2 with N/10 HCl.
 Deliver 20 μl (0.02 ml) of blood from a Hb pipette into it.
„
 Stir with a stirrer and wait for 10 minutes. „
 Add distilled water drop by drop and stir till color
matches with the comparator.
 Take the reading at upper meniscus.

16.  ADVANTAGES:
◦ Simple bedside test no colorimeter is
required.
◦ Reagents and apparatus are cheap.
 DISADVANTAGES
◦ There can be visual error.
◦ Carboxy, met and sulfhaemoglobins cannot
be converted to acid hematin.
◦ Comparator can fade over the years.
◦ Color appearing of acid hematin takes long
time and also fades quickly.
◦ Source of light (day light or artificial)
influences the color comparison.

17. Hemoglobin Color Scale
 This is rapid, simple, inexpensive and
reliable.
 Procedure:
 A drop of blood is placed on strip of
chromatography paper and the color
developed is matched visually against
the printed color scale.
 Color scale consists of printed set of
colors
 corresponding to Hb values.

19.  Tallquist Hb Chart
 This chart consists of a series of
lithographic colors said to correspond
to Hb values ranging from 10% to
100%. It is obsolete.
 B. Photoelectric Method
 Alkaline haematin
 Oxyhaemoglobin
 Cyanmethemoglobin

20. Alkaline haematin
 N/10 NaOH solution is used to convert
alkaline haematin read under
Spectrometer
 Advantages-
Stable solution reched.
Disadvantage-
Not reliable in infant and newborn.
Alkalin resistant. Less accurate.

21. Oxyhaemoglobin method
 Hb is converted into oxyhaemoglobin
by 0.04% ammonia solution .
Measured by spectrophotometer at
540 nm.
 Advantage-
 Reading done in few seconds. And
stable for 3 days.
 Disadvantage - derivatives other than
oxyHb are not measured.

22. Cyanmethemoglobin method:This is method
of choice for estimation of Hb, recommended
by ICSH (International committee for
standardisation in haematology) .
 Principle – Blood is diluted in a
solution called drabkin’s fluid
containing
 Pot. Ferricyanide converts Hb to
methamoglobin.
 Methamoglobin reacts with Pot.
Cyanide to form cyanmetHb.
 All forms are converted except SULF-
Hb.

23.  Sample: Blood from Skin Puncture or
EDTA anti-coagulated blood
◦ Take 5 ml of Drabkin solution in two test
tubes each.
◦ Add 20 micro liter of blood and mix well
◦ Allow the tube to stand for at least 5 min.
or more.
◦ Read in Spectrometer at 540 nm.

24.  Advantage-
 Less error,
 all forms of hemoglobin estimated
except sulphaemoglobin.
 Disadvantage-
 Hyperbilirubinemia affect value.
 Turbidity effect.

25. Gasometric method
 It is indirect method, which estimates
the amount of haemoglobin from the
amount of oxygen it will absorb. It is
accurate method
but too complicated for clinical work.

26. Specific Gravity Method
 This method gives approximate value of Hb.
 Method
 A drop of blood is allowed to fall in copper
sulphate solution of specific gravity 1.053
from a height of 1 cm.
 Specific gravity of 1.053 is equivalent to Hb of
12.5 grams.
 If drop sinks within this time its specific
gravity is higher.
 If it floats- specific gravity is low hence low
Hb.
• Normal specific gravity of blood ranges from
1.048-1.066.