UPH-DR. JOSE G.

TAMAYO MEDICAL UNIVERSITY

STO. NIÑO, CITY OF BIÑAN, LAGUNA
COLLEGE OF MEDICAL TECHNOLOGY

Activity #8&9
Hemoglobin and
Hematocrit
Determination

E.M.LICUDAN,RMT
 OBJECTIVES

➢ Name two main components of hemoglobin

➢ State the function of hemoglobin
➢ Measure hemoglobin levels in blood using the
Sahli-Hellige and Cyanmethemoglobin method
➢ Observe the precautions when performing
hemoglobin determination
➢ Measure the packed cell volume
➢ State the reference values of the hematocrit and
hemoglobin
➢ State the clinical significance that affect the
hemoglobin and hematocrit value
E.M.LICUDAN,RMT
Composition of blood
HEMOGLOBIN

• The primary
component of
erythrocytes
• Within each rbc are ~
200-300 million
molecules of
hemoglobin
• Composed of 4 globin
chains (α and β)
attached to a heme
molecule
E.M.LICUDAN,RMT
 COMPONENTS OF HEMOGLOBIN

• A PROTEIN COMPONENT called globin

composed of two sets (dimers)
of two different polypeptide
chains
• Four molecules of NITROGENOUS
SUBSTANCE protoporphyrin IX
• Four IRON ATOMS IN THE FERROUS
STATE that combine with
protoporphyrin IX to form 4
heme molecules
• One 2,3 – DIPHOSPHOGLYCERATE
molecule as a sometimes
resident molecule in the center
of the Hb unit
E.M.LICUDAN,RMT
 NORMAL HEMOGLOBIN VARIANTS

HEMOGLOBIN MOLECULAR STAGE OF LIFE PROPORTION (%)

STRUCTURE
NEWBORNS ADULT

PORTLAND z2g2 Embryonic 0 0

GOWER I z2e2 Embryonic 0 0
GOWER II a2 e 2 Embryonic 0 0
FETAL (F) a2 g 2 NB and adult 80 <1
A1 a2 b 2 NB and adult 20 97
A2 a2 d 2 NB and adult <0.5 2.5
 FUNCTIONS OF HEMOGLOBIN

• Transport of molecular oxygen from the lungs to the

tissues
• Transport of carbon dioxide from the tissues to the lungs
• Buffering of the blood

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

A. ACID HEMATIN TECHNIQUE OF SAHLI-HELLIGE

Principle: Hemoglobin in blood when mixed with diluted HCl

in a Sahli-Hellige hemometer tube is converted to acid
hematin. The mixture is then diluted with distilled water until
the color matches with the color of the standard then
hemoglobin value is measured directly on the gram scale of
the tube.

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

A. ACID HEMATIN TECHNIQUE OF SAHLI-HELLIGE

Materials:
1. Sahli-Hellige Hemoglobinomter Set (Sahli-Hellige pipet,
comparator block, dilution tube, stirrer and 0.1N HCl)
2. Distilled water
3. Sterile blood lancet
4. Alcohol
5. Cotton balls

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

A. ACID HEMATIN TECHNIQUE OF SAHLI-HELLIGE

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES
Principle: Blood is diluted in a solution of K3Fe(CN)6 and KCN.
The potassium ferricyanide oxidizes hemoglobin to
hemiglobin (or methomoglobin-Hi) and potassium cyanide
provides cyanide ions to form HiCN (hemiglobin-cyanide or
cyanmethemoglobin). The absorbance of the solution is
measured in a spectrophotometer at 540nm and is compared
in a standard HiCN solution.

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES
Materials:
1. Drabkin’s reagent (composed of 1g NaHCO3, 50mg KCN,
200mg K3Fe(CN)6 in distilled water)
2. Sterile blood lancet
3. Alcohol
4. Cotton
5. Hemoglobin pipet
6. Big test tube
7. Spectrophotometer

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

NOTE:
1. The Drabkin’s reagent must be clear and pale yellow
2. It must have a pH of 7.0-7.4
3. It must give a reading of zero when measured at
photometer at 540nm against the water blank
4. Disposal of reagent and samples in running water is
advisable
5. Care must be taken with KCN, as salts or solutions of
cyanide is poisonous.
E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

B. CYANMETHEMOGLOBIN METHOD OF HEMOGLOBIN

ESTIMATES

Reasons why Modified Drabkin’s reagent better than original

Drabkin’s reagent
1. Dihydrogen Potassium Phosphate (KH2PO4) instead of
NaHCO3 shortens the time to convert hemoglobiin into
hemiglobin cyanide from 10 minutes to 3 minutes.
2. Non-ionic detergent e.g. 0.5mL Sterox S.E. (Harleco or
1.0mL Triton X-100 (Rohm &Haas) – this detergent
enhances lysis of erythrocytes and decreases turbidity
from protein precipitation
E.M.LICUDAN,RMT
 HEMOGLOBIN DETERMINATION

NORMAL RANGE
Conventional unit SI unit
Male 14.0-17.5 g/dL 140-175 g/L
Female 12.5-15.3 g/dL 125-153 g/L
Ref: Henry’s Clinical Diagnosis 21st edition, 2007

Conventional unit SI unit

Male 13.5-17.5 g/dL 135-175 g/L
Female 12.0-16.0 g/dL 120-160 g/L
At birth 13.5-19.5 g/dL 135-195 g/L
Ref: Steininger Clinical Hematology

E.M.LICUDAN,RMT
HEMOGLOBIN DETERMINATION

E.M.LICUDAN,RMT
HEMOGLOBIN DETERMINATION

E.M.LICUDAN,RMT
 HEMATOCRIT

• Defined as the volume of

erythrocytes expressed as a
percentage of the volume of
whole blood in a sample
• Measures the proportion of RBC to plasma in peripheral
blood but NOT in the entire circulation
• Dried heparin and EDTA are satisfactory anticoagulants
• Standard 10 to 14 x 75mm tubes, containing 5mL of
blood and an air bubble constitutes at least 20% of the
tube volume, require at least eight inversions

E.M.LICUDAN,RMT
 HEMATOCRIT DETERMINATION

A. MICRO-METHOD OF ADAMS

Principle: The hematocrit of a sample of blood is the ratio of

the volume of erythrocytes to that of the whole blood. It may
be expressed as a percentage (conventional) or as a decimal
fraction (SI units). The units L/L are implied

E.M.LICUDAN,RMT
 HEMATOCRIT DETERMINATION

A. MICRO-METHOD OF ADAMS

Materials:
1. Heparinized capillary tube (about 7cm long with a uniform
bore of about 1mm for skin puncture)
2. Plain hematocrit tube for anticoagulated blood
3. Clay sealer
4. Paraffin
5. Micro-hematocrit reader
6. Blood lancet
7. Cotton
8. Alcohol
E.M.LICUDAN,RMT
 HEMATOCRIT DETERMINATION

A. MICRO-METHOD OF ADAMS
Procedure:

E.M.LICUDAN,RMT
 HEMATOCRIT DETERMINATION

A. MICRO-METHOD OF ADAMS
Procedure:
4. Centrifuge at 10,000 – 12,000g for
five (5) minutes in a micro-hematocrit
centrifuge.
5. After centrifugation, read the
packed cell layer using micro-
hematocrit reader. (DO NOT include
the buffy coat in reading the PCV)

E.M.LICUDAN,RMT
HEMATOCRIT DETERMINATION

E.M.LICUDAN,RMT
HEMATOCRIT DETERMINATION

E.M.LICUDAN,RMT
HEMATOCRIT DETERMINATION

E.M.LICUDAN,RMT