Central Philippine University

College of Medical Laboratory Science

Laboratory Activity No. 1

Hemoglobin Determination
JESSIE D. SAJO, RMT

Learning Outcomes:
At the end of this activity the student must be able to:
1. Demonstrate accurately blood collection procedure through venipuncture or skin
puncture technique.
2. Perform accurately the methods of hemoglobin determination.
3. List completely the normal values associated with hemoglobin both in conventional
and SI units.
4. Relate correctly results of hemoglobin to human diseases.
5. Perform correctly laboratory safety and waste management at all times.
Contents
01 Introduction
The Hemoglobin Molecule

02 Methods of Hemoglobin Determination

Sahli Method
Cyanmethemoglobin Method
Materials and Equipments
Specimen
Principle
Procedure
Physiologic and Pathologic Variations of
03 Hemoglobin
Conditions with Increased or Decreased Hemoglobin levels

04 Reference Values
Introduction

The Hemoglobin Molecule

Synthesis
The primary function of the red blood cell is to
manufacture hemoglobin
Main component of RBC (95% of its cytoplasmic
content)
Functions
Its main function is to transport oxygen from the lungs
to tissues and transport carbon dioxide from the
tissues to the lungs for exhalation.
• Oxyhemoglobin – Hb + O2
• Carbaminohemoglobin – Hb + CO2

From: https://myhealth.alberta.ca/Health/pages/conditions.aspx?hwid=tp10337&
Introduction

The Hemoglobin Molecule

Composition
Composed of four subunits, each containing heme
and the protein globin. Each heme molecule has a
central atom of divalent ferrous iron (Fe2+)
Methods of Hemoglobin Determination

• Hemoglobin measurement is one of the components of a

Complete Blood Count (CBC) aka Full Blood Count
(FBC):
RBC Parameters
 Hemoglobin Concentration (Hemoglobin)
 Erythrocyte Volume Fraction (Hematocrit)
 Erythrocyte Number Concentration (RBC Count)
• Hemoglobin concentration is directly proportional
 RBC indices to the oxygen-combining capacity of the blood.
WBC Parameters • It is important as a screening test for diseases
 Leukocyte Number Concentration (WBC Count) associated with anemia and for following the
response of these diseases to treatment
 Leukocyte Type Number Fraction (Differential Count)
Platelet Parameters
 Thrombocyte Number Concentration (Platelet Count)
Methods of Hemoglobin Determination

A. Sahli Method
Reagents and Equipments
1. Sahli hemoglobinometer 3
2. Sahli pipet 6
3. 0.1 N HCl 1
4. Lancet
5. Rubber tubing
6. Distilled Water

Specimen
Capillary Blood

Principle 4
5
Blood is diluted in an acid solution, converting the
hemoglobin to acid hematin. The test solution is matched 2
against a colored glass reference or standard.
 Methods of Hemoglobin Determination

A. Sahli Method - Procedure

1 Fill the graduated tube to

the 20 mark with 0.1N
HCl.

20 mark
 Methods of Hemoglobin Determination

A. Sahli Method - Procedure

2 Sterilize the area with

sterile cotton moistened
with 70% alcohol. Using a
sterile lancet, draw blood
by pricking the third or
fourth finger, lobe of the
ear or the heel (for 0.02 mark
infants). Wipe away the
first drop of blood with dry
sterile cotton.

3 Draw capillary blood to

0.02 mark of the Sahli
pipet. Do not allow air
bubbles to enter.
 Methods of Hemoglobin Determination

A. Sahli Method - Procedure

4 Wipe the outside tip of the

pipet with absorbent paper
or cloth. Check that blood
is still on the mark.

5 Blow the blood from the

pipet into the graduated
tube of the acid solution.
Rinse pipet by drawing in
and blowing out the acid
solution 3 times. The
mixture of the blood and
acid gives a brownish
color. Allow to stand for 5
minutes
 Methods of Hemoglobin Determination

A. Sahli Method - Procedure

6 Place the graduated tube

in the hemoglobinometer.
Stand facing a window.
Compare the color of the
tube containing diluted
blood with the color of the
reference tube.

7 Dilute by adding distilled

water drop by drop. Stir
with glass rod after adding
each drop. Remove the
rod and compare the
color. Stop when the color
matches.
 Methods of Hemoglobin Determination

A. Sahli Method - Procedure

7 (cont) Remove the rod

and compare the color.
Stop when the color
matches. 15 mark

8 Note the mark reach.

Depending on the type of
hemoglobinometer, this
gives hemoglobin
concentration either in
g/100 mL or as grams %.
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method 1. Methemoglobin (Hi)

• Hemoglobin in which the ferrous iron (Fe+2) is
• Also known as Hemiglobincyanide method oxidized to ferric state (Fe+3)
• Methemoglobinemia - ↑methemoglobin in the
• Reference method approved by CLSI
blood
• Automated cell counters use some modification of this • Chocolate brown discoloration of the blood
method • Reversible
• All forms of hemoglobin are measured except for 2. Carboxyhemoglobin (HbCO)
sulfhemoglobin • Hemoglobin (Hb) + Carbon Monoxide (CO)
• Hb has 210x more affinity to CO than O2
• Cherry red in color
• Hemoglobin Derivatives • Carboxyhemoglobinemia - ↑carboxyhemoglobin
1. Methemoglobin in the blood
• Reversible
2. Carboxyhemoglobin 3. Sulfhemoglobin (SHb)
3. Sulfhemoglobin • Oxidized Hb + Sulfur
• Sulfhemogloninemia - ↑sulfhemoglobin in the
blood
• Mauve lavender discoloration of the blood
• Irreversible
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method 1

Reagents and Equipments

1. Spectrophotometer (or colorimeter) 5
2. Cuvettes
3. Blood (Sahli) Pipettes (0.2 mL)
4. Rubber Tubing
5. Test tubes
6. Test tube rack 6
7. Reference and control solutions (not shown)
8. Drabkin’s reagent

3 2

Specimen
Capillary Blood or EDTA whole blood
4
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method

Principle
Whole blood is added to
cyanmethemoglobin reagent. The
potassium ferricyanide in the reagent
converts the hemoglobin iron from the
Hemoglobin (Fe++) + Potassium FerricyanideMethemoglobin (Fe+++)
ferrous state to ferric state to form the
stable pigment, cyanmethemoglobin
Methemoglobin (Fe+++) + Potassium Cyanide  Cyanmethemoglobin
(HiCN). The non- ionic reagent present in
(HiCN)
the reagent improves the lysis of the red
blood cells and decreases the amount of
turbidity resulting from abnormal proteins
such as lipoprotein. The color intensity is
measured in a spectrophotometer at a
wavelength of 540 nm.
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method

Drabkin’s Reagent
Composition
Potassium ferricyanide [K3Fe(CN)6] – converts hemoglobin into methemoglobin
Potassium cyanide (KCN) – converts methemoglobin to cyanomethemoglobin
Dihydrogen potassium phosphate (KH2PO4)* – shortens conversion time from 10-15 minutes to 3 minutes
*Original Drabkin’s reagent uses NaHCO3
Nonionic detergent – enhances lysis of RBCs (e.g. Steroz and Triton X)
Distilled water

Note
•Stable for at least 6 months @ RT, protected from light by using an amber bottle.
•Should be clear and pale yellow. If it becomes turbid, or loses its color, it should be discarded.
•Potassium cyanide is very poisonous. It must be kept in a locked cabinet at all times when not in use.
 Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method -
Procedure

1 Place 5.0 mL of HiCN

reagent into an
appropriate labelled 10 mL
test tube.

2 Place 5.0 mL of HiCN

reagent to be used as
blank
 Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method -
Procedure

3 Add 0.02 mL of well mixed

whole blood to the tube.

4 Rinse the pipet 3-5 times

with the HiCN reagent by
drawing in and blowing out
until all blood is removed
from the pipet.
 Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method -
Procedure

5 Mix well and allow to

stand at room temperature
for 15 minutes to allow the
reaction of blood and
HiCN reagent.

6 Read at 540 nm and set

OD at zero. Refer to the
prepared curve or chart
for hemoglobin.
 Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method -
Procedure

Compare OD
reading against the
Hb curve
Placing the cuvette
into the spectro
Sources of Error
Turbidity may lead to increased light scattering and apparent absorbance thus leading to falsely high results.
• Lipemia – defined as visible turbidity in serum or plasma samples due to the presence of lipoprotein particles; can
be corrected using a patient blank
• Increased WBCs and platelets – can be corrected by centrifuging test mixture and testing hemoglobin on the
supernatant fluid
• HbS and HbC – dilute hemoglobin with distilled water
• Increased globulins – use of dihydrogen potassium phosphate
Overanticoagulation causes no effect on hemoglobin determinations
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method

Calibration Curve

•A calibration curve must be prepared before the spectrophotometer can be used for hemoglobin
determination.
•Procedure
1. Calculate the hemoglobin value of the reference solution in grams/liter by using the following formula:

Hb value of Reference Solution (g/L) = Concentration in mg/100 ml X 2.5

Example
Concentration of Reference Solution = 60mg/100mL
Solving
Hb value of Reference Solution (g/L) = 60mg/100mL X 2.5 = 150g/L
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method

Calibration Curve
Procedure
2. Prepare a series of dilutions of the reference solution in four test tubes (labeled 1-4). Pipette into each tube
the amounts shown in the table below

Volume of
Volume of Drabkin
Tube No. Reference Dilution
diluting fluid (mL)
Solution (mL)
1 4.0 0.0 Undiluted
2 2.0 2.0 1:2
3 1.3 2.7 1:3
4 1.0 3.0 1:4
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method
Calibration Curve
Procedure
3. Mix the contents of the tubes and allow to stand for 5 minutes.
4. Set the wavelength of the spectrophotometer at 540 nm
5. Fill the cuvette with Drabkin diluting fluid and place in the spectrophotometer. Set the spectrophotometer at
zero.
6. Read the contents of tubes 1 to 4 using a cuvette:

Tube Absorbance
Dilution Hb Concentration (g/L)
No. (@540 nm)
1 Undiluted 150 35
2 1:2 (150/2) 75 17.5
3 1:3 (150/3) 50 11.5
4 1:4 (150/4) 37.5 8.5
Methods of Hemoglobin Determination

B. Cyanmethemoglobin Method
Calibration Curve
Procedure
7. Prepare a graph, plotting
the readings of the
diluted reference
solution against their
respective hemoglobin
concentrations

8. From the graph, make a

table of hemoglobin
values from 20 to 180
g/L

Ex. If a sample has an

absorbance (OD) of 35,
its Hb concentration is
150 g/L
 Physiologic and Pathologic Variations of
Hemoglobin
Physiologic - Something that is normal, that is due neither to anything pathologic nor significant in terms of causing illness

Pathologic - Indicative of or caused by disease

Increased Hgb Decreased Hgb

 Polycythemia vera
Marked increase in the  In the morning
red blood cell (RBC)  Anemia Decrease to below
 In smokers normal Hb
count, hematocrit,  If the patient is lying
 Strenuous concentration,
hemoglobin, white exercise down (slight decrease)
erythrocyte count, or
blood cell count,  High altitudes hematocrit
platelet count, and red
blood cell mass
Reference Values

REFERENCE RANGE*
Conventional** SI
Newborn 13.6-19.6 g/dL 136-196 g/L
Infant 11.3-13.0 g/dL 113-130 g/L
Children 11.5-14.5 g/dL 115-148 g/L
Women 12.0-16.0 g/dL 120-160 g/L
Men 13.0-18.0 g/dL 130-180 g/L
*Philippine Council for Quality Assurance in Clinical Laboratories
**Conversion Factor=10
References

Textbooks

Brown, B. A. (1993). Hematology (6th ed.). Lea & Febiger.

Henry, J., McPherson, R. & Pincus, M. (2011). Henry's clinical diagnosis and management by laboratory
methods. Philadelphia, PA: Elsevier/Saunders.

Keohane, E. M., Smith, L. J., Walenga, J. M., & Rodak, B. F. (2016). Rodak's hematology: Clinical
principles and applications. St. Louis, MO: Elsevier.

Lo, R. W., Liu, E. B., Orillaza, M. A., Faundo, A. C., & Calzada, G. J. D. (2012). PCQACL’S
Standardization and Harmonization of Complete Blood Count in the Philippines (1st ed.). Van Haren
Publishing.

Online Resources
https://myhealth.alberta.ca/Health/pages/conditions.aspx?hwid=tp10337&
https://www.medicinenet.com/script/main/art.asp?articlekey=25870