Laboratory Activity No.

1
HEMOGLOBIN DETERMINATION
Score

Name Date

OBJECTIVES Specimen
At the end of this activity the student must be able to: Capillary blood
1. Demonstrate accurately blood collection
Principle
procedure through venipuncture or skin puncture Blood is diluted in an acid solution, converting the
technique. hemoglobin to acid hematin. The test solution is
2. Perform accurately the methods of hemoglobin matched against a colored glass reference or standard.
determination.
3. List completely the normal values associated Procedure:
with hemoglobin both in conventional and SI 1. Fill the graduated tube to the 20 mark with 0.1N
HCl.
units.
2. Sterilize the area with sterile cotton moistened
4. Relate correctly results of hemoglobin to human with 70% alcohol. Using a sterile lancet, draw
diseases. blood by pricking the third or fourth finger, lobe
5. Perform correctly laboratory safety and waste of the ear or the heel (for infants).
management at all times. 3. Wipe away the first drop of blood with dry sterile
cotton
INTRODUCTION 4. Draw capillary blood to 0.02 mark of the Sahli
pipet. Do not allow air bubbles to enter.
The primary function of the red blood cell is to
5. Wipe the outside tip of the pipet with absorbent
manufacture hemoglobin, which in turn, transports
paper or cloth. Check that blood is still on the
oxygen to and carbon dioxide from the tissues. The
mark.
hemoglobin molecule is composed of four subunits, each
6. Blow the blood from the pipet into the graduated
containing heme and the protein globin.
tube of the acid solution. Rinse pipet by drawing
in and blowing out the acid solution 3 times. The
Hemoglobin concentration is directly proportional to the mixture of the blood and acid gives a brownish
oxygen-combining capacity of the blood. Therefore, color. Allow to stand for 5 minutes.
measurement of the hemoglobin concentration in the 7. Place the graduated tube in the
blood is important as a screening test for diseases hemoglobinometer. Stand facing a window.
associated with anemia and for following the response of Compare the color of the tube containing diluted
these diseases to treatment. Shown below are some blood with the color of the reference tube.
conditions wherein hemoglobin values are usually 8. Dilute by adding distilled water drop by drop. Stir
increased or decreased. with glass rod after adding each drop. Remove
the rod and compare the color. Stop when the
Increased Decreased color matches.
9. Note the mark reach. Depending on the type of
hemoglobinometer, this gives hemoglobin
 Polycythemia vera
concentration either in g/100 mL or as grams %.
 In the morning  Anemia
 In smokers  In the evening B. CYANMETHEMOGLOBIN METHOD
 Strenuous exercise  If the patient is lying
 High altitudes down (slight decrease)
Reagents and Equipments
1. Spectrophotometer (or colorimeter)
2. Cuvettes
A. SAHLI METHOD 3. Blood (Sahli) Pipettes (0.2 mL)
4. Rubber Tubing
Reagents and Equipments 5. Test tubes
1. Sahli hemoglobinometer 6. Test tube rack
2. Sahli pipet 7. Drabkin’s reagent
3. 0.1 N HCl Composition:Sodium bicarbonate, potassium
4. Lancet
cyanide, potassium ferricyanide
5. Rubber tubing
6. Distilled Water 8. Reference and control solutions

MLS 3102 Hematology I - Laboratory

Specimen
Capillary blood or EDTA whole blood

Principle
Whole blood is added to cyanmethemoglobin reagent.
The potassium ferricyanide in the reagent converts the
hemoglobin iron from the ferrous state to ferric state to
form the stable pigment, cyanmethemoglobin (HiCN).
The non- ionic reagent present in 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. All forms
of hemoglobin are measured except for sulfhemoglobin.
2+
Hemoglobin (Fe ) + Potassium
3+
FerricyanideMethemoglobin (Fe )
3+
Methemoglobin (Fe ) + Potassium Cyanide 
Cyanmethemoglobin (HiCN)

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.
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.
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.

REFERENCE VALUES* References:

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

Conventional** SI
Henry, J., McPherson, R. & Pincus, M. (2011). Henry's
Newborn 13.6-19.6 g/dL 136-196 g/L clinical diagnosis and management by laboratory
Infant 11.3-13.0 g/dL 113-130 g/L methods. Philadelphia, PA: Elsevier/Saunders.
Children 11.5-14.5 g/dL 115-148 g/L
Keohane, E. M., Smith, L. J., Walenga, J. M., & Rodak, B.
Women 12.0-16.0 g/dL 120-160 g/L F. (2016). Rodak's hematology: Clinical principles and
Men 13.0-18.0 g/dL 130-180 g/L applications. St. Louis, MO: Elsevier.
*Philippine Council for Quality Assurance in Clinical Laboratories
**Conversion Factor=10 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.

MLS 3102 Hematology I - Laboratory