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Lab Activity No. 1 - Slide Presentation
Lab Activity No. 1 - Slide Presentation
Lab Activity No. 1 - Slide Presentation
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
04 Reference Values
Introduction
From: https://myhealth.alberta.ca/Health/pages/conditions.aspx?hwid=tp10337&
Introduction
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
20 mark
Methods of Hemoglobin Determination
B. Cyanmethemoglobin Method 1
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 FerricyanideMethemoglobin (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
B. Cyanmethemoglobin Method -
Procedure
B. Cyanmethemoglobin Method -
Procedure
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:
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
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
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