HAEMOGLOBIN

Ready For Use

INTENDED USE METHOD
Vitro haemoglobin reagent is intended for the in vitro Colorimetric endpoint cyanmethaemoglobin method1.
quantitative determination of haemoglobin in whole blood on
manual systems.
BACKGROUND
Haemoglobin the main component of the red blood cells, functions in the transportation of oxygen and CO2. Haemoglobin consists of one molecule of
globin and four molecules of haeme (each containing one molecule of iron in the ferrous state). Globin consists of two pairs of polypeptide chains. In
the haemoglobin molecule, each polypeptide chain is associated with one haeme group; each haeme group can combine with one molecule of
oxygen or CO2. Haemoglobin carries oxygen from places of high oxygen pressure (lung) to places of low oxygen pressure (tissues), where it readily
releases the oxygen. Haemoglobin also returns CO 2 from the tissues to the lungs. Haemoglobin in circulating blood is a mixture of haemoglobin,
oxyhaemoglobin, carboxyhaemoglobin and minor amounts of other forms of this pigment. Measurements of haemoglobin from venous or capillary
blood aids in the detection of a variety of conditions that alter the normal haemoglobin concentration of the blood, e.g. anemia or polycythemia2,3.
ASSAY PRINCIPLE
The methods used for estimating haemoglobin can be either visual, or photo-electric. In visual methods changes of colour are judged by eye, whereas
in photo-electric methods they are estimated by a photo-electric colorimeter. The visual color comparison methods include the use of the Tallqvist
haemoglobin chart, Sahli haemoglobinometer, and Lovibond comparator. Previous methods used for the determination of blood haemoglobin were
based on estimation of oxygen or carbon monoxide capacity or iron content. Of all methods, only the cyanmethemoglobin has gained popular
acceptance. The original cyanmethemoglobin technique was proposed by Stadie4 in 1920. This method used separated alkaline ferricyanide and
cyanide reagents. A single reagent was introduced by Drabkin and Austin5 in 1935. In 1958 the National Research Council (NRC) recommended
adoption of the cyanmethemoglobin procedure based on field trials conducted by the Army Medical Department.6,7 In 1966 the International
Committee on Standardization in Hematology approved the proposal that all clinical laboratories should adopt this method exclusively8.
It is necessary to make a stable derivative involving all forms of haemoglobin in the blood in order to measure this compound accurately. The
cyanmethemoglobin derivative can be conveniently and reproducibly prepared and is widely used for haemoglobin determinations. All forms of
circulating haemoglobin are readily converted to cyanmethemoglobin except for sulfhemoglobin which is rarely present in significant amounts.

1. In a reagent solution the ferrous ions of haemoglobin are oxidized to the ferric state by potassium ferricyanide to form methemoglobin.
2. Methemoglobin subsequently reacts with the cyanide ions provided by potassium cyanide to form cyanmethemoglobin.

Haemoglobin + Cyanide + Ferricyanide Cyanmethemoglobin

The amount of cyanmethemoglobin can be measured spectrophotometrically at a wavelength of 546 nm. The intensity of color is directly proportional
to haemoglobin concentration in the specimen9.
EXPECTED VALUES SPECIMEN
• Use whole blood with EDTA as an anticoagulant.
Adults 10,11 • Oxalate, citrate, or heparin may also be used as anticoagulants.
• Capillary or venous blood may be collected if used before clotting
Males 13.0 – 18.0 g/dl
occurs.
Females 11.0 – 16.0 g/dl
• Specimen Preparation & Stability
Whole blood mixed well with an anticoagulant appears stable for one
Children week at room temperature.
At birth 13.6 – 19.6 g/dl
PROCEDURE
At one year 11.3 – 13.0 g/dl
Wavelength 520 - 560 nm
10 – 12 years 11.5 – 14.8 g/dl
Cuvette 1 cm light path
Temperature 20 - 25 0C
Factors such as age, race, exercise, season and altitude are reported to Zero adjustment against reagent blank
influence the values of normal ranges. The above ranges should serve Label test tubes: blank, and specimens.
only as a guideline. Each laboratory should establish its own range.

REAGENTS Pipette into test tube or cuvette

Drabkin's Reagent Working solution 2.5 ml

62 mmol/l
Potassium ferricyanide
R1 76 mmol/l
Potassium cyanide
60 mmol/l Sample 10 l
Potassium phosphate
5 ml/l
Non ionic surfactant
Rinse the pipette used for the blood
Cyanmethemoglobin standard specimen few times with the reaction mixture.
R2
Equivalent to 15 g/dl hemoglobin
Shake reagent solution slightly after adding the blood to avoid clumping
of the erythrocytes.
• Reagent Preparation & Stability Incubate for 5 minutes at room temperature.
All reagents are stable up to the expiry date given on label when stored at Measure the absorbance of specimen (Aspecimen) against reagent blank.
room temperature protected from light. The color is stable for 60 minutes. Don’t expose to strong light. The test
tubes must be kept stoppered when stored for several hours.
The cyanmethemoglobin standard is stable up to expiry date provided CALCULATION
that contamination is avoided. Store at 2 - 8 0C Method (1)
Never return standard to original bottle after use. Calculate the haemoglobin concentration by using the following formulae:
Haemoglobin Concentration (g/dl)= Specimen absorbance x 36.77

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Article # :127-EN
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Date of Revision : 2/2018
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 BIBLIOGRAPHY
Method (2) 1. Drabkin, D.L et al., (1932): J. Biol. Chem. 98, 719.
Hemoglobin Concentration= 2. Turgeon, Mary Louise: Clinical Hematology-Theories and Procedures, 3rd
edition, pp317-318.
Absorbance of Specimen X Standard value* 3. Lotspech-Steininger, Cheryl A, et al.,: Clinical Hematology-Principles,
Absorbance of Standard procedures and Correlations,1st edition, pp.108-110.
* Hemoglobin standard concentration given on label. 4. Stadie, W.C. (1920): J. Biol. Chem., 41:237.
LINEARITY 5. Drabkin, D.L. & Austin, J.H, (1935): J. Biol. Chem., 112: 51.
When run as recommended, the assay is linear up to 20 g/dl. 6. Crosby, W.H., et al., (1954): U.S. Armed Forces Med J. 5: 693.
Specimens with values above 20.0 g/dl must be re-run using one half the 7. Crosby, W.H., et al., (1957): Blood, 12: 1132.
sample volumes. Multiply final result by 2.
8. Eilers, R.J. (1967): Am. J. Clin. Pathol., 47: 212.
SENSITIVITY 9. Tietz, N.W. (1976): Fundamentals of Clinical Chemistry, 2nd ed., W.B.
Saunders Co., Philadelphia, p.4111.
The sensitivity is defined as the change of analytical response per unit
change in analyte concentration at a path length of 1 cm. 10. Henry, R.F., et al., (1974): Principles and techniques in clinical chemistry,
2nd ed., Harper & Row, Hagerstown, MD, pp. 1128:1135.
When run as recommended the sensitivity of this assay is 2.0 g/dl. 11. Wolf, P.L., (1973): Practical Clinical Hematology, John Wiley & Sons, NY,
p.144.
QUALITY CONTROL 12. Young, DS (1990): Effects of Drugs on Clinical Laboratory Tests. Third
Edition. 1990: 3: 6-12.
It is recommended that controls (normal and abnormal) be included in:
• Each set of assays, or SYMBOL DECLARATION
• At least once a shift, or
• When a new bottle of reagent is used, or
Manufacturer
• After preventive maintenance is performed or a clinical component
is replaced.
Commercially available control material with established haemoglobin Consult instructions for use
values may be routinely used for quality control.
Failure to obtain the proper range of values in the assay of control material Batch code (Lot #)
may indicate:
• Reagent deterioration, Catalog number
• Instrument malfunction, or
• Procedure errors.
Temperature limitation
Control results falling outside the upper or lower limits of the established
ranges indicate that the assay may be out of control. The following In vitro diagnostic medical
corrective actions are recommended in such situations: device
• Repeat the same controls.
• If repeated control results are outside the limits, prepare fresh Use by
control blood and repeat the test.
• If results on fresh control material still remain outside the limits, then
Caution. Consult instructions
repeat the test with fresh reagent.
• If results are still out of control, contact Vitro Technical Services.
Keep away from light
INTERFERING SUBSTANCES
• Substances that cause turbidity10: ORDERING INFORMATION
These substances will falsely elevate the haemoglobin value. These
include lipids, abnormal plasma proteins (macroglobulinemia) or REF SIZE
erythrocyte stroma.
• Drugs: 12701 1 X 500 ml
A review by Young et al12 reveals the numerous drugs that exert an 12702 1 x 1000 ml
in vitro effect to decrease blood haemoglobin values.
• Others: 12703 2 x 500 ml
Extremely high leukocyte counts or platelets.

WARNING & PRECAUTION

• Vitro haemoglobin reagent is for in vitro diagnostic use only. Normal

precautions exercised in handling laboratory reagents should be
followed.
• Reagent contains cyanide. Poison may be fatal if swallowed. DON’T
PIPETTE BY MOUTH.
Don’t mix reagent with acids. Discard by flushing with large volumes of
water.
• Don’t use haemoglobin reagent if it has a different color than yellow,
or reagent becomes cloudy.

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© 2012 Vitro Scient Co Article # :127-EN

Date of Revision : 2/2018