HEMOGLOBIN PROCEDURE

Intended for the Quantitative Determination of Cyanmethemoglobin in the blood

SUMMARY AND EXPLANATION

Hemoglobin is a red pigmented protein which serves to transport oxygen from the lungs to body tissues. It gives blood the characteristic red color. In anemia, hemoglobin levels are abnormally low and this condition suggests an underlying disease. Miale (1) has described the various anemias. Polycythemia and erythrocytosis increase hemoglobin levels.

The EAGLE HEMOGLOBIN PROCEDURE is based on the determination of cyanmethemoglobin, which has become the internationally adopted method (2).

PRINCIPLE

Ferricyanide oxidizes oxyhemoglobin to methemoglobin, and cyanide converts methemoglobin to cyanmethemoglobin (3). Absorbance measurements are made at 540 nm. The CYANMETHEMOGLOBIN REAGENT contains a surfactant to promote rapid hemolysis and to accelerate formation of cyanmethemoglobin. The reaction is completed in 3 minutes.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 6200 provides:

CYANMETHEMOGLOBIN REAGENT - (Cat. No. 6200)

REACTIVE INGREDIENTS:

0.75 mM potassium cyanide and 0.6 mM potassium ferricyanide.

RECONSTITUTION:

Reconstitute with 1000mL D-H2O. Mix gently to dissolve.

PRECAUTIONS:

Caution - contains cyanide. Do not mix with acids. Avoid contact with skin. If ingested, perform gastric lavage and call a physician . Discard by flushing with large amounts of water.

STORAGE AND STABILITY:

After reconstitution, store at 15 - 30ºC. PROTECT FROM LIGHT. Stable for at least six months if properly protected.

DETERIORATION:

The reagent should be a clear solution which is yellow in color. Turbidity would indicate deterioration and the reagent should not be used. If the reagent has been frozen, falsely low values may result.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 540 nm.

SPECIMEN COLLECTION

PRECAUTION:

1. Whole blood with EDTA as an anticoagulant is recommended.

2. The specimens may be collected also with heparin, citrate or oxalate as anticoagulants.

3. If capillary blood is used, exercise care to avoid coagulation.

4. Young et al (4) have reviewed drug effects on hemoglobin assay.

SAMPLE STORAGE:

Hemoglobin in whole blood collected with EDTA appears stable for one week at room temperature (15 - 30ºC).

ADDITIVES:

No special additives or preservatives other than anticoagulants are needed.

INTERFERING SUBSTANCES:

Gross lipemia. leukocytosis and macroglobulinemia may falsely elevate the hemoglobin value.

PROCEDURE

MATERIALS PROVIDED:

CYANTHEMETHEMOGLOBIN REAGENT (Cat. No. 6200)

MATERIALS REQUIRED BUT NOT PROVIDED:

1. CYANMETHEMOGLOBIN STANDARD (Cat. No. 6210)

2. 0.02 mL micropipettor

3. 5.0 mL pipette or dispenser

4. Test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 540 nm

Reaction Type Endpoint

Incubation Temperature 15 - 30ºC

Incubation Time 3 minutes

Sample Volume 0.02 mL

Reagent Volume 5.0 mL

Total Volume 5.02 mL

PERFORMANCE OF TEST - AUTOMATED PROCEDURE:

This is a manual test procedure only.

PERFORMANCE OF TEST - MANUAL PROCEDURE:

1. Place 2 mL of CYANMETHEMOGLOBIN STANDARD in tube

labeled Standard.

2. Dispense 5.0 mL CYANMETHEMOGLOBIN REAGENT into tubes labeled Reagent Blank, Control, Sample 1, etc.

3. Place 0.02 mL specimen into the appropriately labeled tube. Use deionized water as specimen for Reagent Blank. Mix well.

4. Allow the test samples to stand at room temperature (15 - 30ºC) for at least 3 minutes.

5. Adjust instrument to zero absorbance at 540 nm using Reagent Blank.

6. Read and record absorbance values for Standard, Controls  and Unknowns.

NOTE: For a direct read-out instrument, set read-out concentration value of the Standard and read the unknown concentrations directly.

STABILITY OF FINAL REACTION:

Cyanmethemoglobin appears quite stable. However, the test samples should be read within an hour before evaporation of the reaction solutions becomes significant.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using quality control materials (normal and abnormal) analyzed in the same manner employed.

CALIBRATION:

CYANMETHEMOGLOBIN STANDARD (Cat. No. 6210), which is available in a ready to use form and is recommended. The assigned value of this standard is 20 g/dL hemoglobin, and a calibration curve may be prepared.

CALCULATION OF RESULTS

The following equation is used to determine unknown concentrations:

                                          Abs. Unk.

        Unknown (g/dL) =  ---------------     X   Std. Conc. (g/dL)

                                         Abs. Std.

EXAMPLE:

A 20 g/dL Standard had an Abs. = 0.550; the Unknown Abs.. = 0.395. The hemoglobin concentration of the unknown is:

           0.395

                                ------------     X    20 g/dL  =   14.4 g/dL

                 0.550

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner as the Unknowns. EAGLE DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

LIMITATIONS

Sulfhemoglobin is not measured by this procedure.

EXPECTED VALUES

ADULT MALE: 12.7 - 18.3 g/dL

ADULT FEMALE: 11.0 - 15.8 g/dL

This range represents the 95% confidence interval from a clinically normal population. Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 20 g/dL hemoglobin.

PRECISION:

Hemoglobin controls at three levels were assayed 20 times each for within run precision, and for 10 working days to establish run to run precision.

WITHIN RUN

LEVEL      MEAN    ST. DEV.       %CV

I  8.1   0.13   1.6 II 11.1     0.17   1.5

III 16.6             0.12     0.7

RUN TO RUN

LEVEL      MEAN     ST. DEV.       %CV

    I  8.2          0.20    2.4  II 11.1             0.18    1.6

  III 16.5     0.19            1.2

SPECIFICITY:

The cyanmethemoglobin procedure is the most widely used and preferred method for hemoglobin assay (2). A comparative study of the HEMOGLOBIN PROCEDURE and another widely used commercial method showed a 99% correlation.

SENSITIVITY:

This procedure has a sensitivity of 0.04g/dL per 0.001 absorbance unit.

REFERENCES

1. Miale, J.B., Laboratory Medicine: Hemotology, C.V. Mosby Co., St. Louis, 1972, p. 494.

2. Van Kampen, E.J., and Zijlstra, W.G., Clin. Chem. Acta 6. 538 (1961).

3. Tiez, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders Co., Philadelphia, 1976, p. 411.

4. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin. Chem., Vol. 21, p. 316 D (1975).