GLYCOHEMOGLOBIN  UNITEST  PROCEDURE

For the Quantitative Determination of Glycohemoglobin in Blood

Protected By U.S. Patent No. 4407961

SUMMARY AND EXPLANATION

Throughout the circulatory life of the red cell, glycohemoglobin is formed continuously by adduction of glucose to the N-terminal of the hemoglobin beta chain. This process, which is non enzymatic, reflects the mean blood glucose over an extended period. In a classical study, Trivelli, et al (1) showed glycohemoglobin in diabetic subjects to be elevated 2-3 fold over levels found in normal individuals. Several investigators have recommended that glycohemoglobin serve as an indicator of diabetic control since glycohemoglobin levels approach normal values for diabetics responding to treatment (2-4).

Glycohemoglobin has been defined operationally as the "fast fraction" hemoglobins (HbA1a, A1b, A1c) which elute first during column chromatography with cation-exchange resin. The non-glycosylated hemoglobin, which consists of the bulk of hemoglobin, has been designated HbAo. The Eagle Glycohemoglobin Procedure employs a weekly binding cation-exchange resin for rapid separation of glycohemoglobin (fast fraction) from non-glycosylated hemoglobin.

PRINCIPAL

A hemolyzed preparation of whole blood is mixed continuously for 5 minutes with a weakly binding cation-exchange resin. During this time, HbAo binds to the resin. After the mixing period. a filter separator is used to remove the resin from the supernatant liquid which contains glycohemoglobin. The percent glycohemoglobin is determined by measuring absorbances at 415 nm for the glycohemoglobin fraction and the total hemoglobin fraction, calculating the ratio of the absorbances, and comparing this ratio to that of a calibrator carried through the separation procedure.

REAGENTS:  FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. GH-330 provides:

GLYCOHEMOGLOBIN ION - EXCHANGE RESIN TUBES

REACTIVE INGREDIENTS:

8 mg/mL cation-exchange resin buffered at pH 6.9.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date on bottle.

DETERIORATION:

The supernatant liquid above the resin should be clear and colorless. Turbidity or discoloration would indicate deterioration, and the reagent should not be used.

GLYCOHEMOGLOBIN LYSING REAGENT - (Cat. No. GH-20)

REACTIVE INGREDIENTS:

10mM potassium cyanide. Surfactant added.

PRECAUTIONS:

Contains Cyanide. Do not mix with acids. Wash hands after handling. discard by flushing with large amounts of water.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date on bottle.

DETERIORATION:

The reagent should be clear and colorless. Turbidity or discoloration would indicate deterioration, and the reagent should not be used.

FILTER SEPARATORS -  (Cat. No. GH-30)  

30 each

GLYCOHEMOGLOBIN CALIBRATOR - (Cat. No. GH-40)

REACTIVE INGREDIENTS:

Glycohemoglobin prepared from human erythrocytes in a stable, lyophilized form. Preservatives added. CONSULT VIAL FOR ASSIGNED VALUE.

PRECAUTIONS:

Prepared from human erythrocytes that tested non-reactive for Hepatitis B Surface Antigen, Hepatitis B Core Antigen and HIV Antibody. Handle with same precautions used for human blood samples.

STORAGE AND STABILITY:

Store at 2-8ºC. Stable until expiration date until reconstituted.

CALIBRATOR RECONSTITUTION:

Add 1.0 mL deionized water. Gently mix for 10 minutes. The reconstituted calibrator is stable for at least two weeks if stored at 2-8ºC and sealed tightly or it may be aliquoted and frozen for eight weeks. Do not warm to room temperature. Remove from refrigerator and use immediately.

DETERIORATION:

A dark brown coloration and clumping after reconstitution would indicate deterioration for the calibrator. Failure to achieve assay values of freshly reconstituted controls could also indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 415 nm. Eagle Diagnostics has available the GLYCO-METER instrument which is designed to provide a direct calculation of this glycohemoglobin method.

SPECIMEN COLLECTION

Procedure requires the use of whole blood collected with EDTA as an anticoagulant.

PRECAUTIONS:

Handle with same precautions used for all human blood samples.

SAMPLE STORAGE:

Glycohemoglobin in whole blood collected with EDTA appears stable for one week at 2-8ºC.

ADDITIVES:

No special additives or preservatives other than anticoagulants are required.

INTERFERING SUBSTANCES:

Gross lipemia may cause falsely high results. For grossly lipemic samples, centrifuge the red cells and remove the lipemic plasma replacing it with an approximately equal amount of saline. Resuspend the red cells in the saline and proceed with performance of test. Glycosylated HbS and HbC bind tightly to the resin which produces falsely low results. Fetal hemoglobin (HbF) does not interfere significantly in the assay. The unstable fraction (aldimine) is eliminated during resin mixing and does not contribute to the glycohemoglobin value.

PROCEDURE

MATERIALS PROVIDED:

GLYCOHEMOGLOBIN  UNITEST  SET (Cat. No. GH-330) provides ION-EXCHANGE RESIN TUBES and LYSING REAGENT, along with the FILTER SEPARATORS (Cat. No. GH-30) and the GLYCOHEMOGLOBIN  CALIBRATOR (Cat. No. GH-40) which are required for performance of the test.

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.02 mL and 0.10 mL micropipettors

2. 0.50 mL and 5.0 mL pipettes

3. 13 x 100 mm test tubes

4. Rocker or rotator

5. Timer and test tube rack

6. Instrument

PERFORMANCE OF TEST

Note: 1)   Resin Tubes / Lysing Reagent must be at room temperature before

starting the test procedure.

         2)   Each sample to be tested (including Calibrator and Controls require

a tube for Lysing, Resin Separation and Total Hemoglobin.

A. HEMOLYSATE PREPARATION:

1. Dispense 0.5 mL Lysing Reagent into tubes labeled: CALIBRATOR,

CONTROL, SAMPLE 1, etc.

2. Place 0.1 mL of well-mixed blood sample into the appropriately labeled

tube.

Mix until complete lysis is evident.

3. Allow to stand for 5 minutes.

B. GLYCOHEMOGLOBIN SEPARATION:

1. Remove cap from Glycohemoglobin Ion-Exchange Resin Tubes and label: CALIBRATOR, CONTROL, SAMPLE 1, etc.

2. Place 0.1 mL of the hemolysate from Step A into the appropriately labeled

resin tube.

3. Position the Filter Separator in the tube so that the rubber sleeve is

approximately 2 cm above the liquid level.

4. Fix the tubes on the rocker or rotator and mix continuously for 5 minutes.

5. Remove the tubes from the rocker or rotator.

6. For ease of separation, let the tubes stand upright in the test tube rack for a

few minutes, then push the Filter Separator into the tube until the resin is

firmly packed.

7. Pour the supernatant liquid into another tube or directly into a cuvette for

absorbance measurement.

8. Adjust the instrument to zero absorbance at 415 nm using deionized water

as the blank.

9. Read and record the absorbance values for CALIBRATOR, CONTROL,

SAMPLE 1, etc. These readings are for glycohemoglobin.

C. Total Hemoglobin Fraction

1. Dispense 5.0 mL deionized water into tubes labeled: CALIBRATOR,

  CONTROL, SAMPLE 1, etc.

2. Place 0.02 mL of hemolysate from Step A into the appropriately labeled tube. Mix will.

3. Adjust the instrument to zero absorbance at 415 nm using deionized water as the blank.

4. Read and record the absorbance value for CALIBRATOR, CONTROL,

SAMPLE 1, etc. These readings are for total hemoglobin.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within an hour.

CALIBRATION:

Glycohemoglobin Calibrator (Cat. No. GH-40) has an assigned value established by an accepted reference method (1) and must be used for calibration of this method. Refer to instruction sheet with calibrator for specific handling directions.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials analyzed in the same manner as the Unknowns. EAGLE DIAGNOSTICS makes available three levels of controls: Normal (GH-50), Midrange (GH-70) and elevated ( GH-60) for this purpose. Failure to achieve assayed values of freshly prepared control materials should be thoroughly investigated before patient values are reported.

CALCULATION OF RESULTS

Users of the Eagle GLYCO-METER must refer to the Instrument manual for complete instructions. Manual calculation of results for the unknowns should be determined in the following manner.

For each sample, calculate the ratio (R)  of the glycohemoglobin absorbance to the total hemoglobin absorbance. Use the following equation to determine unknown concentrations:

1) Absorbance Calibrator Glyco

--------------------------------------- = R  (Ratio of Calibrator)

    Absorbance Calibrator Total

2) Absorbance Unknown Glyco

---------------------------------------- = R ( Ratio of Unknown)

   Absorbance Unknown Total

3) R (Ratio of Unknown)

  ----------------------------- X Value of Calibrator (%) = Glyco %of Unknown

    R (Ratio of Calibrator

EXAMPLE:

A calibrator containing 10.0 % glycohemoglobin had Abs. = 0.610 for the glycohemoglobin fraction and Abs. = 0.635 for the total hemoglobin fraction. An unknown sample had glycohemoglobin Abs. = 0.775 and total; hemoglobin Abs. = 0.710. The glycohemoglobin concentration of the unknown is:

        0.610                                                                           0.775

Calibrator R   = -------------    =   0.961                     Unknown R   =    --------- =   1.09

0.635                                                                            0.710

                                         1.09

                              Unknown %   = --------------  X    10.0%    = 11.3%

    0.961

LIMITATIONS

Blood samples with total hemoglobin greater than 18 g/dL should be diluted x 2 with saline before assay. Samples from patients with hemoglobinopathies or decreased red cell survival times may show incorrect results. - See Section on SPECIMEN COLLECTION.

EXPECTED VALUES

This Glycohemoglobin Procedure reports values as a Hemoglobin A1 (a+b+c):

NORMAL 6.0 - 8.3 %

GOOD CONTROL 7.5 - 9.0 %

FAIR CONTROL 9.0 - 10.0 %

POOR CONTROL >>10.0 %

Recently, experts have suggested that all glycohemoglobin methods should report values as related to Hemoglobin A1c. When compared to a reference A1c method, the Eagle method showed a 98% correlation with an equation of

                         Y (A1c value)    =    0.838    X    (Eagle value)        0.732.

The value obtained by the Eagle method may be converted to a Calculated A1c value by use of this formulation. For a direct Calculated A1c value, the value of the Calibrator may be changed to = 7.6% in lieu of 10.0% and the results reported will be A1c (calculated) values.

If results are reported as Calculated A1c, then use as EXPECTED VALUES:

NORMAL 4.2 -6.2 %

GOOD CONTROL 5.5 -6.8 %

FAIR CONTROL 6.8-7.6 %

POOR CONTROL   >>7.6%

MEAN BLOOD GLUCOSE ESTIMATE:

In a test study performed by Dr. D.M. Nathan (5), the glycohemoglobin test was shown to have a linear correlation with mean blood glucose results from patients performing frequent self-monitoring of their blood glucose levels. This relationship is best described as a straight line between glycohemoglobin A1 values in the range of 6.5 - 13.0 % with the equation:

      MBG    (mg/dL) =    36.7    X     A1 value       185

Using this equation, Eagle Diagnostics has generated a table to convert glycohemoglobin A1% results to an estimated MBG value. Contact Eagle Diagnostics Customer Service for a copy of this conversion table.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This Glycohemoglobin Procedure shows linearity for glycohemoglobin levels in the range of 4.0%-20.0%.

PRECISION:

Within-Run - The within-run precision was established by assaying bloods with normal and elevated glycohemoglobin levels twenty times each.

       MEAN                STD.DEV.        %CV

Normal  7.8 0.21 2.7

Elevated 13.4 0.23 1.7

Run-to Run - The run-to-run precision was established by assaying bloods with normal and elevated glycohemoglobin levels for ten runs each conducted over a five day period.

Normal  7.6 0.31 4.1

Elevated 13.0 0.60 4.6

SPECIFICITY:

A comparative study of the Eagle Glycohemoglobin Procedure and another widely used commercial method showed a correlation of 0.96.

SENSITIVITY:

This Glycohemoglobin Procedure has a sensitivity of 0.02% glycohemoglobin per 0.001 units of absorption.

REFERENCES

1. Trivelli, L.A., Ranney, H.M. and Lai, H.T., New Eng. J. Med. 284, 353 (1971).

2. Goen, B., and Rubenstein, A.H., Diabetologia 15, 1 (1978).

3. Gabby, K.H., Hasty, K., Breslow, J.L.,Ellison. R.C., Bunn, H.F., and Gallop, P.M., J. Clin. Endocrinol. Metab. 44, 859 (1977).

4. Bates, H.M., Lab Manag., Vol 16 (Jan. 1978).

5. Nathan, D.M., et al., The Clinical Information Value of the Glycoslyated Hemoglobin Assay, The New England Journal of Medicine 310,  341-346 (1984).