3.
Initial use of control is advised to check test system
Glycosylated Hemo- performance within limits.
EXPECTED RANGE :
(Ion Exchange Resin Method) Non Diabetic : 4.5 to 8.0 %
Code : 10009/10/11 (10 / 20 / 50 Tests) Good Control : 8.0 to 9.0 %
Fair Control : 9.0 to 10.0 %
GLYCOSYLATED HEMOGLOBIN kit is intended for the In
Poor Control : 10.0 and above
Vitro Quantitative determination of percent Glycosylated
Hemoglobin (GHb%) in human whole blood. LINEARITY : This method is linear upto 20%
SUMMARY & EXPLANATION OF TEST : PROCEDURE :
Glycosylated Hemoglobin (GHb) is a normal adult hemoglobin Assay temperature : 23+1OC or 30+1OC
(HbA1) which is covalently bonded to a glucose molecule. GHb Wave length : 415 nm
concentration is dependent on the average blood glucose
concentration. It is formed progressively and irreversibly over Step I - Hemolysate preparation
a period of time and is stable till the life of the RBC. 1. Pipette 0.25 ml of lysing reagent (2) in a test tube.
2. Add to it 0.05 ml of well mixed sample/control.
A Single glucose determination gives a value which is true
only at the time the blood sample is drawn. GHb on the other 3. Mix well and allow to stand at room temperature for 5
hand is unaffected by diet, insulin or exercise on the day minutes.
of testing and thus reflects the average glucose level over Step II - GHb separation and assay
the last several weeks. Hence, it reflects on the long term 1. Bring a Resin Tube (1) to assay temperature by incubating
metabolic control of glucose in individuals. GHb is now widely the tube in water bath.
recognised as an important test for the diagnosis of Diabetes 2. Add to it 0.1 ml of hemolysate (from step 1)
mellitus and is a reliable indicator of the efficacy of therapy. 3. Position a Resin Separator in the tube, so that the rubber
Abraham et al reported excellent correlation between HbA1 sleeve is approximately 3 cms, above the resin level.
concentration and diabetic control and concluded that the 4. Mix the contents on vortex mixer continuously for 5
determination of HbA1 rather than HbA1c (a fraction of HbA1) minutes
be used for clinical purpose. 5. Allow the Resin to settle at assay temperature for 5 minutes
There are several acceptable methods of GHb measurement push down the Resin separator in the tube until the Resin
like electrophoresis, ion-exchange chromatography, affinity is firmly packed.
chromatography, HPLC and colorimetry. Excel’s GHb kit based 6. Pour the supernatant directly into a cuvette and measure
upon the property of non-glycosylated hemoglobin to bind the absorbance against deionized water.
with a weak cation exchange resin leaving GHb free in the Step III - Total Hemoglobin (THb) assay
supernatant. 1. Pipette 5.0 ml of deionized water into a test tube.
2. Add to it 0.02 ml of hemolysate (from step 1).
PRINCIPLE :
3. Mix and read absorbance against deionized water.
Whole blood is mixed with lysing reagent to prepare a
hemolysate. This is then mixed with a weakly binding cation- CALCULATION :
exchange resin. the non-glycosylated hemoglobin binds to GHb% = A of GHb x 10 x Temp. factor (Tf)
the resin leaving GHb free in the supernatant. The GHb A of THb
percentage is determined by measuring the absorbance of For assay at 23OC Tf = 1.0; at 30OC Tf = 0.9
the GHb fraction and of the total Hb. NOTES :
REAGENTS & ACCESSORIES PROVIDED : H Due to variations in inter - laboratory assay conditions,
10 Tests 20 Tests 50 Tests instruments and demography, it is recommended that each
laboratory should establish its own normal range. To ensure
1. Resin Tubes 10 x 3 ml 20 x 3 ml 50 x 3 ml adequate quality control, each run should include a normal
2. Lysing Reagent 5 ml 10 ml 2 x 10 ml and abnormal assayed controls. The assigned value of the
3. Control 0.5 ml 0.5 ml 0.5 ml control must be confirmed by this methodology.
4. Resin Separators 10 Nos 20 Nos 50 Nos H Final diagnosis should be based on a co-relation of test
All reagents are stable at 2-8OC till the expiry date mentioned results with other clinical observations / Diagnostic tools.
on the label. Do not freeze. BIBLIOGRAPHY :
SPECIMEN : 1. Trivelli, LIA. et al (1971) New Eng. J. Med. 284:353.
Whole blood collected with EDTA/Heparin. GHb in blood is 2. Gonen, B (1978) Diabetologia 15:1
found to be stable for one week at 2-8OC 3. Gabby, K.H. et al (1977) J.Clin. End. Met. 44:859
4. Abraham, E.c. et al (1978) Diabetes 27:931.
REAGENT PREPARATION : 5. Bunn, H.F. (1981) Diabetes 30:613.
Dissolve the control (3) with 0.5 ml of deionized water by
inverting / swirling. Do not shake vigorously. Reconstituted
control is stable for 3 days at 2-8OC or 21 days at - 20OC.
PRECAUTIONS : Manufactured in India by :
1. Ion exchange Resin is prepipetted and securely sealed to M/s Excel Diagnostics Pvt. Ltd.
ensure accuracy and reproducibility of results. Do not use Plot NO. 89, Road No.8, ALEAP I.E., Near Pragathi Nagar,
resin Tube in case of visible and significant leakage. Opp. Kukatpally JNTU, Hyderabad - 500 090 (A.P.) INDIA.
2. Ensure constant assay temperature (preferable 23OC) of E-mail : edpl@rediffmail.com Visit us at - www.exceldiag.com
Resin during step II of the assay.
CONVERSION CHART OF GLYCOSYLATED HEMOGLOBIN A1%
TO MEAN BLOOD GLUCOSE AND GLYCOSYLATED HEMOGLOBIN A1c%
The glycosylated hemoglobin assay has been validated as a reliable indicator of mean blood glucose (MBG) levels for a period of
8-12 week period prior to determination. This assay provides valuable information for the physician’s clinical assessment of long
term diabetic control. Physicians have conventionally used information such as symptoms, urine tests and random blood glucose
determination to evaluate the metabolic state of their diabetic patient and to estimate roughly the average blood glucose of the
patient. Recently, the glycohemoglobin test has been shown to have a linear correlation with MBG results from patients performing
frequent self-monitoring of blood glucose levels.
Using this correlation, a table of the glycosylated Hemoglobin A1% from the Glycosylated Hemoglobin assay A1c% & Mean Blood
Glucose is obtained.
A1 A1c MBG A1 A1c MBG
6.0 4.30 35 9.9 7.56 178
6.1 4.38 39 10.0 7.64 182
6.2 4.46 43 10.1 7.73 186
6.3 4.54 46 10.2 7.81 189
6.4 4.63 50 10.3 7.89 193
6.5 4.71 54 10.4 7.98 197
6.6 4.79 58 10.5 8.06 200
6.7 4.88 61 10.6 8.15 204
6.8 4.96 65 10.7 8.23 207
6.9 5.05 68 10.8 8.31 211
7.0 5.13 72 10.9 8.40 215
7.1 5.21 76 11.0 8.48 219
7.2 5.30 79 11.1 8.56 222
7.3 5.38 83 11.2 8.65 226
7.4 5.46 87 11.3 8.73 230
7.5 5.55 90 11.4 8.82 233
7.6 5.63 94 11.5 8.90 237
7.7 5.72 98 11.6 8.98 241
7.8 5.80 101 11.7 9.07 244
7.9 5.88 105 11.8 9.15 248
8.0 5.97 109 11.9 9.24 252
8.1 6.05 112 12.0 9.32 255
8.2 6.14 116 12.1 9.40 259
8.3 6.22 120 12.2 9.49 263
8.4 6.30 123 12.3 9.57 266
8.5 6.39 127 12.4 9.65 270
8.6 6.47 131 12.5 9.74 274
8.7 6.55 134 12.6 9.82 277
8.8 6.64 138 12.7 9.91 281
8.9 6.72 142 12.8 9.99 285
9.0 6.81 145 12.9 10.07 288
9.1 6.89 149 13.0 10.16 292
9.2 6.97 153 13.1 10.24 295
9.3 7.06 156 13.2 10.33 299
9.4 7.14 160 13.3 10.41 304
9.5 7.22 164 13.4 10.49 309
9.6 7.31 167 13.5 10.58 314
9.7 7.39 171 13.6 10.66 320
9.8 7.48 175 13.7 10.74 326
It should be noted that glycosylated hemoglobin values below 6.0% may indicate the presence of abnormal hemoglobins (S and C)
of hemolytic disorders. Additional tests are the recommended.
REFERENCES :
(1) Nathan, D.M., et al., The Clinical information value of the Glycosylated Assay, The New England Journal of Medicine, 310 : 346
(1984).
