HbA1c test

Hey (:

Subject Title: Clinical Chemistry
Topic: HbA1c test

Haemoglobin A1c (HbA1c) Test using Bio-Rad D-10

Note: I will just be focusing on the main procedure of running the samples. I will talk about the Calibration next week.


Principle

This test is based on ion- exchange high-performance liquid chromatography. Samples are automatically diluted on the D-10, injected into the analytical flow path, and applied to the analytical cartridge. The D-10 provides a programmed buffer gradient of increasing ionic strength to the cartridge, where the haemoglobins are separated based on their ionic interactions with the cartridge material. The separated haemoglobins then pass through the flow cell of the filter photometer, where changes in the absorbance at 415 nm are measured.

An accurate index of the mean blood glucose concentration maybe established by the measurement of haemoglobin A1c every 2 to 3 months. HbA1c is formed in 2 steps by non-enzymatic glycation of HbA. The first step is the formation of an unstable aldimine (pre- A1c), a reversible reaction between the carbonyl group of glucose and the N –Terminal valine of the b-chain of haemoglobin. During red blood cell circulation, some of the pre- A1c is converted to form a stable ketoamine, HbA1c. The level of HbA1c is proportional to both the average glucose concentration and the life span of the red blood cell in the circulation. The measurement of HbA1c has therefore been accepted as the clinical management of diabetes.


Materials

1.Bio-Rad D-10 Machine
2.D-10- Haemoglobin A1cRecorder Pack
a) Elution Buffer 1
b) Elution Buffer 2
c) Wash/Diluent Solution
d) Analytical Cartridge
e) Floppy Diskette
f) Calibrator/Diluent Set
g) Whole Blood Primer
h) Samples Vials
I) Thermal Paper
3.Sample Vial Adapter, 10 x 1.5mL
4.Lymphochek Diabetes Bi-level Control, 6 x 0.5 mL
5.Pipettes, 5mL, 0.5mL, 1mL, 7mL
6.Deionised water
7.Disposable gloves



Method

1.Start up the machine if it is in “Sleep” mode.
2.Collect EDTA.
3.Check the amount of blood present.
4.If there is less than about 2 mL of blood in the tube, the sample will be pre-diluted.
5.To pre-dilute, pipette 1500 mL of Wash/Diluent Solution into a vial, followed by 5 mL of the whole blood sample. Use a sample vial adapter for 1.5 mL vials.
6.Invert the tubes about 6 times.
7.Load the EDTA tube(s) into D-10 sample rack and place the rack in D-10. Must ensure that the sample barcodes are facing towards the back of the instrument.
8.Wait for the sample’s laboratory request number to appear on the screen (if they are not pre-diluted) before pressing “Start”.
9.When more than 1 tube is tested, press “Edit” and then, press “Done” for all the tubes and finally, press “Start”.


Results and Interpretation

1. Go to “data” and click “details’’
2. The graph must be above 0.02. If below 0.02, repeat test
3. F must not be more than 3%
4. Total area: 1- 4 million (cannot be more than 5 million)
5. P3 cannot be more than 3
6. Cannot have “unknown” below A1c, if not reject the test
7. The overall results must not be more than 10, otherwise, rerun the test and record “R:” in the worksheet.
8. If got variant window, record “variant window” in worksheet and then
- select sample with variant window
- choose “selected samples”
- Press ‘’Print”
9. At the end of the day, go to “data”, daily summary and press ‘‘Print”
10. If the total area is low (lesser than 1 million), do Manual run (dilution). If total area is very low, take more of sample and vice versa.
a. Take the diluent bottle (ensure that lot number on the bottle is the same with main diluent bottle located behind of the machine)
b. Take the patient’s sample and pipette out 5ul to 1500ul of diluent in the sample vial
c. Invert a few times to mix it
d. Manually key in sample number and run HbA1c test again


Samples from patients with haemolytic anaemia will exhibit decreased glycated haemoglobin values due to shortened lifespan of the red blood cells.

Do not report results if the peak shape of the graph is abnormal.

Expected Value Range

Factors such as duration of diabetes, adherence to therapy, and the age of the patient should also be considered in assessing the degree of blood glucose control.