1. Blood Glucose Test By Fifth Stage Student 18 Dr. Sakar Karem Abdulla
M.Sc. Clinical Biochemistry

2. Glucose
Glucose is a major tissue energy source especially for brain which is highly dependent upon the extracellular glucose concentration for its energy supply.
indeed, hypoglycaemia is likely to impair cerebral function or even lead to irreversible neuronald amaqe.
o metabolizes ubstrates other than glucose and ketones plasma ketonec oncentrations are usually very low and ketonesa re of little importance as an energys ource underp hysiological conditions, o extracte nough glucose from the extracellularfl uid at low concentrationfso r its metabolicn eeds, because entry into brain cells is not facilitated by insulin

3. Hormones involved in glucose homeostasis
Insulin
Glucagon
Somatostatin
Other hormones such as growth hormone, glucocorticoids and adrenaline which oppose the normal action of insulin.

4. Renal threshold for glucose
The proximal tubule can only reabsorb a limited amount of glucose this point is called the renal threshold for glucose (RTG).
Normaly renal tubular cells reabsorb almost all the glucose filtered by the glomeruli, and urinary glucose concentration is normally too low to be detected by the test.
When the plasma glucose concentration exceeds the renal threshold the proximal tubule becomes overwhelmed and begins to excrete glucose in the urine which known as glycosuria

6. Hyperglycemia
Hyperglycemia which is high level of blood glucose concentration may be due to:
Intravenous infusion of glucose-containing fluids
Severe stress
Diabetes mellitus

7. Blood glucose tests Random blood sugar test Fasting blood sugar test
Glycated heamoglobin (HbA1c) test
Glucose tolerance test (GTT)

8. HbA1c test
HbA1c refers to glycated haemoglobin which is formed by non enzymatic glycation of haemoglobin and is depend on mean plasma glucose concentrations and on the lifespan of RBC.
HbA1c test measures the amount of glucose bound to hemoglobin over days.
HbA1c test does not require fasting
HbA1c test used for diagnosis and follow-up of diabetes mellitus.
The result value below 6.5% represents healthy subject and above that value indicates diabetes.

9. HbA1c test
For HbA1c test we drew blood from a vein of the patients and put in EDTA tube
We dispensed 500µl hemolysin reagent into tubes labeled. Then added 10µl of well mixed whole blood into the tube. It was mixed and allowed to stand for 5 minutes.
These reagents used in automatic analyzers
or until complete lysis is evident, The treated sample may be stored up to 10 days at 2-8oC.

10. OGTT
The patient should be fasting ( for at least 10h but not more than 16h)
A venous sample is withdrawn for fasting plasma glucose estimation
A solution containing 75 g of anhydrous glucose in 300 mL of water should be drunk slowly over a few minutes
Further blood is taken 2h after the ingestion of glucose then interpret the result.

11. OGTT
In normal individual fasting blood glucose levels <110 mg/dL and a rise to < 140 mg/dL after a glucose load.
In diabetic patients fasting blood glucose ≥126 mg/dL, it rises to ≥200 mg/dL following the oral administration of glucose.

12. Diagnosis of diabetes
FPG ≥126 mg/dL (on more than one occasion or once in the presence of diabetes symptoms)
Random plasma glucose ≥200 mg/dL
HbA1c ≥6.5%
In oral glucose tolerance test FPG ≥126 mg/dL 2-h after PG ≥200 mg/dL

13. Reagents of blood glucose test
R1: Phenol
R2: Glucose oxidase (GOD), Peroxidase (POD), 4 – Aminophenazone
Glucose standard 100 mg/dL
Working reagent: Dissolve the contents of one vial R2 enzymes in one bottle of R1. Cap and mix gently to dissolve contents. The reagent is stable 1 month after reconstitution in the refrigerator (2-8ºC).

14. Principle of blood glucose test
Colorimetric, enzymatic method with glucose oxidase
Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide.
The formed hydrogen peroxide (H2O2), react with phenol and aminophenazone in the presence of peroxidase (POD) to form a color complex quinone.
The absorbance of coloured complex proportional to the concentration of glucose in the sample

15. Principle of blood glucose test
Glucose + H2O + O2 → gluconic acid + H2O2 (by glucose oxidase)
2H2O2 +phenol + 4-aminophenazone → quinone+ 4 H2O (by peroxidase)
The intensity of the color formed is proportional to the glucose concentration in the sample.

16. Test procedure Standard Assay 1000 µl Working reagent 10 µl Serum
Mix and incubate for 20 min at room temperature
Read the absorbance of the samples and standard at 500 nm
The color is stable for at least 30 minutes.

17. calculation
S.glucose=Abs.Assay/Abs.standard  x conc.standard (100 mg/dl)