Analytical Methods of Determining Diabetes MellitusHANISHA ERICA P. VILLAESTER

A. Blood Test

Sample Collection Venipuncture Capillary Blood by Skin Puncture

Done when venipuncture cannot be performed or when reducing frequency of needle sticks is desired and less painful

A.1 FASTING BLOOD GLUCOSE TEST

Person to be tested should be on normal diet for at least 3 days prior to testing Fasting for 8 – 12 hours Plasma or serum is used instead of whole blood

More readily measured on automated equipment Glucose concentration is 10 – 15% higher than in whole blood

A.1 FASTING BLOOD GLUCOSE

FASTING BLOOD GLUCOSE

< 110 mg/dL Normal

110 – 126 mg/dL Pre-Diabetes

> 126 mg/dL Diabetes

A.2.a ORAL GLUCOSE TOLERANCE TEST

Fasting for 8 – 12 hours Patients requested to drink anhydrous glucose dissolved in 250 – 300mL water

Adults: 75g anhydrous glucose Children: 1.75g/kg of body weight anhydrous glucose

Blood tested regularly in 30 minutes interval In conditions of insulin defiency, blood glucose levels got elevated due to impaired

utilization of glucose

Required for confirmation Glucose Tolerance Curve is then plotted

NORMAL GLUCOSE TOLERANCE CURVE

DIABETIC GLUCOSE TOLERANCE CURVE

NORMAL VS DIABETIC

A.2.b Intravenous Glucose Tolerance Test

Undertaken for patients with malabsorption Patient is given 25g of glucose dissolved in 100mL distilled water as intravenous

injection within 5 minutes Blood samples drawn at 10 minutes interval for the next 2 hours Interpretation:

Normal individuals, blood glucose level returns to normal within 60 minutes Diabetes mellitus – decline is slow

A.2.b Intravenous Glucose Tolerance Test

Factors Affecting Glucose Tolerance Test Acute Infection Liver Disease Hyperthyroidism Hypothyroidism Starvation

A.2.c Mini or Modern GTT

Two samples are collected Fasting (zero hour) 2 Hour Post Glucose Load

Zero Hour After 2 Hours

Normal Person < 110 mg/dL < 140 mg/dL

Diabetic Person > 126 mg/dL > 200 mg/dL

Increase Glucose Tolerance 110 – 126 mg/dL 140 – 199 mg/dL

A.3 Post Prandial Blood Sugar

Patient advised to have normal meal Blood collected 2 hours after meal

Glucose Level

Normal < 140 mg/dL

Pre-diabetic 140 – 200 mg/dL

Diabetic > 200 mg/dL

A.4 Random Blood Glucose

Only required during emergency Diabetis Mellitus: > 200 mg/dL

A.5 HbA1c

Test that measures amount of glycated haemoglobin in your blood Glycated haemoglobin is a substance in red blood cell that is formed when blood

sugar attaches to haemoglobin Generally reflect the state of glycemia over the preceding 8 – 12 weeks thereby

providing an improved method of assessing diabetic control

Analytical Methods of Determining Diabetes MellitusKEZIA AIJELETH SHAHAR CABANGAL

Self monitoring blood glucose (SMBG)

People who take insulin should regularly self monitor blood glucose.

For people with non-insulin treated type 2 diabetes testing is most useful if patients use the results to learn and alter behaviour, or medication.

“...SMBG is most useful if patients use the results to learn, as part of an overall diabetes education package….”

Laboratory tests to prevent and delay complications of diabetes

People with diabetes usually die from macrovascular complications of their diabetes; namely cardiovascular disease. This is influenced by all of the commonly recognised risk factors for cardiovascular disease as well as glycaemic control. Fasting lipid levels are measured three monthly until stable and then 6 - 12 monthly thereafter.

It is important that management should be individualised

Parameter Optimal value

Total cholesterol < 4 mmol/L

LDL cholesterol

< 2.5 mmol/L

HDL cholesterol > 1 mmol/L

TC:HDL ratio < 4.5

Triglycerides < 1.7 mmol/L

HbA1C < 7 mmol/L

Diabetic renal disease

The best way of testing for diabetic renal disease is by urinary albumin:creatinine ratio (ACR) and serum creatinine with estimated glomerular filtration rate (eGFR). These tests are performed on everyone with diabetes at diagnosis and repeated at least annually – more frequently if there is proteinuria, microalbuminuria or reduced eGFR.Albumin:creatinine ratio

ACR provides an estimate of daily urinary albumin excretion. Microalbuminuria cannot be detected on a conventional urinary protein dip stick. Microalbuminuria is urinary albumin excretion between 30 and 300 mg/day; above

300mg/day represents proteinuria. ACR is best measured in the laboratory using a first morning urine sample where

possible when the patient is well. An abnormal initial test requires confirmation by testing on two further occasions.

If at least one of these tests is positive microalbuminuria has been confirmed.

Renal testing in diabetes

*Non-diabetic renal disease is suspected when there is absence of diabetic retinopathy in a person with renal disease, there are urinary abnormalities such as haematuria or casts, or when there is renal disease without microalbuminuria or proteinuria.

ACRmg/mmol(confirme

d)

eGFRmL/

min/1.732

Risk Management

men < 2.5women <

3.5and > 60

2 - 4% per year progress to microalbuminuria.

Annual ACR and eGFR. Good diabetes & BP management.

men ≥ 2.5women ≥

3.5or < 60

One third progress to overt nephropathy.CVD risk doubled.

Review ACR and eGFR at each visit.Intensive management of glycaemia and CVD risk factors.Use ACE inhibitor and low-dose aspirin.Avoid nephrotoxic drugs.Investigate if suspicious of causes other than diabetes*

> 30 or < 30

Almost all proceed to end stage renal disease or die prematurely of CVD.

Overt nephropathyRefer specialist

Other tests

Testing of LFTs is recommended for people with diabetes: at diagnosis, at the start of antidiabetic drug therapy, and at any other time indicated by clinical judgement

Other laboratory testsIn patients with type 1 diabetes, intermittent checks for other autoimmune conditions may be useful. This could include testing for thyroid dysfunction or coeliac disease.

22Urinary Glucose

-Glucose can be detected in urine using the specific test strips that contain glucose

oxidase, peroxidase, and a chromagen.

-Other carbohydrates using Benedict's and Febling's reagents.

23Urinary Ketones

-Acetone and acetoacetic acid can be detected in urine using the AcetesTM or KetostixTM systems.

-These tablets or strips use nitroprusside (sodium nitroferricyanide) to detect ketones.

24Urinary Ketones

-Because beta-hydroxybutyric acid lacks a ketone group is not detected by this assay.

-Quantitative assays for acetoacetate and beta-hydroxybutyric acid are available using beta-hydroxybutyrate dehydrogenase and either NADH or NAD.

25Urinary Ketones

-If NAD is used as the cofactor and the reaction is buffered at around pH 9.0, beta-hydroxyburyric acid is measured.

-On the other hand, a separate reaction using NADH and buffered around pH 7.0 would measure acetoacetic acid.

26Microalbuminuria

-Diabetes mellitus causes progressive changes to the kidneys and ultimately results in diabetic renal nephropathy.

-This complication progresses over a period of years and may be delayed by aggressive glycaemic control.

-An early sign that nephropathy is occurring is an increase in urinary albumin.

27Microalbuminuria

-Microalbumin measurements are useful to assist in diagnosis at an early stage and

prior to the development of proteinuria. -Microalbumin concentrations are between 20 to 300 mg/d. -Proteinuria is typically greater than 0.5 g/d.

28Proteinuria in Diabetes

- Many people excrete small quantities of protein in urine, typically around 10 mg/day of mainly low molecular weight proteins such as albumin.

-Some diabetic patients develop albumin excretion rates 30 µg/min this range classed as microalbuminuria.

29

METHODS FOR THE DETERMINATION OF GLUCOSE

The most used methods of

glucose analysis employ the enzymes glucose oxidase or hexokinas.

A) Glucose Oxidase

B) Hexokinase

30

SELECTED METHODS FOR THE MEASUREMENTS OF GLYCATED HAEMOGLOBINS

Interference Measurement Method Carbamyl HbHbFTemperature-sensitive

HbA1c Cation exchange

HbA1c Monoclonal antibody

Glycated Hb Affinity chromatographyPhenyl boronate matrixLatex agglutinationFluorescence quenching

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