diagnosis tessts in diabetes mellitus_ dr selim

Diagnostic Testsof Diabetes

Dr Shahjada Selim Assistant Professor

Department of EndocrinologyBangabandhu Sheikh Mujib Medical University

Email: [email protected], [email protected]

mailto:[email protected]

mailto:[email protected]

GLUCOSE TOLERANCE GLUCOSE TOLERANCE TEST [GTT]TEST [GTT]

A glucose tolerance test is the A glucose tolerance test is the administration of glucose in a controlled administration of glucose in a controlled and defined environment to determine how and defined environment to determine how quickly it is cleared from the blood. The quickly it is cleared from the blood. The test is usually used to test for diabetes, test is usually used to test for diabetes, insulin resistance, and sometimes reactive insulin resistance, and sometimes reactive hypoglycemia. The glucose is most often hypoglycemia. The glucose is most often given orally. given orally.

Types of GTTTypes of GTT

Standard Oral glucose tolerance testStandard Oral glucose tolerance test

I/V Glucose tolerance testI/V Glucose tolerance test

Mini Glucose tolerance testMini Glucose tolerance test

GGlucose tolerance testlucose tolerance test Glucose tolerance means the ability of the Glucose tolerance means the ability of the

body to utilize glucose in blood circulation.body to utilize glucose in blood circulation.

American Diabetes Association -------- For American Diabetes Association -------- For routine diagnosisroutine diagnosis

WHO ------------For those with impaired fasting WHO ------------For those with impaired fasting glucose.glucose.

American Diabetes Association and WHOAmerican Diabetes Association and WHO

Gestational Diabetes Gestational Diabetes

Indication of OGTTIndication of OGTT In In asymptomaticasymptomatic persons with sustained or transient persons with sustained or transient

glycosuriaglycosuria.. In persons with In persons with symptoms of diabetes but no symptoms of diabetes but no

glycosuria or hyperglycemiaglycosuria or hyperglycemia.. Persons with Persons with family history but no symptoms or family history but no symptoms or

positive blood findingspositive blood findings.. In persons with or without symptoms of diabetes In persons with or without symptoms of diabetes

mellitus mellitus showing one abnormal blood findingsshowing one abnormal blood findings.. In patients with neuropathies or retinopathies of In patients with neuropathies or retinopathies of

unknown unknown origin.origin.

Contraindications of Contraindications of glucose tolerance testglucose tolerance test There is no indication for doing GTT in There is no indication for doing GTT in

a person with a person with confirmedconfirmed diabetics diabetics mellitus.mellitus.

GTT has no role in GTT has no role in follow-upfollow-up of of diabetics.diabetics.

The test should not be done in The test should not be done in ill ill patientspatients..

Patient should on carbohydrate rich Patient should on carbohydrate rich unrestricted diet for 3 days.unrestricted diet for 3 days.

Patient should be ambulatory with normal Patient should be ambulatory with normal physical activity.physical activity.

Medications should be discontinued on the Medications should be discontinued on the day of testing.day of testing.

Exercise, smoking and tea or coffee are not Exercise, smoking and tea or coffee are not allowed during test period.allowed during test period.

OGTT carried out in the morning after patient OGTT carried out in the morning after patient has has fasted overnight for 8-12 hours.fasted overnight for 8-12 hours.

Preparation of patientPreparation of patient

TestTest

A fasting venous A fasting venous blood sample is blood sample is collected in the collected in the morning.morning.

Patients ingest 75 g Patients ingest 75 g of anhydrous of anhydrous glucose in 250-300 glucose in 250-300 ml of water over 5 ml of water over 5 minutes. ( for minutes. ( for children, the dose is children, the dose is 1.75 g of glucose 1.75 g of glucose per kg).per kg).

Test Test

In the classical procedures, the blood In the classical procedures, the blood and urine samples are collected at half and urine samples are collected at half hourly interval of the next three hours.hourly interval of the next three hours.

A curve is plotted with the blood glucose A curve is plotted with the blood glucose levels on the vertical axis against the levels on the vertical axis against the time of collection on the horizontal axis. time of collection on the horizontal axis.

The curve so obtained is called glucose The curve so obtained is called glucose tolerance curve. tolerance curve.

Normal Glucose Normal Glucose tolerance curvetolerance curve

Diabetic curveDiabetic curve

Intravenous GTTIntravenous GTT

• This test is undertaken for patients with This test is undertaken for patients with malabsorption (Celiac disease or malabsorption (Celiac disease or enteropathies).enteropathies).

• Under these conditions oral glucose Under these conditions oral glucose load is not well absorbed and the results load is not well absorbed and the results of oral glucose tolerance test become of oral glucose tolerance test become inconclusive. inconclusive.

I/V GTT- ProcedureI/V GTT- Procedure• I/V glucose tolerance test is carried I/V glucose tolerance test is carried

out by giving 25 g of glucose out by giving 25 g of glucose dissolved in 100 ml distilled water as dissolved in 100 ml distilled water as intravenous injection within 5 intravenous injection within 5 minutes. minutes.

• Completion of infusion is taken as Completion of infusion is taken as time zero.time zero.

• Blood samples are taken at 10 Blood samples are taken at 10 minutes interval for the next hour. minutes interval for the next hour.

• The peak value is reached within a The peak value is reached within a few minutes. few minutes.

I/V GTTI/V GTT

Interpretation:Interpretation:• Normally, blood glucose level returns to Normally, blood glucose level returns to

normal range within 60 minutes.normal range within 60 minutes.• In diabetes mellitus, this decline is slow.In diabetes mellitus, this decline is slow.

Mini or Modern GTTMini or Modern GTT

As per current WHO recommendations, in As per current WHO recommendations, in the mini or modern glucose tolerance test, the mini or modern glucose tolerance test, only two samples are collected: Fasting only two samples are collected: Fasting (zero hour) and 2 hour post glucose load. (zero hour) and 2 hour post glucose load.

Urine samples are also collected during Urine samples are also collected during the same time. the same time.

The diagnosis is made from the variations The diagnosis is made from the variations observed in these results. observed in these results.

Mini or Modern GTTMini or Modern GTT

Zero Hour After 2 Hours

Normal Person

< 110 mg/dL < 140 mg/dL

Increase Glucose

Tolerance110 – 126

mg/dL140 – 199

mg/dL

GTT Under special conditions

Cortisone stress test- used for Cortisone stress test- used for detecting pre diabetes or Latent detecting pre diabetes or Latent diabetesdiabetes

Extended GTT- To diagnose the Extended GTT- To diagnose the cause of hypoglycemia especially 2-cause of hypoglycemia especially 2-3 hours after meals.3 hours after meals.

Factors affecting Factors affecting GTTGTT

a)a) Acute infections- Cortisol is secreted, the Acute infections- Cortisol is secreted, the curve is elevated and prolonged. curve is elevated and prolonged.

b)b) Hypothyroidism-A flat curve is obtained in Hypothyroidism-A flat curve is obtained in hypothyroidism. Thyroid hormone hypothyroidism. Thyroid hormone increases the absorption of glucose from increases the absorption of glucose from the gut. the gut.

c)c) Starvation- There is rise of counter Starvation- There is rise of counter regulatory hormones, which show regulatory hormones, which show increased glucose tolerance.increased glucose tolerance.

Gestational Gestational diabetesdiabetes

Gestational diabetes is high Gestational diabetes is high blood sugar that develops at blood sugar that develops at any time during pregnancy in a any time during pregnancy in a woman who does not have woman who does not have diabetes.diabetes.

OGTT in gestational OGTT in gestational DiabetesDiabetes Impairment of glucose tolerance develops Impairment of glucose tolerance develops

normally during pregnancy, particularly in normally during pregnancy, particularly in 22ndnd and 3 and 3rdrd trimester. trimester.

Repeat testing on subsequent day OGTT indicated for average risk and high risk pregnant female

OGTT for GDM

One step approach Two step approach

100 gm glucose is administered

3- hours OGTT is performed

50 gm glucose is administered irrespective of time of last meal

After one hour, venous blood sample collected

If glucose level exceeds 140 mg/dl

Otherwise GDM is excluded

Gestational diabetes is diagnosed if the woman Gestational diabetes is diagnosed if the woman is at or exceeds any two of the following four is at or exceeds any two of the following four plasma glucose levels during 100 gm testplasma glucose levels during 100 gm test

Fasting – 95 mg/dl1 hr – 180 mg/dl2 hr – 155 mg/dl3 hr – 140 mg/dl

Laboratory Test for Laboratory Test for ScreeningScreening

Laboratory test for Laboratory test for screeningscreening

Recommended screening test is fasting Recommended screening test is fasting plasma glucose.plasma glucose.

American Diabetes Association American Diabetes Association recommends screening for Type 2 DM recommends screening for Type 2 DM in all asymptomatic individuals ≥ 45 yrs in all asymptomatic individuals ≥ 45 yrs of age using fasting plasma glucose.of age using fasting plasma glucose.

Laboratory test for Laboratory test for screeningscreening

If fasting test is normal, screening If fasting test is normal, screening test should be repeated every three test should be repeated every three years.years.

If fasting blood glucose level is If fasting blood glucose level is normal but there is strong clinical normal but there is strong clinical suspicion then OGTT.suspicion then OGTT.

Selective screeningSelective screening High risk individuals ---Obese High risk individuals ---Obese Family h/o DMFamily h/o DM Hypertension Hypertension DyslipidemiaDyslipidemia Impaired glucose Impaired glucose

tolerancetolerance

Screening test is performed at earlier age ( 30 yrs ) and repeated more frequently

Laboratory Test to Laboratory Test to Assess Glycemic Assess Glycemic

ControlControl

Laboratory test to assess Laboratory test to assess glycemic controlglycemic control

Periodic measurement of glycated Periodic measurement of glycated hemoglobin.hemoglobin.

Daily self assessment of blood Daily self assessment of blood glucose.glucose.

Others.Others.

Glycated HemoglobinGlycated Hemoglobin Glycated hemoglobin Glycated hemoglobin

covers a number of covers a number of chemically different chemically different modification resulting modification resulting from the from the non-enzymaticnon-enzymatic and and irreversiblyirreversibly binding of different binding of different sugars to different sugars to different amino groups in the amino groups in the hemoglobin molecule. hemoglobin molecule.

(Maillard Reaction )(Maillard Reaction )Hemoglobin + glucose Aldimine Glycated hemoglobin

Glycated hemoglobinGlycated hemoglobin HbA₁HbA₁сс gives information about the gives information about the

average blood glucose concentration average blood glucose concentration over a retrospective period of time.over a retrospective period of time.

Reflects the mean glucose Reflects the mean glucose concentration.concentration.

Normally, less than 5% of hemoglobin Normally, less than 5% of hemoglobin is glycated.is glycated.

Glycated hemoglobinGlycated hemoglobin

About 50% HbA₁About 50% HbA₁сс values results from values results from the blood glucose of the preceding 30 the blood glucose of the preceding 30 days , 40% from the preceding 31 -90 days , 40% from the preceding 31 -90 days and only 10% from the period days and only 10% from the period between the 91 – 120 days.between the 91 – 120 days.

No effect of diet, exercise & insulin on No effect of diet, exercise & insulin on test results.test results.

More informative. More informative.

HbA1CHbA1C

Blood sample can be drawn at any Blood sample can be drawn at any time of day.time of day.

HbA1c of 6 % corresponds to mean HbA1c of 6 % corresponds to mean serum glucose level of 135 mg/dl. serum glucose level of 135 mg/dl.

With every rise of 1.0%, serum With every rise of 1.0%, serum glucose increases by 35 mg/dl.glucose increases by 35 mg/dl.

INDICATIONSINDICATIONS In all diabetes to monitor long term In all diabetes to monitor long term

blood glucose level control, index of blood glucose level control, index of diabetic control:diabetic control:

7% HbA₁7% HbA₁сс – good – good 10% HbA₁10% HbA₁сс- Moderate uncontrolled- Moderate uncontrolled > 13% HbA₁> 13% HbA₁сс- Very poor.- Very poor. To monitor patient compliance.To monitor patient compliance.

INDICATIONSINDICATIONS To predict development & progression To predict development & progression

of microvascular complication.of microvascular complication. For determining the therapeutic For determining the therapeutic

option whether to use oral agents, option whether to use oral agents, insulin or insulin or ββ cell transplantation. cell transplantation.

Also increasingly used for primary Also increasingly used for primary diagnosis of DM.diagnosis of DM.

Methods used for Methods used for determination of HbA₁determination of HbA₁сс

HbAHbA1c1c electrophoresis. electrophoresis.

Cation-exchange Cation-exchange chromatography, chromatography,

Boronate affinity Boronate affinity ChromatographyChromatography

Immunoassays. Immunoassays. Colorimetric methodColorimetric method

At what interval should At what interval should HbA₁с be determined?HbA₁с be determined?

Treatment by time of diabetes

Recommended frequency

Type-1 DM( minimal /conventional therapy)

4 times a year

Type – 1 DM (intensified therapy)

Every (1) -2 months.

Type-2 DM Twice a year in stable patients.

Glycated hemoglobinGlycated hemoglobin High Values Low ValuesDiabetes Mellitus Haemolysed

specimenPCOS Hereditary HbFHyperglycemia Neonate &

PregnancyGlycosuria Fetal maternal

transfusion

Glycated hemoglobinGlycated hemoglobinFalsely high values

Falsely low values

Iron deficiency anemia

Hemolytic anemia

Post spleenectomy Chronic blood lossAlcohol poisoning Chronic Renal

FailureLead toxicity Pregnancy

The goal of therapy should be to achieve The goal of therapy should be to achieve HbA₁с values as close as possible to the HbA₁с values as close as possible to the refrence range but without losing sight of refrence range but without losing sight of the increased risk of hypoglycemia.the increased risk of hypoglycemia.

Guideline by ADA:-Guideline by ADA:- HbA₁с values <7% indicate good HbA₁с values <7% indicate good

glycemic control.(normal range: 4.5% - glycemic control.(normal range: 4.5% - 6.3%).6.3%).

If HbA₁с values > 8% the treatment If HbA₁с values > 8% the treatment should be reconsidered.should be reconsidered.

Fructosamine assayFructosamine assay Generic term for measurement of all Generic term for measurement of all

serum glycated protein though the serum glycated protein though the bulk being albumin.bulk being albumin.

Does not appear to be influenced by Does not appear to be influenced by transient (stress) hyperglycaemia.transient (stress) hyperglycaemia.

Unable to detect short term or transient Unable to detect short term or transient abnormalities in the blood glucose abnormalities in the blood glucose concentration. Ex: hypoglycemia.concentration. Ex: hypoglycemia.

Reference range – in non diabetic- 2.4-Reference range – in non diabetic- 2.4-3.4 mmol/l.3.4 mmol/l.

Fructosamine / albumin ratio:- 54- 86 Fructosamine / albumin ratio:- 54- 86 µmol/gm.µmol/gm.

Self Monitoring of Blood Glucose- Self Monitoring of Blood Glucose- SMBGSMBG

Regular use of SMBG devices by Regular use of SMBG devices by diabetic patients has improved the diabetic patients has improved the management of DM.management of DM.

SMBG devices measure capillary SMBG devices measure capillary whole blood glucose obtained by whole blood glucose obtained by finger prick and use test strips that finger prick and use test strips that incorporate glucose oxidase or incorporate glucose oxidase or hexokinase.hexokinase.

SMBG devices yield unreliable SMBG devices yield unreliable results at very high and very low results at very high and very low glucose levels.glucose levels.

It is necessary to periodically check It is necessary to periodically check the performance of glucometer by the performance of glucometer by measuring parallel venous plasma measuring parallel venous plasma glucose in the laboratory.glucose in the laboratory.

Fructosamine assayFructosamine assay

Fructosamine test HbA1c

Measures average blood glucose level over the past two or

three weeks

Measures average blood glucose level over the past two to three months.

Glycosylated Albumin

Half -life of albumin is approximately Half -life of albumin is approximately 15 days.15 days.

Glycated albumin level is believed to Glycated albumin level is believed to reflect the glycemic change over a 2-reflect the glycemic change over a 2-week period.week period.

GA can be useful in evaluating the GA can be useful in evaluating the therapeutic effect of recently therapeutic effect of recently substituted hypoglycemic agents at an substituted hypoglycemic agents at an early stage.early stage.

Glycosylated Albumin

GA can also act as a valuable GA can also act as a valuable glycemic control marker in glycemic control marker in diabetic patients with various diabetic patients with various comorbidities since it is unrelated comorbidities since it is unrelated to the metabolism of hemoglobin.to the metabolism of hemoglobin.

Insulin assayInsulin assay

Measurement of Measurement of insulin level by insulin level by radioimmunoassay & radioimmunoassay & ELISA.ELISA.

Crucial for type I DM.Crucial for type I DM.

Proinsulin AssayProinsulin Assay It is precursor molecule for insulin. Most proinsulin is converted to insulin and

C-Peptide, which are secreted in equimolar amounts into the blood.

The biological activity of proinsulin is only about 10% of insulin, but the half life of proinsulin is three times as long as insulin.

Proinsulin AssayElevated in:Elevated in:At onset of T1DM and in healthy sliblings of T1DM At onset of T1DM and in healthy sliblings of T1DM patients.patients.With established T2DM.With established T2DM.Older patients.Older patients.Pregnant .Pregnant .Obese pt with diabetes. Obese pt with diabetes. Insulinomas.Insulinomas.Functional hypoglycemia.Functional hypoglycemia.Hyperinsulinemia.Hyperinsulinemia.

Released in circulation during Released in circulation during conversion of proinsulin to insulin conversion of proinsulin to insulin in equimolar quantities to insulin.in equimolar quantities to insulin.

Its level correlate with insulin Its level correlate with insulin level in blood. level in blood.

Low C Low C –– peptide levels are peptide levels are characteristic of type I DM.characteristic of type I DM.

C-peptide levels are measured C-peptide levels are measured instead of insulin levels because instead of insulin levels because C- peptide can assess a person’s C- peptide can assess a person’s own insulin secretion even if they own insulin secretion even if they receive insulin injections.receive insulin injections.

The test may be used to help determine the cause of hypoglycaemia, values will be low if a person has taken an overdose of insulin but not suppressed if hypoglycaemia is due to an insulinomas..

Factitious hypoglycemia may occur secondary to the surreptious use of insulin. Measuring C-peptide levels will help differentiate a healthy patients from a diabetic one.

Urine glucose estimation

Presence of chemically detectable amount of glucose in urine is called glycosuria.

Urine glucose test results correlate well with plasma or serum glucose values.

Presence of glucose in urine indicates that blood glucose level of the patient could have elevated > 180 mg/dl.

Normally less than 500mg/24 hrs or less than 15 mg/dl of glucose is present in urine.

Renal Glycosuria Blood Glucose level is normal but there is defect in the

reabsorptive ability of renal tubule. Non pathological causes Pregnancy Stress Anxiety Pathological causes Cystinosis Heavy metal poisoning Fanconi’s syndrome Galactosemia

Alimentary glycosuria

Lag storage disorder. Occur in gastrectomy, gastrojejunostomy,

hyperthyroidism. Glucose tolerance test reveals a peak at 1

hour above renal threshold. Fasting and 2 hours glucose value are normal.

Qualitative test.Benedicts.Clintest tablet test.Reagent strip test Quantitative test. Benedicts.

Benedict’s testBenedict’s test Based on copper reduction methodBased on copper reduction method Detect any reducing sugar in urineDetect any reducing sugar in urine PrinciplePrinciple

Cu Cu 2+2+ CuCu + +

CuCu + + + OH+ OH -- CuOH CuOH

2CuOH2CuOH Cu Cu22O + HO + H22OO

Hot alkaline solution

Heat

Add 8 drops

of urine

Boil for 2

to 3 min

CoolTake 5.0ml of

Benedict’s

reagent

Observe

Benedict reagent : sodium citrate 173 gm, sodium carbonate 100 gm, cupric sulphate 17.3 gm and distill water 900 ml.

Procedure

Observations Observations Color Sugar

Blue Absent

Green without

precipitate

Present, trace

Green with

precipitate

1+ (0.5 g/dl)

Brown precipitate 2+ (1.0 g/dl)

Yellow - Orange

precipitate

3+ (1.5 g/dl)

Brick red precipitate 4+ (≥ 2.0 g/dl)

False positive False positive testtest

Ascorbic acid Creatinine Uric acid Homogentisic acid Cephalosporins Salicylates Radiographic media

Clinitest tablet method

Modified form of Benedicts test in which reagents are present in tablet form.

Contains copper sulfate, citric acid, sodium carbonate and anhydrous sodium hydroxide.

Reagent strip method

Based on specific glucose oxidase and peroxidase method.Based on specific glucose oxidase and peroxidase method. Specific for glucose.Specific for glucose. Principle - Principle -

Glucose + OGlucose + O22 Gluconic acid + H Gluconic acid + H22OO22

HH22OO22 + Chromogen + Chromogen oxidized chromogen oxidized chromogen + + HH22OO

False positive :False positive :

- Oxidizing cleaning agent in urine - Oxidizing cleaning agent in urine container.container.

False negative :False negative :

- Ascorbic acid- Ascorbic acid

Contents Contents –– Potassiun Thiocyanate , Potassium Potassiun Thiocyanate , Potassium Ferrocyanide , Sodium Citrate , Sodium Ferrocyanide , Sodium Citrate , Sodium

Carbonate , Copper SulfateCarbonate , Copper Sulfate. . Principle :Principle :

Cu Cu 2+2+ CuCu + +

CuCu + + +potassium thiocyanate +potassium thiocyanate Cu Cu thiocynatethiocynate

Hot alkaline solution

White precipitate

2-3g of

NaCO3

Keep Boiling

Add Urine drop

by drop using

5 ml ppt till

blue color

disappear

Take 5.0ml of

Benedict’s Qt

reagent

Chalky white

Method – titration

Calculation Glucose in urine = 5(g/100ml) urine

used

10 g glucose reduces 5 ml of reagent

Procedure

Benedicts qualitative tests

Positive

Glucose oxidase strip method

Positive Negative

Glucose Lactose

Fructose

Galactose

Benedicts quantitative test

Semiquantitative urine glucose testing for monitoring of diabetes mellitus in home setting is not recommended.

This is because(1)Even if glucose is absent in urine, no

information about blood glucose concentration below the renal threshold is obtained.

(2)Urinary glucose testing cannot detect hypoglycemia

(3)Concentration of glucose in urine is affected by urinary concentration.

Laboratory Tests to Laboratory Tests to Assess Long Term Assess Long Term

RisksRisks

Laboratory tests to assess long term

risks

1. Urinary albumin excretion.1. Urinary albumin excretion.

2. Lipid profile. 2. Lipid profile.

Screening for proteinuria should be performed yearly in the following patients:

Type 1 DM : 5 yrs after diagnosis of DM, or earlier in the presence of other cardiovascular risk factors.

Type 2 DM : at the time of diagnosis of diabetes.

Urine should be screened for proteinuria with conventional dipstick on an early morning urine specimen.

If urine dipstick for proteinuria is negative, screening for microalbuminuria should be performed.

If microalbuminuria is detected, confirmation should be made with two further tests within 3 to 6 month period.

Frank proteinuriaFrank proteinuria Precipitation test.Precipitation test. Heat test- precipitation of protein by heat.Heat test- precipitation of protein by heat. Not affected by radiographic contrast media.Not affected by radiographic contrast media. Sulfosalicylic acid method- precipitation of Sulfosalicylic acid method- precipitation of

protein by protein by acid. acid. False positive results are obtained in False positive results are obtained in

presence of radiographic contrast media.presence of radiographic contrast media. Reagent strip test.Reagent strip test.

Fill the supernatant urine upto 2/3 clean test tube

Boil the upper portion

PROCEDURE OF HEAT TEST

If turbidity develops add 1 to 2 drops of glacial acidic acid

Phosphates will clear

No turbidity – Proteins absent

Presence of turbidity – Proteins present

Transfer about 5ml urine to a centrifuge tube

Centrifuge Transfer 3.0 ml of supernatant urine in a clean test tube

Add 2-3 drops of 30% sulfosalicylic acid or equal amount of 3%

Mix well and Wait for 10 minutes

Observe the degree of turbidity and flocculation

PROCEDURE OF SULFOSALICYLIC ACID METHOD

1.1. NegativeNegative – No turbidity – No turbidity (~5mg/dl or less)(~5mg/dl or less)

2.2. TraceTrace – Perceptible turbidity – Perceptible turbidity (~20 mg/dl)(~20 mg/dl)

3.3. 1+1+ - Distinct turbidity but no - Distinct turbidity but no discrete granulation(~50mg/dl) discrete granulation(~50mg/dl)

4.4. 2+2+ - Turbidity with granulation - Turbidity with granulation but no flocculation(~200mg/dl) but no flocculation(~200mg/dl)

5.5. 3+3+ - Turbidity with granulation - Turbidity with granulation and flocculation(~500mg/dl) and flocculation(~500mg/dl)

6.6. 4+4+ - Clumps of precipitated - Clumps of precipitated protein, or solid precipitate protein, or solid precipitate (~1.0g/dl or more)(~1.0g/dl or more)

Reagent strip Reagent strip method method

Principle :Impregnated with bromphenol blue buffered to pH 3 with

citrate

30 to 60 second urine application

Variable sheds of green color formed

MicroalbuminuriaMicroalbuminuria Normally, only a small amount of albumin is

filtered at the glomerulus, and most of that albumin is degraded and reabsorbed by the proximal tubule.

Defined as persistent proteinuria that cannot be detected by routine reagent strips but greater than normal.

Present in the very early stage of diabetes, at a time when GFR may be normal and when there is no evidence of glomerular lesion.

MicroalbuminuriaMicroalbuminuria

Normalbuminuria- <20 microgram/minute or <30 mg/24 hrs.

Microalbuminuria- the range in between: Urinary excretion of albumin of 20-200

microgram/minute or 30-300 mg/24 hrs.• Macroalbuminuria- More than 200

microgram/minute or more than 300 mg/24 hrs.

MicroalbuminuriaMicroalbuminuriaLower limit

Upper limit

unit

24 hour urine collection

30 300 mg/24 hr

Short time urine

collection

20 200 ug/min

Spot urine albumin sample

30 300 mg/l

Urine albumin creatinin ratio Wome

n

3.5

30

35

300

mg/mmol

mg/gm

Men 2.5

30

35

300

mg/mmol

mg/gm

MicroalbuminuriaMicroalbuminuria It indicates increase in capillary

permeability to albumin. Albumin is the first protein to enter the

urine after the kidney is damaged. Appearance of microalbuminuria is

predictor of progression to overt proteinuria.(incipient nephropathy)

It is an independent risk factor for cardiovascular disease in diabetes mellitus.

Methods of detection include1. Measurement of albumin creatinine ratio

in a random urine sample2. Measurement of albumin in an early

morning and random3. Measurement of albumin in 24 hr sample.Test strips that screen for

microalbuminuria are available commercially.

Detection of microalbuminuriaDetection of microalbuminuria

Albumin to Creatinine Albumin to Creatinine ratioratio

Reagent Strips are firm plastic strips that contain two reagent areas that test for albumin and creatinine in urine.

An albumin-to-creatinine ratio is also determined, which allows for the use of single-void specimens in testing. The ratio is given in milligrams albumin per gram or millimole creatinine (mg/g or mg/mmol).

This product provides semi-quantitative results and can be used for screening samples for microalbuminuria; positive results should be confirmed with quantitative methods for albumin.

Microalbuminuria Microalbuminuria strips strips

The strip is an immunochemical strip specific for albumin.

Albumin in the sample get bound to soluble conjugate of antibodies and marker enzyme b-galactosidase.

Conjugate-albumin complexes are separated and enzyme b-galactosidase reacts with a substrate to produce a red dye.

The reagent part of the test strip should be dipped into the urine for 5 seconds and then laid down horizontally and read after 5 minutes.

Microalbuminuria Microalbuminuria strips strips

The intensity of the colour produced is proportional to the albumin concentration in the urine.

The colour formed is compared with the reference chart on the vial.

Quantitative test for Quantitative test for microalbuminuriamicroalbuminuria

Colorimetric testColorimetric test ELISA.ELISA. Radioimmune assay.Radioimmune assay. Immunoturbidiometric assay.Immunoturbidiometric assay. Nephelometry.Nephelometry. Chemiluminescence.Chemiluminescence.

DYE BINDING DYE BINDING COLORIMETRIC COLORIMETRIC

METHODMETHOD Pyrogallol red molybdate reagent complex

react with protein to form a blue purple colour.

Optical density of the coloured complex can be measured at 600nm.

The measured O.D. is propotional to the protein concentration in the specimen.

ELISAELISA

Uses antibodies and colour Uses antibodies and colour change to identify the change to identify the substance. substance.

The intensity of the color The intensity of the color measured with microwell measured with microwell reader at 450 nm.reader at 450 nm.

RADIOIMMUNOASSARADIOIMMUNOASSAYY

Technique used for the detection of Technique used for the detection of antibody or antigen.antibody or antigen.

Uses radioactive label or tracer .Uses radioactive label or tracer . Tritium, I-131, I-125 are commonly Tritium, I-131, I-125 are commonly

used tracers.used tracers. PRINCIPLE – competitive binding PRINCIPLE – competitive binding

between radiolabelled & unlabelled between radiolabelled & unlabelled molecules of antigen to bind with a molecules of antigen to bind with a high affinity , specific antibody.high affinity , specific antibody.

The amount of unlabelled antigen is The amount of unlabelled antigen is measured by its competitive effect measured by its competitive effect on the labelled antigen for limited on the labelled antigen for limited antibody sites.antibody sites.

TURBIDIMETRYTURBIDIMETRY Measurement of reduction in light Measurement of reduction in light

transmission caused by particle formation.transmission caused by particle formation. Light transmitted in forward direction is Light transmitted in forward direction is

detected.detected. Amount of light absorbed by a suspension Amount of light absorbed by a suspension

of particles depends on the specimen of particles depends on the specimen concentration & on particle size.concentration & on particle size.

Not specific to protein . Nucleic acid can Not specific to protein . Nucleic acid can also precipitate.also precipitate.

NEPHELOMETRYNEPHELOMETRY

Measurement of light Measurement of light scattered by the scattered by the particulate solution.particulate solution.

Nephelometer measure Nephelometer measure scattered light at 90 to scattered light at 90 to the incident light.the incident light.

CHEMILUMINESENCCHEMILUMINESENCEE

Chemiluminescence is the Chemiluminescence is the emission of energy with emission of energy with limited emission of heat limited emission of heat (luminescence), as the result (luminescence), as the result of a chemical reaction. of a chemical reaction.

In immunoassay In immunoassay technology , the light technology , the light produced by the reaction produced by the reaction indicates the amount of indicates the amount of analyate in the sample.analyate in the sample.

Triglycerides Triglycerides (mg/dl)(mg/dl) CategoryCategory

<150 <150 Low riskLow risk

150-199150-199 Intermediate riskIntermediate risk

≥ ≥ 200200 High riskHigh risk

LDL cholesterolLDL cholesterol<100<100 Low riskLow risk


≥≥130130 High riskHigh risk

HDL cholesterolHDL cholesterol<35<35 High riskHigh risk


>45>45 Low riskLow risk

Diagnostic Testing With 3 Diagnostic Testing With 3 Different TestsDifferent Tests

Dealing with Discordance•Many people identified as having diabetes using A1C will not be identified as having diabetes by traditional glucose criteria, and vice versa.

• When results of more than one test are available (amongst FPG, A1C, 2hPG in a 75-g OGTT) and the results are discordant, the test whose result is above diagnostic cut-point should be repeated, and the diagnosis made on basis of the repeat test.

FPG 2hPG

A1C

Fasting plasma glucose (FPG)≥126 mg/dL (7.0 mmol/L)

OR2-h plasma glucose ≥200 mg/dL(11.1 mmol/L) during an OGTT

ORA1C ≥6.5%

ORRandom plasma glucose

≥200 mg/dL (11.1 mmol/L)

Criteria for the Diagnosis of Diabetes ADA 2017

American Diabetes Association Standards of Medical Care in Diabetes. Classification and diagnosis of diabetes. Diabetes Care 2016; 39 (Suppl. 1): S13-S22

A1C ≥6.5% *A1C ≥6.5% *

Performed in a laboratory using a method that is NGSP certified and standardized to the DCCT assay - www.ngsp.org

POC testing not recommended Greater convenience, preanalytical

stability, and less day-to-day perturbations than FPG and OGTT

Consider cost, age, race/ethnicity, anemia, etc.


Testing should begin at age 45 for Testing should begin at age 45 for all patients, particularly those who all patients, particularly those who are overweight or obese. are overweight or obese. BB

Recommendations: PrediabetesRecommendations: Prediabetes


FPG, 2-h PG after 75-g OGTT, and A1C, are equally appropriate for prediabetes testing. B

In patients with prediabetes, identify and, if appropriate, treat other CVD risk factors. B

Consider prediabetes testing in overweight/obese children and adolescents with 2 or more add’l diabetes risk factors. E

Recommendations: Prediabetes Recommendations: Prediabetes (2)(2)


FPG 100–125 mg/dL (5.6–6.9 mmol/L): IFG

OR2-h plasma glucose 140–199 mg/dL

(7.8–11.0 mmol/L): IGTOR

A1C 5.7–6.4%

Prediabetes*Prediabetes*

* For all three tests, risk is continuous, extending below the lower limit of a range and becoming disproportionately

greater at higher ends of the range.


Blood glucose rather than A1C should be used to dx type 1 diabetes in symptomatic individuals. E

Inform relatives of patients with T1D of the opportunity to be tested for type 1 diabetes risk, but only in the setting of a clinical research study. E

Recommendation: Screening

for Type 1 Diabetes


Screening for prediabetes and risk for future diabetes with an infor- mal assessment of risk factors or validated tools should be considered in asymptomatic adults. B

Testing for prediabetes and risk for future diabetes in asymptomatic people should be considered in adults of any age who are overweight or obese (BMI $25 kg/m2 BMI $23 kg/m2 in Asian Americans). and who have one or more additional risk factors for diabetes. B

Recommendations: Screening

for Type 2 Diabetes


For all people, testing should begin at age 45 years. B c If tests are normal, repeat testing carried out at a minimum of 3-year intervals is reasonable. C

Recommendations: Screening

for Type 2 Diabetes (2)


Criteria for Testing forT2DM in Children &

Adolescents Overweight plus any 2 :

Family history of type 2 diabetes in 1st or 2nd degree relative

Race/ethnicity Signs of insulin resistance or conditions

associated with insulin resistance Maternal history of diabetes or GDM

Age of initiation 10 years or at onset of puberty

Frequency: every 3 years Screen with A1C


Limitations of OGTT

•The OGTT is less accurate than the hyperinsulinemic-

euglycemic clamp technique (the "gold standard" for

measuring insulin resistance), or the insulin tolerance test,

but is technically less difficult. Neither of the two technically

demanding tests can be easily applied in a clinical setting or

used in epidemiological studies.

•HOMA-IR (homeostatic model assessment) is a convenient

way of measuring insulin resistance in normal subjects, which

can be used in epidemiological studies, but can give

erroneous results for diabetic patients.

THANKS

diagnosis tessts in diabetes mellitus_ dr selim

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