Laboratory diagnosis of

Diabetes MellitusPresented by Dr. Monika Nema

Dr. Monika Nema

Dr. Monika Nema

Introduction

Etiology

Impaired insulin

secretion

Impaired insulin

function

Diabetes is a group of metabolic disorder sharing the common features of hyperglycemia.

Dr. Monika Nema

Dr. Monika Nema

Laboratory diagnosis Urine analysis

Blood chemistry

Immunological Assays

• Glucose • Ketone • Microalbuminuria• Blood glucose estimation

• Glucose tolerance test• Glycated hemoglobin measurement

• Lipid profile • Serum insulin or C- peptide level

Dr. Monika Nema

Laboratory diagnosis

Diagnosis

Screening

Assessment of glycemic control

Assessment of association of long

term risk

Dr. Monika Nema

Current criteria

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Laboratory test for diagnosis

Dr. Monika Nema

Laboratory test for diagnosis 1. Estimation of blood glucose.2. Oral glucose tolerance test.

Dr. Monika Nema

Estimation of blood glucoseMeasurement of blood glucose is indicative of

current state of carbohydrate metabolism.Depending on time of collection:Fasting blood glucose- after an overnight fast.Post meal or postprandial blood glucose-2 hrs

after the subject has taken a normal meal.Random blood glucose – Any time of the day.

Dr. Monika Nema

General considerationTotal glucose in 100 ml of plasma is about 15%

greater than in 100 ml of whole blood.

Plasma is prefered as whole blood is affected by concentration of proteins (especially haemoglobin).

In capillary blood the value of blood glucose at rest is about 5 % higher than venous blood.

Dr. Monika Nema

Whole blood left at room temperature

Glycolysis @ 7mg/dl/hr

Sodium fluoride

Leucocytosis and bacterial contamination

(-) (+)

Dr. Monika Nema

Blood glucose estimation

Glucose oxidase/Peroxidase.

Hexokinase. Glucose dehydrogenase

Folin wu

Somogyi – nelson method

Orthotoluidine method

Dr. Monika Nema

Folin-Wu and the Somogyi-Nelson Based on the same principles.

Principle-

Cupric ions ( alkaline cupric sulphate )

Intensity of colour is proportional to glucose present in the blood

Blood glucose

cuprous ionsphosphomolybdic acid

Blue coloured compound.

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Orthotoluidine methodPrinciple

Glucose + orthotoluidine green coloured complex

The intensity of the final colour is measured at 620 – 660 nm.

The measured colour intensity is directly proportional to the concentration of glucose .

hot acidic medium

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Enzymatic methods Glucose Oxidase/Peroxidase methodGlucose + O2 gluconic acid + H2O2

2 H2O2 2H2O +2 O2

Intensity measured at 530 nm.

peroxidase pink coloured compoundPhenol

4 aminophenazone

GOD

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Principle –

Glucose + ATP G6P + ADP

G6P + NADP 6 PG + NADPH

Glucose concentration proportional to

rate of production of NADPH.

HexoKinase

G6PD

Hexokinase method

Dr. Monika Nema

Advantages of enzymatic method

Accurate, sensitive, specific and precise.

Reagents are safe to handle. Very small serum & reagents are

required.Mono step method, carried out at room

temperature.Linear upto 700 mg/dl.Sodium fluoride do not interfere in the

assay.

Dr. Monika Nema

Glucose tolerance testGlucose tolerance means the ability of the body to

utilize glucose in blood circulation.

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

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

American Diabetes Association and WHO

Gestational Diabetes

Dr. Monika Nema

Indication of Glucose tolerance test In asymptomatic persons with sustained or

transient glycosuria.In persons with symptoms of diabetes but no

glycosuria or hyperglycemia.Persons with family history but no symptoms

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

diabetes mellitus showing one abnormal blood findings.

In patients with neuropathies or retinopathies of unknown origin.

Dr. Monika Nema

Contraindications of glucose tolerance test

There is no indication for doing GTT in a person with confirmed diabetics mellitus.

GTT has no role in follow-up of diabetics.The test should not be done in ill patients.

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Types of glucose tolerance testStandard Oral glucose tolerance test

I/V Glucose tolerance test

Mini Glucose tolerance test

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Patient should on carbohydrate rich unrestricted diet for 3 days.

Patient should be ambulatory with normal physical activity.

Medications should be discontinued on the day of testing.

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

OGTT carried out in the morning after patient has fasted overnight for 8-14 hours.

Preparation of patient

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TestA fasting venous

blood sample is collected in the morning.

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

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Test In the classical procedures, the blood and

urine samples are collected at half hourly interval of the next three hours.

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

The curve so obtained is called glucose tolerance curve.

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Normal Glucose tolerance curve

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Diabetic curve

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

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

Dr. Monika Nema

I/V Glucose tolerance test- Procedure• I/V glucose tolerance test is carried out by

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

• Completion of infusion is taken as time zero.• Blood samples are taken at 10 minutes

interval for the next hour. • The peak value is reached within a few

minutes.

Dr. Monika Nema

I/V Glucose tolerance testInterpretation• Normally, blood glucose level returns to

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

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Mini or Modern GTT

As per current WHO recommendations, in the mini or modern glucose tolerance test, only two samples are collected.

Fasting (zero hour) and 2 hour post glucose load.

Urine samples are also collected during the same time.

The diagnosis is made from the variations observed in these results. Zero Hour After 2 Hours

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

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

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GTT Under special conditionsCortisone stress test- used for detecting pre

diabetes or Latent diabetesExtended GTT- To diagnose the cause of

hypoglycemia especially 2-3 hours after meals.

Dr. Monika Nema

Factors affecting GTTa) Acute infections- Cortisol is secreted, the

curve is elevated and prolonged. b) Hypothyroidism-A flat curve is obtained in

hypothyroidism. Thyroid hormone increases the absorption of glucose from the gut.

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

Dr. Monika Nema

Gestational diabetesGestational diabetes is high blood sugar

that develops at any time during pregnancy in a woman who does not have diabetes.

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OGTT in gestational DiabetesImpairment of glucose tolerance develops

normally during pregnancy, particularly in 2nd and 3rd trimester.

•<25 yrs age, Normal body weight before pregnancy, absence of DM in first degree relative, no h/0 poor obstetric outcome, no h/o abnormal glucose tolerance

Low risk

•Tested at 24-28 weeks of gestation.

Average risk

•Marked obesity, strong family history of DM, glycosuria, personal history of GDM.

High risk

Dr. Monika Nema

Fasting plasma glucose or random plasma glucose

Deranged Normal

Repeat testing on subsequent day

OGTT indicated for average risk and high risk pregnant female

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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

Dr. Monika Nema

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

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

Dr. Monika Nema

Laboratory test for screening

Dr. Monika Nema

Laboratory test for screeningRecommended screening test is fasting plasma glucose.

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

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

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

Dr. Monika Nema

Selective screeningHigh risk individuals ---Obese Family h/o DM Hypertension Dyslipidemia Impaired glucose

tolerance

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

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Laboratory test to assess glycemic control

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Laboratory test to assess glycemic controlPeriodic measurement of glycated

haemoglobin.Daily self assessment of blood glucose.Others.

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Glycated hemoglobin Glycated

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

( Maillard Reaction )Hemoglobin + glucose Aldimine

Glycated hemoglobin

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TYPE COMPONENTSGlycated haemoglobin (Ghb) Haemoglobin in which glucose &/

any other carbohydrates are bound to free amino groups.

HbA1( fast haemoglobin) Carbohydrate bound to N- terminal of the β- chain.

HbA₁а₁ Fructose-1, 6-biphosphate bound to the N-terminal valine of the β-chain

HbA₁а₂ Glucose-6-phosphate bound to the N-terminal valine of the β-chain

HbA₁ь Unknown carbohydrate residue bound to the N-terminal valine of the β-chain

HbA₁с Glucose bound to the N-terminal valine of the β-chain

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Glycated hemoglobinHbA₁с gives information about the average blood

glucose concentration over a retrospective period of time.

Reflects the mean glucose concentration.Normally, less than 5% of hemoglobin is glycated.

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Glycated hemoglobinAbout 50% HbA₁с values results from the blood

glucose of the preceding 30 days , 40% from the preceding 31 -90 days and only 10% from the period between the 91 – 120 days.

No effect of diet, exercise & insulin on test results.More informative. Blood sample can be drawn at any time of day.HbA1c of 6 % corresponds to mean serum glucose

level of 135 mg/dl. With every rise of 1.0%, serum glucose increases

by 35 mg/dl.

Dr. Monika Nema

INDICATIONS In all diabetics to monitor long term blood glucose level control,

index of diabetic control:- 7% HbA₁с – good 10% HbA₁с- fair 13-20% HbA₁с- poor.

To monitor patient compliance.

To predict development & progression of microvascular complication.

For determining the therapeutic option whether to use oral agents, insulin ,or β cell transplantation.

Also increasingly used for primary diagnosis of DM.

Dr. Monika Nema

Methods used for determination of HbA₁с HbA1c

electrophoresis. Cation-exchange

chromatography, Boronate affinity

ChromatographyImmunoassays. Colorimetric method

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At what interval should 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.

Dr. Monika Nema

Glycated hemoglobin High values Low values

Diabetes Mellitus Haemolysed specimen

Polycystic Ovarian Disease Hereditary HbF

Hyperglycemia Neonate&Pregnancy

Glycosuria Fetal maternal transfusion

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Glycated hemoglobinFalsely high values Falsely low values

Iron deficiency anemia Hemolytic anemia

Post spleenectomy Chronic blood loss

Alcohol poisoning Chronic Renal Failure

Lead toxicity Pregnancy

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

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

control.(normal range: 4.5% - 6.3%). If HbA₁с values > 8% the treatment should

be reconsidered.

Dr. Monika Nema

Self monitoring of blood glucoseRegular use of SMBG devices by

diabetic patients has improved the management of DM.

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

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

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

Dr. Monika Nema

Fructosamine assay Generic term for measurement of all serum glycated

protein though the bulk being albumin.

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

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

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

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

Fructosamine test HbA1cMeasures average blood glucose level over the past two or three weeks

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

Dr. Monika Nema

Glycosylated albumin

Half -life of albumin is approximately 15 days.Glycated albumin level is believed to reflect

the glycemic change over a 2-week period.GA can be useful in evaluating the

therapeutic effect of recently substituted hypoglycemic agents at an early stage.

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

Dr. Monika Nema

Insulin assay

Measurement of insulin level by radioimmunoassay & ELISA.

Crucial for type I DM.

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Proinsulin 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.

Dr. Monika Nema

Proinsulin AssayElevated in:-At onset of IDDM and in healthy sliblings of

IDDM patients.With established NIDDM.Older patients.Pregnant .Obese diabetics. Insulinomas.Functional hypoglycemia.Hyperinsulinemia.

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C peptide assay Released in circulation during conversion of proinsulin

to insulin in equimolar quantities to insulin.

Its level correlate with insulin level in blood.

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

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

Dr. Monika Nema

Urine glucose estimationPresence 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.

Dr. Monika Nema

Renal glycosuriaBlood 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

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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.

Dr. Monika Nema

Tests for urine sugarQualitative test.Benedicts.Clintest tablet test.Reagent strip test

Quantitative test. Benedicts.

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Benedict’s test

Based on copper reduction method

Detect any reducing sugar in urine

Principle

Cu 2+ Cu +

Cu + + OH - CuOH

2CuOH Cu2O + H2O

Hot alkaline solution

Heat

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Procedure

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.

Dr. Monika Nema

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)

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False positive test

Ascorbic acid

Creatine

Uric acid

Homogentisic acid

Cephalosporins

Salicylates

Radiographic media

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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.

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Reagent strip methodBased on specific glucose oxidase and peroxidase method.

Specific for glucose.

Principle - Glucose + O2 Gluconic acid + H2O2

H2O2 + Chromogen oxidized chromogen + H2O

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False positive :- Oxidizing cleaning agent in urine container.

False negative :- Ascorbic acid

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Benedicts quantitative testContents – Potassiun Thiocyanate , Potassium

Ferrocyanide , Sodium Citrate , Sodium Carbonate ,

Copper Sulfate. Principle :

Cu 2+ Cu +

Cu + +potassium thiocyanate Cu thiocynate

Hot alkaline solution

White precipitate

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Procedure

2-3g of NaCO3

keep BoilingAddUrine drop by drop using 5 ml pptTill blue colour 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

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Benedicts qualitative tests

Positive

Glucose oxidase strip method

Positive Negative

Glucose Lactose Fructose Galactose

Benedicts quantitative test

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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.

Dr. Monika Nema

Laboratory tests to assess long term risks

Dr. Monika Nema

Laboratory tests to assess long term risks1. Urinary albumin excretion.2. Lipid profile.

Dr. Monika Nema

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.

Dr. Monika Nema

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.

Dr. Monika Nema

Frank proteinuriaPrecipitation test.Heat test- precipitation of protein by heat. Not affected by radiographic

contrast media. Sulfosalicylic acid method- precipitation of

protein by acid. False positive

results are obtained in presence of radiographic contrast media.

Reagent strip test.

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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

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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

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OBSERVATION

1. Negative – No turbidity (~5mg/dl or less)

2. Trace – Perceptible turbidity (~20 mg/dl)

3. 1+ - Distinct turbidity but no discrete granulation(~50mg/dl)

4. 2+ - Turbidity with granulation but no flocculation(~200mg/dl)

5. 3+ - Turbidity with granulation and flocculation(~500mg/dl)

6. 4+ - Clumps of precipitated protein, or solid precipitate (~1.0g/dl or more)

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Reagent strip method

Principle :Impregnated with bromphenol blue buffered to pH 3

with citrate

30 to 60 second urine application

Variable sheds of green color formed

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MicroalbuminuriaNormally, only a small amount of albumin is

fiiltered 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.

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MicroalbuminuriaNormalbuminuria- <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.

Dr. Monika Nema

Microalbuminuria

Lower 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 Women

3.5

30

35

300

mg/mmol

mg/gm

Men 2.5

30

35

300

mg/mmol

mg/gm

Dr. Monika Nema

MicroalbuminuriaIt 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.

Dr. Monika Nema

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 microalbuminuria

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Albumin to Creatinine ratio 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.

Dr. Monika Nema

Microalbuminuria 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.

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.

Dr. Monika Nema

Quantitative test for microalbuminuriaColorimetric testELISA.Radioimmune assay.Immunoturbidiometric assay.Nephelometry.Chemiluminescence.

Dr. Monika Nema

DYE BINDING COLORIMETRIC METHODPyrogallol 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.

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ELISA Uses antibodies and colour change to identify the substance.

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

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RADIOIMMUNOASSAY Technique used for the detection of antibody

or antigen.

Uses radioactive label or tracer .

Tritium, I-131, I-125 are commonly used tracers.

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

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

Dr. Monika Nema

TURBIDIMETRYMeasurement of reduction in light transmission

caused by particle formation.Light transmitted in forward direction is

detected.Amount of light absorbed by a suspension of

particles depends on the specimen concentration & on particle size.

Not specific to protein . Nucleic acid can also precipitate.

Dr. Monika Nema

NEPHELOMETRYMeasurement of light

scattered by the particulate solution.

Nephelometer measure scattered light at 90 to the incident light.

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CHEMILUMINESENCEChemiluminescence  is the

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

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

Dr. Monika Nema

Triglycerides (mg/dl) Category<150 Low risk150-199 Intermediate risk≥ 200 High riskLDL cholesterol<100 Low risk100-129 Intermediate risk≥130 High riskHDL cholesterol<35 High risk35-45 Intermediate risk>45 Low risk

Lipid profile

Dr. Monika Nema

Laboratory test in acute complication of Diabetes Mellitus

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Acute complication of Diabetes MellitusDiabetic ketoacidosis.Hyperglycemic hyperosmolar state.Hypoglycemia.

Dr. Monika Nema

Diabetic ketoacidosisState of absolute or relative insulin deficiency aggravated

by ensuing hyperglycemia, dehydration, and acidosis-producing derangements in intermediary metabolism.

Normally the blood level of ketone bodies is <1 mg/dl & only traces are excreted in urine.

Increased synthesis causes the accumulation of ketone bodies in blood.

More common in case of Type 1 DM.

Dr. Monika Nema

Hyperglycemia

Ketosis

Acidosis

*

Definition of Diabetic Ketoacidosis

The normal gap is <12 mEq/L.

In ketoacidosis, the “delta” of the anion gap above 12 mEq/L is composed of anions derived from keto-acids

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Symptoms of Diabetic Ketoacidosis

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Lab Findings in DKASevere hyperglycemiaIncreased blood and urine ketones (Acetone,.

Acetoacetic acid , 3-hydroxybutyrate ).Low bicarbonateHigh anion gapLow arterial pHLow PCO2 (respiratory compensation)

Dr. Monika Nema

Methods to detect ketone bodies 1. Rothera’s test

2. Reagent strip

3. Gerhardt ferric chloride test

Dr. Monika Nema

Rothera testBased on nitroprusside reaction

ProcedureTake 5.00 ml urine and saturate it with

ammonium sulphate. Add a crystal of sodium nitroprusside.

Slowly pour concentrated ammonium hydroxide(1-2ml) by the side of test tube.

Pink-purple ring

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Reagent strip for ketonuriaBased on nitroprusside reaction

Principle:

Sodium nitroprusside + Glycine

acetoacetic acid and acetone in alkaline medium

Violet colorSensitivity: 25-50mg/dl

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Gerhardt’s testAddition of 10% ferric chloride solution to

urine causes solution to become reddish or purplish if acetoacetic acid is present.

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Hyperglycemic Hyperosmolar StateCompared to DKA, in HHS there is greater

severity of: Dehydration Hyperglycemia HypernatremiaHyperosmolality

Because some insulin typically persists in HHS, ketogenesis is absent to minimal and is insufficient to produce significant acidosis.

More commonly present in Type 2 DM.

Dr. Monika Nema

Hypoglycemia Results from an imbalance between glucose

utilization and production in such a manner that the rate of glucose utilization exceeds the rate at which glucose being produced.

Whipple’s triad:-Symptoms consistent with hypoglycemia.Plasma glucose level < 55 mg/dl.Relief of symptoms with correction of

hypoglycemia.

Dr. Monika Nema

Conclusion Diabetes is a very complicated disease.Anyone at any age can have diabetes despite

of negative family history Laboratory plays an important part in the

diagnosis and care of diabetic patient.

Dr. Monika NemaThank you