Non-Invasive Diagnosis of Peripheral Polyneuropathy in Type-2 Diabetes Mellitus Patients
Laboratory diagnosis of Diabetes mellitus
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Transcript of Laboratory diagnosis of Diabetes mellitus
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
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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.
Dr. Monika Nema
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
Dr. Monika Nema
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
Dr. Monika Nema
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).
Dr. Monika Nema
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.
Dr. Monika Nema
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.
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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
Dr. Monika Nema
Laboratory test to assess glycemic control
Dr. Monika Nema
Laboratory test to assess glycemic controlPeriodic measurement of glycated
haemoglobin.Daily self assessment of blood glucose.Others.
Dr. Monika Nema
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
Dr. Monika Nema
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
Dr. Monika Nema
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.
Dr. Monika Nema
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
Dr. Monika Nema
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
Dr. Monika Nema
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.
Dr. Monika Nema
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.
Dr. Monika Nema
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.
Dr. Monika Nema
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)
Dr. Monika Nema
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.
Dr. Monika Nema
Reagent strip methodBased on specific glucose oxidase and peroxidase method.
Specific for glucose.
Principle - Glucose + O2 Gluconic acid + H2O2
H2O2 + Chromogen oxidized chromogen + H2O
Dr. Monika Nema
False positive :- Oxidizing cleaning agent in urine container.
False negative :- Ascorbic acid
Dr. Monika Nema
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
Dr. Monika Nema
Benedicts qualitative tests
Positive
Glucose oxidase strip method
Positive Negative
Glucose Lactose Fructose Galactose
Benedicts quantitative test
Dr. Monika Nema
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.
Dr. Monika Nema
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
Dr. Monika Nema
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
Dr. Monika Nema
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)
Dr. Monika Nema
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.
Dr. Monika Nema
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
Dr. Monika Nema
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.
Dr. Monika Nema
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.
Dr. Monika Nema
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
Dr. Monika Nema
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
Dr. Monika Nema
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
Dr. Monika Nema
Reagent strip for ketonuriaBased on nitroprusside reaction
Principle:
Sodium nitroprusside + Glycine
acetoacetic acid and acetone in alkaline medium
Violet colorSensitivity: 25-50mg/dl
Dr. Monika Nema
Gerhardt’s testAddition of 10% ferric chloride solution to
urine causes solution to become reddish or purplish if acetoacetic acid is present.
Dr. Monika Nema
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