Diabetes and its lab Investigations

1. Presented by Dr. Monika Nema
Dr. Monika Nema

2. Introduction
Etiology
Dr. Monika Nema
Impaired
insulin
secretion
Impaired
insulin
function
Diabetes is a group of metabolic disorder
sharing the common features of
hyperglycemia.

3. Dr. Monika Nema

4. Laboratory diagnosis
• Glucose
Urine analysis • Ketone
• Microalbuminuria
• Blood glucoseestimation
• Glucose tolerance test
Blood chemistry • Glycated hemoglobin
measurement
• Lipid profile
• Serum insulin or C- peptide
level
Immunological Assays
Dr. Monika Nema

5. Laboratory diagnosis
Diagnosis
Screening
Assessmentof glycemic
control
Assessmentof association
of long term risk
Dr. Monika Nema

6. Current criteria
Dr. Monika Nema

7. Laboratory test for diagnosis
Dr. Monika Nema

8. Laboratory test for diagnosis
Dr. Monika Nema
⚫1. Estimationof blood glucose.
⚫2. Oral glucose tolerance test.

9. Estimation of blood glucose
Dr. Monika Nema
⚫Measurementof blood glucose is indicativeof current
stateof carbohydrate metabolism.
⚫Depending on timeof collection:
Fasting blood glucose- afteran overnight fast.
Post meal or postprandial blood glucose-2 hrs after
the subject has taken a normal meal.
Random blood glucose – Any timeof theday.

10. Total 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

11. Whole blood leftat room temperature
Glycolysis @ 7mg/dl/hr
Sodium fluoride
Leucocytosis and bacterial
contamination
(-) (+)
Dr. Monika Nema

12. Blood
glucose
estimation
Enzymatic
method
Chemical
method
Glucose
oxidase/Peroxidase.
Hexokinase.
Glucose
dehydrogenase
Folin wu
Somogyi – nelson
method
Orthotoluidine
method
Dr. Monika Nema

13.  Based on the same principles.
 Principle-
Cupric ions
( alkalinecupricsulphate )
 Intensityof colour is proportional toglucose present in the
blood
Blood glucose
cuprous ions
phosphomolybdic
acid
Blue
coloured
compound.
Dr. Monika Nema

14. Principle
hotacidic
medium
 Glucose + orthotoluidine green coloured complex
 The intensityof the final colour is measured at 620 – 660 nm.
 The measured colour intensity isdirectly proportional to the
concentration of glucose .
Dr. Monika Nema

15. Glucose Oxidase/Peroxidase method
⚫Glucose + O2 gluconic acid + H2O2
2 H2O2 2H2O +2 O2
⚫Intensity measured at 530 nm.
peroxidase
pink coloured
compound
Phenol
4 aminophenazone
GOD
Dr. Monika Nema

16. G6P + ADP
6 PG + NADPH
⚫Glucose concentration proportional to rateof
production of NADPH.
Hexokinase method
⚫Principle –
HexoKinase
Glucose + ATP
G6PD
G6P + NADP
Dr. Monika Nema

17. ⚫Accurate, sensitive, specific and precise.
⚫Reagentsaresafe to handle.
⚫Very small serum & reagents are required.
⚫Mono step method, carried out at room
temperature.
⚫Linearupto 700 mg/dl.
⚫Sodium fluoride do not interfere in theassay.
Dr. Monika Nema

18. Glucose tolerance test
⚫Glucosetolerance means theabilityof the body toutilize
glucose in blood circulation.
⚫American Diabetes Association -------- Forroutinediagnosis
⚫WHO ------------Forthosewith impaired fasting glucose.
⚫ American Diabetes Associationand WHO
Gestational Diabetes
Dr. Monika Nema

19. Indication of Glucose tolerance test
Dr. Monika Nema
⚫ In asymptomaticpersonswith sustained or transient
glycosuria.
⚫In personswith symptomsof diabetes but no
glycosuriaor hyperglycemia.
⚫Personswith family history but no symptomsor
positive blood findings.
⚫In personswith orwithoutsymptomsof diabetes
mellitusshowing one abnormal blood findings.
⚫In patientswith neuropathiesorretinopathiesof
unknownorigin.

20. Contraindications of glucose
tolerance test
Dr. Monika Nema
⚫There is no indication fordoing GTT in a person with
confirmed diabetics mellitus.
⚫GTT has no role in follow-upof diabetics.
⚫The testshould not bedone in ill patients.

21. Types of glucose tolerance test
Dr. Monika Nema
⚫Standard Oral glucose tolerancetest
⚫I/V Glucose tolerance test
⚫Mini Glucose tolerance test

22. ⚫ Patientshould on carbohydraterich unrestricted diet for 3 days.
⚫ Patientshould beambulatorywith normal physical activity.
⚫ Medicationsshould bediscontinued on thedayof testing.
⚫ Exercise, smoking and teaorcoffee are notallowed during testperiod.
⚫ OGTTcarried out in the morning afterpatient has fasted overnight for
8-14 hours.
Dr. Monika Nema
Preparation of patient

23. Test
⚫A fasting venous blood
sample is collected in the
morning.
⚫Patients ingest 75 g of
anhydrous glucose in
250-300 ml of waterover
5 minutes. ( forchildren,
the dose is 1.75 g of
glucoseper kg).
Dr. Monika Nema

24. Test
Dr. Monika Nema
⚫In the classical procedures, the blood and urine
samplesarecollected at half hourly interval of the next
three hours.
⚫A curve is plotted with the blood glucose levelson the
vertical axis against the time of collection on the
horizontal axis.
⚫Thecurvesoobtained is called glucose tolerance
curve.

25. Normal Glucose tolerance curve
Dr. Monika Nema

26. Diabetic curve
Dr. Monika Nema

27. Intravenous Glucose tolerance test
Dr. Monika Nema
•This test is undertaken for patientswith malabsorption
(Celiacdiseaseorenteropathies).
•Under these conditions oral glucose load is not well
absorbed and the resultsof oral glucose tolerance test
become inconclusive.

28. I/V Glucose tolerance test-
Procedure
Dr. Monika Nema
• I/V glucose tolerance test is carried out bygiving 25 g
of glucosedissolved in 100 ml distilled water as
intravenous injectionwithin 5 minutes.
• Completionof infusion is takenas time zero.
• Blood samplesare takenat 10 minutes interval forthe
next hour.
• The peak value is reached within a few minutes.

29. I/V Glucose tolerance test
Dr. Monika Nema
Interpretation
• Normally, blood glucose level returns to normal range
within 60 minutes.
• In diabetes mellitus, thisdecline is slow.

30. Mini or Modern GTT
⚫As percurrent WHO recommendations, in the mini or
modern glucose tolerance test, only twosamplesare
collected.
⚫ Fasting (zero hour) and 2 hourpostglucose load.
⚫Urine samplesare alsocollected during the same time.
⚫The diagnosis is made from thevariationsobserved in
these results.
Zero Hour After 2 Hours
Normal Person < 110 mg/dL < 140 mg/dL
Increase Glucose
Tolerance Dr. Mon
110 – 126 mg/dL
ika Nema
140 – 199 mg/dL

31. GTT Under special conditions
Dr. Monika Nema
⚫Cortisonestress test- used fordetecting pre diabetesor
Latentdiabetes
⚫Extended GTT- Todiagnose thecauseof hypoglycemia
especially 2-3 hoursafter meals.

32. Factors affecting GTT
Dr. Monika Nema
a) Acute infections- Cortisol is secreted, thecurve is
elevated and prolonged.
b) Hypothyroidism-A flat curve is obtained in
hypothyroidism. Thyroid hormone increases the
absorptionof glucose from thegut.
c) Starvation- There is rise of counter regulatory
hormones, which show increased glucose tolerance.

33. Gestational diabetes
Dr. Monika Nema
⚫Gestational diabetes is high blood sugar that
developsat any timeduring pregnancy in a woman
whodoes not havediabetes.

34. OGTT in gestational Diabetes
⚫Impairment 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/oabnormal glucose tolerance
Lowrisk
• Tested at 24-28 weeks of gestation.
Average
risk
• Marked obesity, strong family history of DM, glycosuria,
personal historyof GDM.
High risk
Dr. Monika Nema

35. Fasting plasma glucose or
random plasma glucose
Deranged Normal
Repeattesting on subsequentday
Dr. Monika Nema
OGTT indicated for
average risk and high
risk pregnantfemale

36. OGTT for GDM
One stepapproach Two stepapproach
100 gm glucose is administered
3- hours OGTT is performed
50 gm glucose is administered
irrespectiveof time of last meal
Afterone hour, venous blood samplecollected
If glucose level exceeds 140 mg/dl
Otherwise GDM is excluded
Dr. Monika Nema

37. ⚫Gestational diabetes is diagnosed if thewoman is ator
exceeds any twoof the following fourplasmaglucose
levelsduring 100 gm test
Dr. Monika Nema
Fasting –95 mg/dl
1 hr –180 mg/dl
2 hr –155 mg/dl
3 hr –140 mg/dl

38. Laboratory test for screening
Dr. Monika Nema

39. Laboratory test for screening
Dr. Monika Nema
⚫Recommended screening test is fasting plasmaglucose.
⚫American Diabetes Association recommends screening for
Type 2 DM in all asymptomatic individuals >= 45 yrs of age
using fasting plasmaglucose.
⚫If fasting test is normal, screening test should be repeated
every threeyears.
⚫If fasting blood glucose level is normal but there is strong
clinical suspicion then OGTT.

40. Selective screening
⚫High risk individuals ---Obese
Family h/o DM
Hypertension
Dyslipidemia
Impaired glucose tolerance
Screening test is performed atearlierage ( 30 yrs )
and repeated more frequently
Dr. Monika Nema

41. Laboratory test to assess glycemic
control
Dr. Monika Nema

42. Laboratory test to assess glycemic
control
Dr. Monika Nema
⚫Periodic measurementof glycated haemoglobin.
⚫Daily self assessmentof blood glucose.
⚫Others.

43. Glycated hemoglobin
⚫ Glycated haemoglobin
covers a number of
chemically different
modification resulting
from the non-enzymatic
and irrevesibly binding of
different sugars todifferent
amino groups in the
haemoglobin molecule.
( Maillard Reaction )
Hemoglobin + glucose Aldimine Glycated hemoglobin
Dr. Monika Nema

44. TYPE COMPONENTS
Glycated 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

45. Glycated hemoglobin
Dr. Monika Nema
⚫HbA₁с gives informationabout theaverage blood glucose
concentration overa retrospectiveperiod of time.
⚫Reflects the mean glucose concentration.
⚫Normally, less than 5% of hemoglobin is glycated.

46. Glycated hemoglobin
Dr. Monika Nema
⚫About 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.
⚫Noeffectof diet, exercise & insulinon test results.
⚫More informative.
⚫Blood samplecan be drawn atany timeof day.
⚫HbA1c of 6 % corresponds to mean serum glucose level of
135 mg/dl.
⚫With everyriseof 1.0%, serum glucose increases by 35
mg/dl.

47. INDICATIONS
Dr. Monika Nema
⚫ In all diabetics to monitorlong term blood glucose level control, index
of diabeticcontrol:-
7% HbA₁с – good
10% HbA₁с- fair
13-20% HbA₁с- poor.
⚫ To monitorpatientcompliance.
⚫ Topredictdevelopment & progressionof microvascularcomplication.
⚫ Fordetermining the therapeuticoptionwhethertouseoral agents,
insulin ,orβ cell transplantation.
⚫ Also increasingly used forprimary diagnosisof DM.

48. Methods used for
determination of HbA₁с
⚫HbA1c electrophoresis.
⚫Cation-exchange
chromatography,
⚫Boronateaffinity
Chromatography
⚫Immunoassays.
⚫Colorimetric method
Dr. Monika Nema

49. At what interval should HbA₁с
be determined?
Dr. Monika Nema
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.

50. Glycated hemoglobin
Dr. Monika Nema
High values Low values
Diabetes Mellitus Haemolysed specimen
Polycystic Ovarian Disease Hereditary HbF
Hyperglycemia Neonate&Pregnancy
Glycosuria Fetal maternal transfusion

51. Glycated hemoglobin
Dr. Monika Nema
Falsely high values Falsely low values
Iron deficiency anemia Hemolytic anemia
Post spleenectomy Chronic blood loss
Alcohol poisoning Chronic Renal Failure
Lead toxicity Pregnancy

52. ⚫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% indicategood glycemic
control.(normal range: 4.5% - 6.3%).
If HbA₁с values > 8% the treatment should be
reconsidered.
Dr. Monika Nema

53. Self monitoring of blood glucose
⚫ Regularuseof SMBG devices by
diabeticpatients has improved the
managementof DM.
⚫ SMBG devices measurecapillarywhole
blood glucoseobtained by fingerprick
and use test strips that incorporate
glucoseoxidaseor hexokinase.
⚫ SMBG devicesyield unreliableresultsat
very high and very lowglucose levels.
⚫ It is necessary toperiodicallycheck the
performance of glucometer by
measuring parallel venous plasma
glucose in the laboratory.
Dr. Monika Nema

54. Fructosamine assay
⚫ Genericterm for measurementof all serum glycated
protein though the bulk being albumin.
⚫ Does notappear to be influenced by transient (stress)
hyperglycaemia.
⚫ Unableto detect short term or transientabnormalities 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 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.
Dr. Monika Nema

55. Glycosylated albumin
Dr. Monika Nema
⚫Half -life of albumin is approximately 15 days.
⚫Glycated albumin level is believed to reflect the
glycemicchangeovera 2-week period.
⚫GA can be useful in evaluating the therapeuticeffectof
recentlysubstituted hypoglycemicagents atan early
stage.
⚫GA can also act as a valuable glycemic control marker
in diabetic patientswith variouscomorbidities since it
is unrelated to the metabolismof hemoglobin.

56. Insulin assay
⚫Measurementof insulin
level by radioimmunoassay
& ELISA.
⚫Crucial for type I DM.
Dr. Monika Nema

57. Proinsulin Assay
Dr. Monika Nema
⚫It is precursor molecule for insulin.
⚫Mostproinsulin is converted to insulinand C-Peptide,
which are secreted in equimolaramounts 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.

58. Proinsulin Assay
Dr. Monika Nema
⚫Elevated in:-
Atonsetof IDDM and in healthysliblingsof IDDM
patients.
With established NIDDM.
Olderpatients.
Pregnant .
Obesediabetics.
Insulinomas.
Functional hypoglycemia.
Hyperinsulinemia.

59. ⚫ Released in circulationduring conversion of proinsulin to
insulin inequimolarquantities to insulin.
⚫ Its level correlate with insulin level in blood.
⚫ Low C – peptide levelsare characteristicof type I DM.
⚫ C-peptide levels are measured instead of insulin levels
because C- peptide can assessa person’sown insulinsecretion
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 useof insulin. Measuring C-peptide levelswill
helpdifferentiate a healthypatients from adiabetic one.
Dr. Monika Nema

60. Urine glucose estimation
Dr. Monika Nema
⚫Presence of chemicallydetectableamountof glucose
in urine is called glycosuria.
⚫Urine glucose test resultscorrelatewell with plasmaor
serum glucosevalues.
⚫Presence of glucose in urine indicates that blood
glucose level of the patientcould haveelevated > 180
mg/dl.
⚫Normally less than 500mg/24 hrs or less than 15 mg/dl
of glucose is present in urine.

61. Renal glycosuria
Dr. Monika Nema
⚫ Blood Glucose level is normal but there is defect in the
reabsorptiveabilityof renal tubule.
⚫ Non pathological causes
Pregnancy
Stress
Anxiety
⚫ Pathological causes
Cystinosis
Heavy metal poisoning
Fanconi’s syndrome
Galactosemia

62. Alimentary glycosuria
Dr. Monika Nema
⚫Lag storagedisorder.
⚫Occurin gastrectomy, gastrojejunostomy
,hyperthyroidism.
⚫Glucose tolerance testrevealsa peak at 1 hourabove
renal threshold.
⚫Fasting and 2 hoursglucosevalueare normal.

63. ⚫Qualitative test.
Benedicts.
Clintest tablet test.
Reagent strip test
⚫Quantitativetest.
 Benedicts.
Dr. Monika Nema

64. Benedict’s test
⚫ Based on copper reduction method
⚫ Detectany reducing sugar in urine
⚫Principle
Cu+
CuOH
Cu 2+
Cu+ + OH -
2CuOH Cu2O + H2O
Hotalkaline solution
Heat
Dr. Monika Nema

65. Procedure
Add 8 drops
of urine
Boil for2
to 3 min
Cool
Take 5.0ml of
Benedict’s
reagent
Observe
Benedict reagent : sodium citrate 173 gm, sodium carbonate 100 gm, cupricsulphate 17.3 gm
and distill water 900 ml. Dr. Monika Nema

66. 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

67. False positive test
Dr. Monika Nema
⚫Ascorbicacid
⚫Creatine
⚫Uric acid
⚫Homogentisicacid
⚫Cephalosporins
⚫Salicylates
⚫Radiographic media

68. Clinitest tablet method
Dr. Monika Nema
⚫Modified form of Benedicts test in which reagentsare
present in tablet form.
⚫Containscoppersulfate, citricacid, sodium carbonate
and anhydrous sodium hydroxide.

69. Reagent strip method
⚫Based on specific glucose oxidase and peroxidase method.
⚫Specific for glucose.
⚫Principle -
Glucose + O2 Gluconic acid + H2O2
H2O2 + Chromogen oxidized chromogen + H2O
Dr. Monika Nema

70. Falsepositive :
- Oxidizing cleaning agent in urinecontainer.
False negative :
- Ascorbicacid
Dr. Monika Nema

71. Cu+
Cu 2+
Cu+ +potassium thiocyanate Cu thiocynate
⚫Contents – Potassiun Thiocyanate , Potassium Ferrocyanide ,
Sodium Citrate , Sodium Carbonate , Copper Sulfate.
⚫Principle :
Hotalkaline solution
White precipitate
Dr. Monika Nema

72. Procedure
2-3g of
NaCO3
keep Boiling
Add
Urine drop by
drop using 5 ml
ppt
Till bluecolour
disappear
Take 5.0ml
of
Benedict’s
Qtreagent
Chalkywhite
⚫ Method – titration
⚫ Calculation
Glucose in urine = 5
(g/100ml) urine used
10 g glucosereduces 5 ml of reagent
Dr. Monika Nema

73. Benedicts qualitative tests
Positive
Glucoseoxidase strip method
Positive Negative
Glucose Lactose
Fructose
Galactose
Benedictsquantitative test
Dr. Monika Nema

74. ⚫Semiquantitativeurineglucose 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 glucoseconcentration below the renal
threshold is obtained.
(2) Urinaryglucose testing cannotdetect hypoglycemia
(3) Concentration of glucose in urine is affected by
urinaryconcentration.
Dr. Monika Nema

75. Laboratory tests to assess long
term risks
Dr. Monika Nema

76. Laboratory tests to assess long
term risks
Dr. Monika Nema
⚫1. Urinary albuminexcretion.
⚫2. Lipid profile.

77. Screening for proteinuria should be performed
yearly in the following patients:
Dr. Monika Nema
⚫Type 1 DM : 5 yrs afterdiagnosis of DM, orearlier in
the presenceof othercardiovascularrisk factors.
⚫Type 2 DM : at the timeof diagnosisof diabetes.

78. ⚫Urine should be screened for proteinuria with
conventional dipstick on an early morning urine
specimen.
⚫If urinedipstick for proteinuria is negative, screening
for microalbuminuriashould be performed.
⚫If microalbuminuria isdetected, confirmation should
be made with two further tests within 3 to 6 month
period.
Dr. Monika Nema

79. Frank proteinuria
Dr. Monika Nema
⚫Precipitation test.
 Heat test- precipitationof protein by heat.
Not affected by radiographiccontrast media.
 Sulfosalicylicacid method- precipitationof protein by
acid.
False positive results are
obtained in presence of
radiographiccontrast media.
⚫Reagentstrip test.

80. Fill the
supernatant
urine upto 2/3
clean test tube
Boil theupper
portion
PROCEDURE OF HEAT TEST
If turbidity
developsadd
1 to 2 dropsof
glacial acidicacid
Phosphates
willclear
No turbidity– Proteinsabsent
Dr. M
Po
rn
ei
k
sa
eN
ne
m
ca
eof turbidity – Proteins present

81. Transferabout
5ml urine toa
centrifuge tube
Centrifuge
Transfer 3.0 ml
of supernatant
urine in a
cleantesttube
Add 2-3 drops
of 30%
sulfosalicylic
acid orequal
amountof 3%
Mix well
and
Wait for 10
minutes
Observe the
degreeof
turbidityand
flocculation
PROCEDURE OF SULFOSALICYLIC ACID METHOD
Dr. Monika Nema

82. 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+ - Turbiditywith granulation but no
flocculation(~200mg/dl)
5. 3+ - Turbiditywith granulationand
flocculation(~500mg/dl)
6. 4+ - Clumps of precipitated protein, orsolid
precipitate (~1.0g/dl or more)
Dr. Monika Nema

83. Reagent strip method
Principle :
Impregnated with bromphenol blue buffered to pH 3 with citrate
30 to 60 second urineapplication
Variable sheds of green color formed
Dr. Monika Nema

84. Microalbuminuria
Dr. Monika Nema
⚫Normally, onlya small amountof albumin is fiiltered
at theglomerulus, and mostof thatalbumin is
degraded and reabsorbed by the proximal tubule.
⚫Defined as persistentproteinuria thatcannot be detected
by routinereagent strips butgreater than normal.
⚫Present in theveryearly stageof diabetes, ata time
when GFR may be normal and when there is no
evidenceof glomerularlesion.

85. Microalbuminuria
Dr. Monika Nema
⚫Normalbuminuria- <20 microgram/minute or <30
mg/24 hrs.
⚫Microalbuminuria- therange 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.

86. Microalb m
i
uria
collection
Lower limit Upper limit unit
24 hour urine
u n30 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

87. Microalbuminuria
Dr. Monika Nema
⚫It indicates increase in capillary permeability to
albumin.
⚫Albumin is the first protein toenter the urineafter the
kidney is damaged.
⚫Appearanceof microalbuminuria is predictorof
progression toovertproteinuria.(incipient
nephropathy)
⚫It is an independent risk factor for cardiovascular
disease in diabetes mellitus.

88. Methodsof detection include
1. Measurementof albumin creatinine ratio in a
random urinesample
2. Measurementof albumin in an early morning and
random
3. Measurementof albumin in 24 hrsample.
Teststrips thatscreen for microalbuminuriaare
availablecommercially
.
Dr. Monika Nema
Detection of microalbuminuria

89. Albumin to Creatinine ratio
⚫ Reagent Strips are firm plastic strips that
contain two reagentareas that test for
albuminand creatinine in urine.
⚫ An albumin-to-creatinineratio is also
determined, which allows for the use of
single-void specimens in testing. The ratio
is given in milligramsalbumin pergram or
millimolecreatinine (mg/g or mg/mmol).
⚫ This productprovides semi-quantitative
results and can be used for screening
samples for microalbuminuria; positive
results should be confirmed with
quantitative methods foralbumin.
Dr. Monika Nema

90. Microalbuminuria strips
⚫ The strip is an immunochemical stripspecific for albumin.
⚫ Albumin in thesample get bound tosolubleconjugate of
antibodiesand markerenzyme b-galactosidase.
⚫ Conjugate-albumin complexes are separated and enzyme
b-galactosidasereacts with a substrate to produce a red dye.
⚫
⚫ The reagent part of the test stripshould bedipped 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.
⚫ Thecolour formed is compared with the referencecharton
thevial.
Dr. Monika Nema

91. Quantitative test for microalbuminuria
Dr. Monika Nema
⚫Colorimetric test
⚫ELISA.
⚫Radioimmuneassay.
⚫Immunoturbidiometricassay.
⚫Nephelometry.
⚫Chemiluminescence.

92. DYE BINDING COLORIMETRIC
METHOD
Dr. Monika Nema
⚫Pyrogallol red molybdate reagentcomplex reactwith
protein to form a bluepurplecolour.
⚫Optical densityof thecoloured complexcan be
measured at 600nm.
⚫The measured O.D. is propotional to the protein
concentration in the specimen.

93. ELISA ⚫Uses antibodies and colour
change to identify the substance.
⚫The intensity of the color
measured with microwell reader
at 450 nm.
Dr. Monika Nema

94. RADIOIMMUNOASSAY
⚫ Technique used for thedetection of antibodyor
antigen.
⚫ Uses radioactive label or tracer.
⚫ Tritium, I-131, I-125 arecommonly used tracers.
⚫ PRINCIPLE – competitive binding between
radiolabelled & unlabelled moleculesof 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 antibodysites.
Dr. Monika Nema

95. TURBIDIMETRY
Dr. Monika Nema
⚫Measurement 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 particlesize.
⚫Not specific to protein . Nucleicacid can also precipitate.

96. NEPHELOMETRY
⚫Measurementof light scattered
by the particulatesolution.
⚫Nephelometer measure
scattered lightat 90 to the
incident light.
Dr. Monika Nema

97. CHEMILUMINESENCE
⚫Chemiluminescence is the
emission of energy with limited
emissionof heat (luminescence),
as the result of a chemical
reaction.
⚫In immunoassaytechnology , the
light produced by the reaction
indicates the amount of analyate
in thesample.
Dr. Monika Nema

98. Triglycerides (mg/dl) Category
Low risk
Intermediate risk
High risk
Low risk
Intermediate risk
High risk
High risk
Intermediate risk
<150
150-199
≥ 200
LDL cholesterol
<100
100-129
≥130
HDL cholesterol
<35
35-45
>45 Low risk
Dr. Monika Nema

99. Laboratory test in acute
complication of Diabetes Mellitus
Dr. Monika Nema

100. Acute complication of Diabetes
Mellitus
Dr. Monika Nema
⚫Diabetic ketoacidosis.
⚫Hyperglycemic hyperosmolarstate.
⚫Hypoglycemia.

101. Diabetic ketoacidosis
Dr. Monika Nema
⚫State of absolute or relative insulin deficiencyaggravated
by ensuing hyperglycemia, dehydration, and acidosis-
producing derangements in intermediary metabolism.
⚫Normally the blood level of ketone bodies is <1 mg/dl
& only tracesareexcreted in urine.
⚫Increased synthesiscauses theaccumulationof ketone
bodies in blood.
⚫Morecommon in caseof Type 1 DM.

102. Hyperglycemia
Dr. Monika Nema
Ketosis
*
Definition of Diabetic Ketoacidosis
Acidosis
⚫ The normal gap is <12
mEq/L.
⚫ In ketoacidosis, the
“delta” of the anion
gapabove 12 mEq/L
is composed of
anionsderived from
keto-acids

103. Symptoms of Diabetic Ketoacidosis
Dr. Monika Nema

104. Lab Findings in DKA
Dr. Monika Nema
⚫Severe hyperglycemia
⚫ Increased blood and urine ketones (Acetone,.
Acetoacetic acid , 3-hydroxybutyrate ).
⚫Low bicarbonate
⚫High anion gap
⚫Low arterial pH
⚫Low PCO2 (respiratory compensation)

105. Methods to detect ketone
bodies
Dr. Monika Nema
1. Rothera’s test
2. Reagentstrip
3. Gerhardt ferricchloride test

106. Rothera test
⚫Based on nitroprussidereaction
 Procedure
Take 5.00 ml urine and saturate itwith ammonium
sulphate. Add a crystal of sodium nitroprusside.
Slowlypourconcentrated ammonium hydroxide(1-2ml)
by the side of test tube.
Pink-purplering
Dr. Monika Nema

107. Reagent strip for ketonuria
⚫Based on nitroprusside reaction
⚫Principle:
Sodium nitroprusside + Glycine
acetoaceticacid and
acetone in alkaline medium
Violetcolor
⚫Sensitivity: 25-50mg/dl
Dr. Monika Nema

108. ⚫Addition of 10% ferricchloridesolution to urinecauses
solution to become reddish or purplish if acetoacetic
acid is present.
Dr. Monika Nema

109. Hyperglycemic Hyperosmolar
State
Dr. Monika Nema
⚫Compared to DKA, in HHS there is greaterseverityof:
⚫Dehydration
⚫Hyperglycemia
⚫Hypernatremia
⚫Hyperosmolality
⚫Because some insulin typically persists in HHS,
ketogenesis is absent to minimal and is insufficient to
producesignificant acidosis.
⚫Morecommonlypresent in Type 2 DM.

110. Hypoglycemia
Dr. Monika Nema
⚫Results from an imbalance between glucoseutilization
and production in such a manner that the rate of
glucose utilization exceeds the rate at which glucose
being produced.
⚫Whipple’striad:-
Symptomsconsistentwith hypoglycemia.
Plasmaglucose level < 55 mg/dl.
Relief of symptomswith correction of hypoglycemia.

111. Conclusion
Dr. Monika Nema
⚫Diabetes is averycomplicated disease.
⚫Anyoneatany age can havediabetesdespiteof
negative family history
⚫Laboratory plays an importantpart in thediagnosis
and careof diabeticpatient.

112. Thank you
Dr. Monika Nema