LABORATORY TESTS FOR

DIABETES MELLITUS

Suzanna Immanuel
Department of Clinical Pathology
Faculty of Medicine University of Indonesia
 Blood Glucose: Metabolism and Disorders

Pathways In Glucose Metabolism

Glycolysis Metabolism of Glucose to pyruvate
or lactate for production of energy
Gluconeogenesis Formation of Glucose-6-phosphate
from noncarbohydrate sources
Glycogenolysis Breakdown of Glycogen to Glucose
for use as energy
Glycogenesis Conversion of Glucose to Glycogen
for storage
Lipogenesis Conversion of Carbohydrates to
fatty acids
Lipolysis Decomposition of fat
 Blood Glucose: Metabolism and Disorders

Postprandial Storage of Glucose

 Blood Glucose: Metabolism and Disorders

DIABETES MELLITUS
Due to absolute/ relative insulin deficiency
DM :
Hyperglycemia
Polydipsia
Polyuria
Polyphagia
Stupor (severe)
 Diabetes Mellitus
Etiologic Classification of Diabetes Mellitus
(American Diabetes Association/ ADA 1997)
I. Type I Diabetes (-cell destruction, usually leading to
absolute insulin deficiency)
A. Immune mediated
B. Idiopathic
II. Type II Diabetes (may range from predominantly insulin
resistance with relative insulin deficiency to
predominantly secretory defect with insulin resistance)
III. Other specific types:
A. Genetic defects of -cell function
- Maturity Onset Diabetes of the Young (MODY) 1,2,3
- Mitochondrial DNA
B. Genetic defects in insulin action
 Diabetes Mellitus
Other Specific Types: (continued) ADA 1997
C. Diseases of the exocrine pancreas
1. Trauma/ Pancreatitis
2. pancreatectomy
3. Fibrocalculous pancreatopathy
D. Endocrinopathies
1. Acromegaly
2. Cushing syndrome
3. Pheochromocytoma
4. Hyperthyroidism
E. Drug- or chemical-induced
1. Vacor, pentamidine, nicotinic acid
2. Glucocorticoids, thyroid hormone
3. Thiazides, dilantin, -interferon
4. Others
 Diabetes Mellitus
ADA 1997
Other specific types: (continued)
F. Infections
1. Congenital rubella
2. Cytomegalovirus (CMV)
G. Uncommon forms of immune-mediated
diabetes
- Anti insulin receptor antibodies
H. Other genetic syndromes sometimes
associated with diabetes
1. Downs syndrome
2. Klinefelters syndrome
3. Turners syndrome
4. others
III. Gestational Diabetes Mellitus (GDM)
 Diabetes Mellitus

IDDM
- Tend to ketoacidosis and coma
- Th/ insulin
- Children
- Adolescent
- Genetic origin : HLA type DR3 & DR4
- Viral infection
Damage cells of pancreas ~ 90%
Absolute insulin deficiency
- Remission after insulin th/
- Complications : blindness, heart disease, renal
disease, amputation.
NIDDM (type 2) Diabetes Mellitus
- > 40 years
- Ketoacidosis <
- Causes : genetic disorders & environmental factors
- Environmental factors include:
Viral infection as a trigger
Less physical activity
Fat meal
Physical stress e.g. surgical operation
- Pancreatic destruction <
- Pathogenesis :
1. Decreased insulin secretion
2. Insulin resistance: receptors <, metabolic disturbances
3. No insulin respons: glucose
4. Abnormal insulin : < biological activity, e.g. mutation
- Complications : < IDDM
 Diabetes Mellitus

IGT (Impaired glucose tolerance)

- FG 110 125 mg/dl & PP G 140 199 mg/dl
- < Patients become DM
- > Obvious
Old people
Obese patients
After taking certain drugs
- Risk to get arterial blood vessel disease
 Diabetes Mellitus
DIAGNOSTIC CRITERIA
Specific symptoms of DM
Symptoms DM + Symptoms DM -

FG 126 <126 FG 126 110-126 <110

or or
RG 200 <200 RG 200
110-199

Repeat FG or RG
OGTT
FG 126 <126 BG 2 hours
or
RG 200 <200

200 140-199 <140

Diabetes Mellitus (DM) IGT IFG Normal

 Diabetes Mellitus

Laboratory Examination
Urine :
- Tests : glucose, albumin, ketone bodies, sediment
- Microalbumin: detect early renal lesion

Blood tests :
- Blood glucose, GTT
- Glucose daily curve
- Fructosamine & HbA1c
- Insulin, C-peptide
- Acid base balance
 Diabetes Mellitus
Blood Glucose
Principles :
Reduction : Cupri cupro/ Ferri ferro
Condensation : o-toluidin
Enzymatic : - Glucose Oxydase
- Hexokinase
- Dehydrogenase
GTT
No diet 3 days before GTT
Tests : FG, 1 hour, 2 hour after taking 75 g glucose
orally.
With modification: FG & 2 hr after 75 g glucose
 Diabetes Mellitus
Glucose daily curve
Blood glucose performed at fasting, 11 a.m. & 4 p.m.
Provides rough information about metabolic situation
Difficult to standardize

HbA1c & Fructosamine

Non enzymatic glycosylation after protein translation
directly influenced by blood glucose level
DM : chronic hyperglycemia glycosylated protein
HbA1c, fructosamine
HbA1c Blood Glucose within 6 8 weeks
N : 5 9 % total Hb
False A. hemolytic
Fructosamine Blood glucose within 2-4 weeks
N : < 285 mmol/L
Cant replace blood glucose test
 Diabetes Mellitus

Insulin
RIA
Cant differentiate between exogenous & endogenous insulin

C-Peptide
~ insulin from pro-insulin
From blood and urine
Reflects the function of -cell of pancreas
Isnt influenced by exogenous insulin / insulin antibody

Acid base measurement

Uncontrolled diabetics:
HCO3 (buffering activities)
PCO2 (hyperventilation due to lung compensation)
DIABETES MELLITUS

Acute Complications

Chronic Complications
 ACUTE COMPLICATIONS

Coma Hyperglycemic
Ketoacidosis (DKA)
Hyperosmolar hyperglycemic state (HHS)
Lactic Acidosis
Coma Hypoglycemia
PATHOPHYSIOLOGY OF DIABETIC KETOACIDOSIS
Biochemistry of Insulin Lock Laboratory Data Clinical I
Organ Involved Result Blood Urine Signs and Symptoms N
C
Muscle 1. Polyuria, nocturia R
Glucose Uptake Hyperglycemia (Dehydration) E
Fat
2. Compensatory A
Liver
Polydipsia S
Glycosuria
3. If # 1 Severe and I
Muscle Osmotic Diuresis
AA # 2 Insufficient N
Liver Gluconeogenesis Plasma Volume G
Glycerol Hypotension Oliguria
Ketonuria S
FFA Lipolysis
Fat Loss Of K, Na 5. Breath Odor of E
Acetone V
Free Fatty Acids
6. Heavy Breathing E
Ketone Bodies
(Kussmauling) R
= Metabolic Acidosis I
7. Vomiting
( pH CO2 Content) T
8. Abdominal Pain
(Leukocytosis) Y
9. Stupor, Coma
Abbreviations : AA = Amino Acids ; FFA = Free Fatty Acids
 Metabolic complications of DM
1. KETOACIDOSIS
- Glucose 400-700 mg/dl
- GFR urea
- Dehydration Hct , protein
- Excretion of K+ in urine K+ (before th/)
After th/
K+ (K+ going back into the cells)
- Phosphate changes ~ K+
- Osmotic diuresis Na+ / N
- Amylase plasma & urine (phenomenon is
not clear)
 Metabolic complications of DM

2. NON KETOTIC HYPEROSMOLAR

- Untreated Patients Osmotic Diuretic
Hyperosmolality
- Some patients develop coma without ketosis
- Th/ hyperglycemia :
* Restoring fluid balance
* Insulin
* K+ given later (K+ , K extracell K intracell)
 KETON BODIES
Keton Bodies :
Ketogenesis
FFA Acetoacetate (ACAC) 20%
Liver
hydroxybutyrate (3 HB) 78%
Aceton 2%
Testing
Urine
Detect Acetoacetate & Aceton
Blood
New Method : Detect 3 HB from Blood
 LACTIC ACIDOSIS
Produced by Anaerobic Glycolysis
Muscle
Glycogen G G P Pyruvate Lactate +H+
TCA Cycle
Require O2
Gluconeogenesis
Pathogenesis : Accumulation Lactate
Production :
Anaerobic Glycolysis
Utilisation :
Impairment TCA Cycle Gluconeogenesis
Acidosis
DM, hypoxia (after cardiac arrest)
D/ Lactate plasma > 20 mg/dL
Laboratory Analysis of Acute Complications DM

Blood Glucose
Arterial Blood Gases (pH, HCO3)
Electrolytes (Calculated Anion gap)
Blood & urine ketones
Lactate
Osmolality
 PRA ANALYTICAL STAGE
Keton Bodies : - Not be on any treatment
- Fresh Urine

Lactate : - Statis free Vein

- Plasma separated by centrifugation
within 30 min

Electrolytes : - Serum separated from blood cell

within 2 hours
- Hemolysis
 PRA ANALYTICAL STAGE
Blood Gases :
Patient must be relax
On oxygen administration, sample is taken 20
minutes after ventilation alteration
Patients temperature should be recorded
Air in the spuit must be removed
Seal the needle hole with rubber cork
Capillary blood : warm the capillary area to
40 0C 50 0C for 10 minutes
 PRA ANALYTICAL STAGE
Blood Gases :
Anticoagulants (NCCLS)
Sodium & lithium heparin 20 100 IU/ml blood :
> : Dilution
< : Damage the instrument
Blood well mixed with heparin
The assay immediately
Without Ice : < 15 min (The Best)
With Ice : < 2 Hours
 ANALYTICAL STAGE
Urinary ketone testing :
Wet Chemistry :
- Rothera
* SO > Nitroprusside
* Detect Acetoacetate & Aceton
- Gerhardt Qualitative
* Ferrichloride
* Detect Acetoacetate
Dry Chemistry :
Test strip Uses Sodium Nitroprusside detect
Tablet Acetoacetate & Aceton
Results : Semiquantitatively
 BLOOD KETONES TESTING
Modification urine methods :
Powder / crushed Sodium Nitroprusside tablet
Weaknesses e.g. : - Fresh speciemen
- Result : Qualitatively or semi
quantitatively
- False : + /
New : detect 3 hydroxybutyrate quantitative
- Enzymatic : Cobas bio centrifugal analyzer
- Keton sensor : Optium system
- N < 0,6 mmol/L
Ketosis : > 1 mmol/L
Ketoacidosis : 3 mmol/L
 ANALYTICAL STAGE

Blood Gases & Electrolytes Analysis :

Ion selective electrode method
Anion gap : Calculation [Na+] [ Cl + HCO3] mEq/L
Osmolality :
Osmometer
Calculation : 2[ Na (mEq/L) + Glucose (mg/dL)/18 ]
mosm/Kg
HOW TO DIAGNOSE DIABETIC COMA
Coma

Hypoglycaemic Hyperglycemic
Blood Glucose
Coma Low High Coma (DM)

Metabolic Acidosis Blood Gases

pH
pH N
Ketoacidosis Ketone Bodies Osmolality
(+)
() Hyperosmolar
Lactate Hyperglycemic state
Lactic Acidosis
 DIAGNOSTIC CRITERIA FOR DKA & HHS
DKA
Mild Moderate Severe HHS
Plasma glucose
> 250 > 250 > 250 > 600
(mg/dL)
Arterial pH 7.25 7.30 7.00 7.24 < 7.00 > 7.30
Serum bicarbonate
15 18 10 < 15 < 10 > 15
(mEq/L)
Urine ketones* Positive Positive Positive Small
Serum ketones* Positive Positive Positive Small
Effective serum
osmolality Variable Variable Variable > 320
(mOsm/Kg)
Anion gap > 10 > 12 > 12 < 12
Alteration in
sensorium or Alert Alert/drowsy Stupor/coma Stupor / coma
mental obtundation
Nitroprusside reaction method ; calculation : 2 [ measured Na (mEq/L) + Glucose (mg/dL) / 18 ;
calculation : [Na+] [Cl + HCO3] mEq/L. See text for details.
Hypoglycemia Due To Overdoses of The Drugs

- Blood glucose < 40 mg/dl/ plasma glucose < 45

mg/dl
- Symptoms :
Dizziness
Soporous/ coma
Sweaty and tachycardia (adrenaline)
- Th/ parenteral glucose
CHRONIC COMPLICATIONS OF DM
Vascular :
Macroangiopathic ( Macrovascular )
Coronary Heart Disease (CHD)
Cerebrovascular Disease
Peripheral Vascular Disease
Microangiopathic ( Microvascular )
Nephropathy
Retinopathy
Neuropathy
Non Vascular :
Sexual Dysfunction
Skin Changes
RISK FACTORS & MECHANISM CHD
OBESITY

Genetic Insulin Resistance Life Style

Hyperinsulinaemia

Glucose HDL LDL VLDL-TG Fibrinogen Hypertension

Fibrinolysis

CHD
 DYSLIPIDEMIA IN DM
Trigl : LPL
CL VLDL
VLDL >>

LDL Chol : Poor Glycaemic Control

CL LDL By LDL Receptor

Insulin
HDL Chol : Inversely VLDL
(Type 2 DM) LPL CL VLDL
Transfer Apo To HDL
 DIABETIC NEPHROPATHY
~ Long Standing Hyperglycaemia Microvascular
Metabolic Compl (The
Environmental Mechanisms is
Genetic Factors Unknown)
Type 1 DM : 20 40%
Before Puberty
Tendency Hypertension
Type 2 DM : 5 10%
DIABETIC NEPHROPATHY ( MOGENSON )
Stage 1 : Renal Hypertrophy
Glomerular Filtration Rate (GFR)
Stage 2 : Asymptomatic
GFR
Stage 3 : Incipient Diabetic Nephropathy
Microalbuminuria ( 30 300 mg/day
or 20 200 g/min )
GFR
Stage 4 : Clinic Diabetic Nephropathy
Macroalbuminuria ( > 300 mg/day or
> 200 g/min )
GFR
Stage 5 : End Stage Renal Disease (ESRD)
 Natural History Of Type I Diabetic Nephropathy
(MOGENSON)
Glomerular Filtration Rate Albuminuria
Incipient Diabetic Over Diabetic EndStage
Pre Nephropathy Nephropathy Renal Disease
1 2 3 4 5
5000
150 GFR
1000
100
200
50
20
0
0 5 10 15 20 25
YEARS
Functional GFR Microalbunuria Proteinuria, Nephrotic
(25-50%) Hypertension Syndrome, GFR

Structural Renal Mesangial expansion Mesangial Nodules (Kimmelsteil

Hypertrophy Glomerular basement - Wilson Lesions)
membrane Thickening Tubulo Interstitial Fibrosis
Arteriolar Hyalinosis
LABORATORY TEST PREVENTS
CHRONIC COMPLICATIONS DM

Blood Gucose
HbA1C
Lipid Profile : Total Chol
Triglyceride
HDL Chol
LDL Chol
Microalbuminuria
 DIABETIC NEPHROPATHY
The Appearance of 30 mg of Albumin ini 24
Hours Urine at Least
2 of 3 Urine Specimens Collected Within a 3
to 6 Month (Because of Variability in Urinary
Albumin Excretion)
Without : Tract Urinarius Infection
Exercise Within 24 Hours
Congestive Heart Failure
Marked Hypertension
Fever
 SCREENING FOR
MICROALBUMINURIA
PRA ANALITICAL STAGE
Ratio Albumin To Creatinine
(Random Spot Collection)
24 Hours Collection Urine
Timed Collection (e.g. 4 Hours or overnight)
 ANALYTICAL STAGE
Macroalbuminuria :
Test Strip
Protein Error Of pH Indicator
Semi Quantitative
Microalbuminuria :
Quantitative : + /
Protein Error Of pH Indicator
Semi Qualitative : / + / ++ / +++
Immuno Chemistry
Quantitative :
Enzyme Linked Immunoassay
Radio Immunoassay
Immuno Turbidimetry
 DEFINITIONS OF
ABNORMALITIES IN ALBUMIN
EXCRETION
Category 24H Timed Spoot
Collection Collection Collection
(mg/24 h) (g/min) (g/mg Creatinine)

Normal < 30 < 20 < 30

Microalbuminuria 30 299 20 199 30 299
Clinical Albuminuria 300 200 300
OPTIMAL PROFILE LIPID ( PERKENI )

LIPID DESIRABLE BODERLINE HIGH HIGH

( mg/dl ) ( mg/dl ) ( mg/dl )

Total Cholesterol < 200 200 239 240

Triglyceride < 150 150 199 200
LDL Cholesterol < 100 100 129 130
HDL Cholesterol > 45 -