ANIK WIDIJANTI

Clinical Pathology Department Saiful Anwar Hospital / Medical Faculty Brawijaya University MALANG

DM : Hyperglycemia because of deficient

insulin secretion and / or abnormal insulin

action

USA : in 2002, 18,2 million DM.

Indonesia : prevalence DM increasing

parallel with obesity. In 1981/1982 :1.5 - 1,6

%. In 2001/2005 : 12.5 - 14.7 %

Type 1 DM

Destruction

-cells

Deficiency

Insulin abs

Gx acute

G.D.M

DM in

pregnancy

Type 2 DM

Secretory

defect insulin

Insulin

resistance

Mixed

Others

Type DM

-cells defectAction defect ExocrineEndocrinopathyDrug inducedInfectionsOthers

Clinical symptoms DM

classic symptoms (+) classic symptoms (-)

FPGor

CPG

126------- 200

< 126--------< 200

FPGor

CPG

126------- 200

100 - 126 < 100

Repeat FPG or CPG

FPGor

CPG

100 - 199

126------- 200

< 126--------< 200

OGTT2-h PG

200 140-199 < 140

DIABETES MELLITUS IGT IFG NORMAL

Evaluation for gizi status, DM complications, dietary planning

Dietary planning, physical activity, Ideal body weight, No medication

Diagnostic

FPG

> 126 mg/dl

Casual PG

> 200 mg/dl

2-h PG

75-g OGTT

> 200 mg/dl

Others

GAD-65

IAA

ICA-512

Insulin

C-peptide

Genetics

Monitoring

A1C

SMBG

CBGM

Fructosamine

1,5-AG

Complication

CVD

Coma

Ketoacidosis

Nephropathy

Diabetic foot

Infections

Others

classic symptoms (+)

FPG

100-125 : IFG>126 : DM < 100 : N

75 g OGTT

> 200 :DM 140-199 : IGT < 140 : N

CPG

> 200 : DM

Type 1 DM : C-peptide (-)Acute symptoms : hyperglycemia

Auto-Ab screening : non recommended

Type 2 DM : C-peptide (+)

Insulin deficiency (+) /Insulin resistance

75 g OGTT more sensitive & specific than FPG

Poorly reproducible & difficult to perform in practice.

People who do not meet diagnostic criteria for DM

by FPG, but would by the OGTT : A1C < 7.0 %

OGTT not recommended for routine

Useful for further evaluation in whom DM is strongly

suspected but normal FPG or IFG

Fasting PG : 8-h

Enzymatic not SMBG

2-h Plasma G

Enzymatic, not SMBG

75-g glucose load + water( maximum < 15 minute )

Normal

I.F.G

I.G.T

D.M

Adults are BMI 25 kg/mm2 + 1 ore more risk

factors for DM

Risk factors (-) : test begin no later than age 45

If normal, repeat testing at least at 3-years

Test either FPG or 2-h 75-g OGTT

In pre-DM, treated other CVD risk factors

Dx consistent with recommendations for adults

BMI 25 kg/mm2

Family history of type 2 DM in 1st or 2nd degree

relative

Race / ethnicity

Insulin resistance or conditions associated

hypertension, dyslipidemia or PCOS

Maternal history of DM or GDM

Screen GDM using risk factor analysis, use OGTT

Carry out GDM risk assessment at the 1st prenatal visit

Screening / diagnosis at this stage of pregnancy use

standard diagnostic testing of DM

Two approach may be followed for GDM screening at

24-28 weeks

GDM should be screened for DM 6 -12 weeks post

partum, & followed with screening for DM / pre-DM

Screen GDM using risk factor analysis, use OGTT

Carry out GDM risk assessment at the 1st prenatal visit

Screening / diagnosis at this stage of pregnancy use

standard diagnostic testing of DM

Two approach may be followed for GDM screening at

24-28 weeks

GDM should be screened for DM 6 -12 weeks post

partum, & followed with screening for DM / pre-DM

Severe obese

Prior history of GDM < or delivery of large

for gestational-age infant

Presence of glucosuria

Diagnosis of PCOS

Strongly family history of type 2 diabetes

50-g OGTT

1-h PG 140 mg/dlIdentifies ~ 80 % GDM

1-h PG 130 mg/dlIdentifies ~ 90 % GDM

100-g OGTT

100-g OGTT

Initial screening

One step approach :

high prevalence

GDM

All woman at 24-28 weeks

gestation

Two step approach

1

2

Fasting : 95 mg/dl ( 5.3 mmol/L )

1 hour : 180 mg/dl ( 10.0

mmol/L )

2 hour : 155 mg/dl ( 8.6

mmol/L )

3 hour : 140 mg/dl ( 7.8

mmol/L )

Performed in the morning

Overnight fast for at least 8 h

GDM : 2 criteria

A1c ( NGSP-DCCT)

≥ 6.5 % 5.7-6.4 %

Diabetes Mellitus Pre diabetes

Diagnostic Criteria HbA1c (A1c) (A.D.A 2010)

PARAMETER Hb A1C Fructosamine 1,5-AGTime required for significance change

1-3 months 1-2 weeks 1-3 days

Reflection of mean glucose

++ ++ +

Reflection of glucose excursions/postprandial glucose

+ + ++

Association with complications

++ NA NA

Variance Small Small LargeGreatest degree of change is found during

Moderate to severe hyperglycemia

Moderate to severe hyperglycemia

Mild to moderate hyperglycemia

HPLC (reference)

Ion-Exchanges C : satisfactorily correlate with HPLC

Affinity binding C : little effect Hb variant . higher value than

HPLC ( because measure total glycated Hb)

Electrophoresis and isoelectric focusing

Immunoassay : using monoclonal Ab exhibit excellent

precision, Hb variant and carbamylated Hb are not

detected. Correlate well with HPLC, but exhibit low value

Cassette based Immunoassay

Enzyme methods /Chemical analysis : total glycated Hb

Hemoglobinopathy & Hb Variant → affect reliability test :

altering glycation, abnormal peak on chromatography, RBC

more prone to hemolysis

Transfusion, splenectomy, turnover RBC

Alcoholic chronic, opiate, iron deficiency, lead poisoning

Vitamin C and E can falsely lower level by inhibiting glyco-

sylation, but vitamine C also increase levels for some assays

Carbamylated Hb in Uremia, Hyper-triglyceridemia, hyper-

bilirubinemia

Measure of glycosilation of serum protein : interference by

albumin levels→ reflecting glycemia over 1-2 weeks

Whether fructosamine should be corrected for serum protein &

albumin : controversial ???

Not affected by Hb variants, moderate to strong correlation

With A1C → recommended for use in Hb pathy. Cheaper than

A1C, a lack of consensus exists as to it clinical value

Alternative to A1C for estimating glycemic control, particularly

where there are discrepancies SMBG and A1C and where

short-term glycemic monitoring is desired

Using serum sample & automatic equipment, has excellent

batch analytical precision.

In carbonate buffer, fructosamine rearranges to the eneaminol

form, which reduces NBT to a formasan. Absorbance at 530

nm is measured at two time points, and the absorbance

change is proportional to concentrations of fructosamine

Hb > 100 mg/dl and bilirubine > 4 mg/dl interfere : → grossly

hemolyzed and icteric samples should not be used

Vitamine C > 5 mg/dl : negative interfference

Is validated marker of short-term glycemic control

Metabolically inert polyol that competes with glucose in the

kidneys, otherwise stable levels of 1,5-AG are rapidly

depleted as blood glucose levels exceed the renal threshold

for glucosuria.

More accurately predicts rapid changes in glycemia than A1C

& fructosamine. Can not use as an index for glycemic control

in uremic patients.

More tightly associated with glucose fluctuations and

postprandial glucose

1,5-AG may offer complementary information to A1C

1.Measure fasting lipid profile at least annually.

2. In adults with low risk lipid value : LDL

cholesterol < 100 mg/dl, HDL cholesterol > 50

mg/dl, Triglycerides < 150 mg/dl) →

assessments may be repeated every 2 years

Total Cholesterol : enzymatic colorimetric, accurate and

easily automated

HDL Cholesterol : ultracentrifugation, precipitation, ion

exchange chromatography, electrophoresis.

LDL-Cholesterol : Formula Fridewald (not valid in TG > 400

mg/dl) → direct immunoassay, electrophoresis

Triglyceride : enzymatic colorimetric, free glycerol enzymatic

colorimetric, automated, good sensitivity, specifity, precision

UAE annually in Type 1 DM (> 5 years), type 2 DM

starting at diagnosis.

Microalbuminuria by measurement of albumin to

creatinine ratio in a random urine

Serum creatinine at least annually in adults with

DM regardless of the degree of UAE, should use to

estimate GFR and stage CKD if present

Another's test : CBC, Urinalysis, cystatine-C

Plasma Glucose (hypo or hyperglycemia)

Ketone bodies (blood or urine) particularly in

type 1 DM : beta-hydroxyl butyric acid, aceto

acetic acid, acetone. → colorimetric automated

or dipstick

Blood gas analysis

Blood electrolyte analysis

Bacteria : direct smear, culture (Bactec or

conventional)

Mycobacterium tuberculosis : direct smear,

culture

CRP (acute phase reactant), CBC, urinalysis

Sepsis / SIRS : Blood cultures, etc

Urinary keton

Blood keton, Beta hydroxybutarate & others

keton bodies

Plasma Glukosa

Type 1 diabetes Type 2 diabetesC-peptide

levels

Very low or undetectable Detectable

Pre-

diabetes

Auto antibodies (GAD65,

ICA512, IAA) maybe

present

Auto antibodies absent

(testing not indicated)

Medication

Therapy

Insulin absolutely

necessary : multiple daily

injection or insulin pump

Oral agents

Insulin Commonly

needed

Therapy to

prevent or

delay onset

of diabetes

None known

Clinical trial in progress

Lifestyle (weight loss and

physical activity )

Oral medications

Clinical trial in progress

Type 1 diabetes Type 2 diabetes

Frequency 5-10 % 90-95 %

Age of

onset

Any, but most common in

children and young adults

More common with

advancing age, but can

occur in children and

adolescents

Risk

Factor

Genetic, autoimmune,

environmental

Genetic, obesity, race/

ethnicity sedentary

lifestyle,

Pathogene

sis

Destruction of pancreatic

cells, usually autoimmune

No autoimmunity,

insulin resistance and

progressive insulin

deficiency

Pro-insulin → insulin + C-peptide (equivocal)

Ratio C-peptide : insulin serum = 5 : 1 to 15 : 1, due

primarily to hepatic clearance of insulin.

Half life C-peptide & pro-insulin 30 minute, but insulin

4-9 minute. → C-peptide more stabile

C-peptide levels : basal & pos glucagons stimulation

Assay : immunoassay → minor cross reactivity with

pro-insulin, in patients who have circulating Ab to

insulin

Two pairs of basic amino acid used for proteolytic processing

Insulin levels : fasting & 2-h postprandial

Wide variation in assay bias → standardization

Performed for Insulin resistance

1. RIA : cross reaction with pro-insulin & insulin

exogenous, circulating anti insulin antibodies

2. Immuno enzymometric assay specific for insulin :

interference from endogenous insulin & anti insulin Ab

3. Micro particle enzyme immunoassay (MEIA) :

interference from endogenous insulin & anti insulin Ab

Assay

GAD-65, IAA, ICA-512

Antibodies (+) may help in DD type 1 DM from the others

Antibodies (-) does not exclude this diagnosis.

GAD-65 has the highest sensitivity (91 %) as a single

screening marker for detecting multiple Antibodies

IAA : more common in young children who develop type 1

DM

GAD-65 more common in adults

Cut off value for immune marker : not completely

establish / standardized for clinical setting

No consensus as to what follow up testing should

be undertaken when a positive autoantibody test

Incidence DM type 1 is low, testing of healthy

individuals will identify only very small number

(< 0.5 %) who at that moment maybe pre-DM

At diagnosis Type 1 DM : performed Anti TPO & Anti TG

Autoimmune thyroid : 17-30 % in type 1 DM.

Thyroid Ab (+) : predictive of thyroid dysfunction

Check TSH : If normal, rechecked every 1-

2 years, or if patients develop thyroid

dysfunction, thyromegaly, or abnormal

growth rate.

SMBG Insulin therapy : 3 or more times daily

inconvenience, physical discomfort &

disability, along with expense

The procedure is complex for some

patients, advances in technology, errors in

technique

For accuracy & reproducibility : it is

important to routine evaluate maintenance

& standardization for each instrument by

committee POCT especially in hospital

Variation of methods, accuracy, presision

Interferen : temperature, humidity

Used good Calibrator

Strip Storage

Small Volume sample : Whole blood, serum

or plasma

Simple & rapid

For Monitoring, not diagnostic

Diagnostic test : FPG, 2-h PG, OGTT

Monitoring : SMBG, A1C, Fructosamine, 1,5-AG

Complication : as indicated to clinical conditions

GAD-65, IAA, ICA-512 : Auto antibodies may help

to DD Type 1 DM from others, it does not exclude

diagnosis, not recommended for screening.

Each method had false positive and negative

depend on sensitivity & specifity of the test