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 %
 Others
Type 1 DM Type 2 DM Type DM
Destruction Secretory -cells defect G.D.M
defect insulin Action defect
-cells
Exocrine DM in
Deficiency Insulin
Endocrinopathy
Insulin abs pregnancy
resistance Drug induced
Gx acute Mixed Infections
Others
 Clinical symptoms DM

classic symptoms (+) classic symptoms (-)

FPG 126 < 126 FPG 126 100 - 126 < 100
or ------- -------- or -------
CPG  200 < 200 CPG  200

100 - 199
Repeat FPG or CPG

OGTT
FPG 126 < 126 2-h PG
or ------- --------
CPG  200 < 200
 200 140-199 < 140

DIABETES MELLITUS IGT IFG NORMAL

Evaluation for gizi status, DM Dietary planning, physical activity,

complications, dietary planning Ideal body weight, No medication
 Complication
Diagnostic
CVD
Others
FPG Monitoring
Coma GAD-65
> 126 mg/dl
A1C Ketoacidosis IAA
Casual PG
SMBG Nephropathy
> 200 mg/dl ICA-512
CBGM Diabetic foot
2-h PG Insulin
Fructosamine 
75-g OGTT Infections C-peptide
1,5-AG Others
> 200 mg/dl Genetics
 Type 2 DM : > 200 :DM 140-199 : IGT < 140 : N
C-peptide (+)
Insulin deficiency (+) /
Insulin resistance
75 g OGTT
classic symptoms (+)
FPG

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

CPG
Type 1 DM : C-peptide (-)
Acute symptoms : hyperglycemia
> 200 : DM
Auto-Ab screening : non recommended
 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 2-h Plasma G
Enzymatic not SMBG 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

 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

 Initial 50-g OGTT 1 Two step
screening approach

1-h PG  130 mg/dl 1-h PG  140 mg/dl

Identifies ~ 90 % GDM Identifies ~ 80 % GDM

100-g OGTT 2

One step All woman

approach : 100-g OGTT at 24-28
high weeks
prevalence gestation
GDM
 Performed in the morning
Overnight fast for at least 8 h
GDM :  2 criteria

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

A1c ( NGSP-DCCT)

≥ 6.5 % 5.7-6.4 %

Diabetes Mellitus Pre diabetes

PARAMETER Hb A1C Fructosamine 1,5-AG
Time required for 1-3 months 1-2 weeks 1-3 days
significance change
Reflection of mean ++ ++ +
glucose
Reflection of glucose + + ++
excursions/postprandial
glucose
Association with ++ NA NA
complications
Variance Small Small Large
Greatest degree of Moderate to Moderate to Mild to
change is found during severe severe moderate
hyperglycemia hyperglycemia 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, 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 diabetes
C-peptide Very low or undetectable Detectable
levels
Pre- Auto antibodies (GAD65, Auto antibodies absent
diabetes ICA512, IAA) maybe (testing not indicated)
present
Medication Insulin absolutely Oral agents
Therapy necessary : multiple daily Insulin Commonly
injection or insulin pump needed
Therapy to None known Lifestyle (weight loss and
prevent or Clinical trial in progress physical activity )
delay onset Oral medications
of diabetes Clinical trial in progress
 Type 1 diabetes Type 2 diabetes
Frequency 5-10 % 90-95 %

Age of Any, but most common in More common with

onset children and young adults advancing age, but can
occur in children and
adolescents
Risk Genetic, autoimmune, Genetic, obesity, race/
Factor environmental ethnicity sedentary
lifestyle,
Pathogene Destruction of pancreatic  No autoimmunity,
sis cells, usually autoimmune 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
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
A 1. RIA : cross reaction with pro-insulin & insulin
s
exogenous, circulating anti insulin antibodies
s
2. Immuno enzymometric assay specific for insulin :
a
y interference from endogenous insulin & anti insulin Ab
3. Micro particle enzyme immunoassay (MEIA) :
interference from endogenous insulin & anti insulin Ab
 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