14-08-2020

Metabolic syndrome

Chemical pathology of Diabetes Mellitus and its complications

Laboratory management of Diabetes mellitus and its complications

Ramesh R
Professor
JIPMER

Metabolic syndrome

1
 14-08-2020

Definition

Constellation of metabolic abnormalities that

confer increased risk of cardiovascular
disease(CVD) and diabetes mellitus.

2
 14-08-2020

Alternative names

• Metabolic syndrome

• Syndrome X

• Insulin resistance syndrome

• Deadly quartet

• Reaven’s syndrome

Pathogenesis contd…

3
 14-08-2020

The term diabetes mellitus describes a metabolic disorder of. multiple

aetiology characterized by chronic hyperglycaemia. with disturbances of
carbohydrate, fat and protein metabolism. resulting from defects in insulin
secretion, insulin action, or. both.

DIABETES MELLITUS

4
14-08-2020

5
14-08-2020

6
14-08-2020

7
 14-08-2020

CHEMICAL PATHOLOGY OF COMPLICATIONS OF DIABETES MELLITUS

8
14-08-2020

9
14-08-2020

10
 14-08-2020

LABORATORY MANAGEMENT OF DIABETES MELLITUS

GLUCOSE ESTIMATION

11
 14-08-2020

Blood for FBG analysis should be collected in the morning after individuals have
fasted overnight. ( no calorie intake for 8hrs )

Place the sample tube immediately in an ice water slurry and plasma should be
separated from the cells within 30 minutes.

Tube with only enolase inhibitors ( Sodium fluoride) should not be relied to prevent
glycolysis.

FBG is high in the morning than in afternoon indicating that many diabetic
cases would be missed in patients seen in the afternoon.

The rate of glycolysis is about 5 to 7% ( 10mg/hr) which various with glucose

concentration, temperature and Leukocyte count.

12
 14-08-2020

Glucose concentration is stable for 8hrs at 25 degrees and 72hrs at 4 degrees

in separated non haemolysed sterile serum without fluoride.

Acidification of blood with citrate buffer inhibits invite glycolysis better than fluoride.

Plasma is recommended for diagnosis of DM.

Though the molality of glucose in whole blood is identical to that in plasma the
concentration of water in plasma is approximately 11% higher than in whole
blood therefore glucose concentration is 11% higher in plasma than whole
Blood.

Concentration in heparinised plasma is 5% lower than in serum.

Random capillary samples are 20% higher than venous glucose

( not much difference in fasting samples)

13
 14-08-2020

Analytical imprecision ≤ 2.9%

Bias ≤ 2.2%

TAE ≤ 6.9%

GLUCOMETERS

14
 14-08-2020

SMBG is recommended for all insulin treated patient with Diabetes.

In patient with type 2 DM with diet and oral agents SMBG agents SMBG may
help achieve better Control particularly when therapy is initiated or changed.

Comparison between SMBG and concurrent laboratory glucose analysis should

be performed at regular Intervals to evaluate the performance of meters in
patient hand.

Performance Goals

Error rate should be less than 95% at 100mg%

ADA : ≥ 3 times /day with multiple insulin

injection

15
 14-08-2020

End points in studies of SMBG

HBA1c
Frequency of hypoglycaemic episode
Long term outcomes

Factors affecting Glucometers

Changes in haematocrit
Altitude
Temperature
Humidity
Hypotension
Hypoxia
High triglyceride levels
drugs

Continuous monitoring glucose sensors

16
 14-08-2020

It detects unsuspected hypoglycaemia

Strict glucose control is accompanied by a marked increase in the risk

of hypoglycaemia

Patients require extensive training in using the device.

Available devices must be calibrated with SMBG readings

Implanted sensors use multiple detection systems

Enzyme ( glucose oxidase)

Electrode and fluorescence based technique
Artificial glucose receptors
Glucose recognition molecules.

No specific analytical goals

17
 14-08-2020

GESTATIONAL DIABETES MELLITUS

18
14-08-2020

19
 14-08-2020

GDM should be diagnosed a 75g OGTT according to the IADPSG criteria f

rom HAPO study

TYPES

1step : OGTT performed initially

11 step

50g of oral glucose load ( regardless of patient fasting)

Followed by glucose measurement at 1hr ≥ 140mg/dL

Perform definitive OGTT

100g or 75g - OGTT should be performed

20
 14-08-2020

A 55 year old diabetic patient who was on irregular treatment

Presented to emergency department in a comatose state.
His breath had a sweety odour and his breathing was shallow.
His lab findings are as follows:

Blood glucose ( Random) : 640mg%

A, Based on the above history, physical findings and lab results

Give your probable diagnosis.

B. What test will you perform with the urine sample of the
Above patient to confirm your diagnosis.

A self proclaimed politician declared indefinite fasting

Along with his followers. He called for a press meet and
Informed that he was fasting for the past 18 days. The
News was flashed as “ Breaking news “ in all the leading
Channels. But one of the reporter suspected and contacted
A laboratory scientist who happens to be his good friend
And enquired is there any test to find out whether he was
Truly fasting for 18 days. The reporter somehow collected a
urine sample of the politician and handed over it to the lab.
Next day the headlines changed

“ Cheating by a famous politician”

“ Nagged down with evidence for faking fasting.”

21
 14-08-2020

DIABETIC KETOACIDOSIS

KETONE BODIES
The entry of acetyl CoA into the citric acid cycle
depends on the availability of oxaloacetate.
The concentration of oxaloacetate is lowered if
carbohydrate is unavailable (starvation) or improperly
utilized (diabetes).

Oxaloacetate is
normally formed from
pyruvate by pyruvate
carboxylase
(anaplerotic reaction).

Fats burn in the flame

of carbohydrates.

22
 14-08-2020

KETOSIS
The absence of insulin in diabetes mellitus
 liver cannot absorb glucose  activation of fatty
 inhibition of glycolysis acid mobilization by
 activation of adipose tissue
gluconeogenesis
 deficit of oxaloacetate
 large amounts of acetyl CoA which can
not be utilized in Krebs cycle

 large amounts of ketone bodies (moderately strong acids)

 severe acidosis (ketosis)

Impairment of the tissue function, most importantly
in the central nervous system

Normally present in blood and urine at concentration less than 0.5mmol/L

False positive results

High coloured urine

Sulfhydryl containing drugs
ACE inhibitors
Exposure of reagents to air
Highly acidic urine

23
 14-08-2020

Assay methods

Nitroprusside

Sodium nitroferricyanide

Purple colour

This reagents cannot be used to measure beta hydroxybutyrate

Whole blood samples are stable at 4degree up to 24hrs

Serum/plasma stable for upto 1 week at 4degree and more at – 20 degree

Beta hydroxyl butyrate assays

Enzymatic widely used

Gas chromatography

Capillary electrophoresis

24
 14-08-2020

Beta hydroxy butyrate dehydrogenase converts beta hydroxy butyrate to

acetoacetate and NADH

Measured at 340nm

Urine ketone measurement should not be used diagnose or monitor the course
of DKA

Normal levels : less than 0,5mmol/l

More than 2mmol/l ( ketoacidosis)

GLYCATED HEMOGLOBIN

25
 14-08-2020

Target > 7%

26
 14-08-2020

Potential benefit for long term complications should be balanced against the
risk for hypoglycaemia

Note: Laboratories should use HBA1c assay methods that are certified by
NGSP program as traceable to DCCT Reference.

Also should show traceability to IFCC reference methods

Methods of estimation

I . Basis of charge differences between glycated and non glycated components

Cation exchange chromatography

Agar gel electrophoresis

II. Structural difference between glycated and non glycated compounds

Borate affinity chromatography

Immunoassay

27
 14-08-2020

CV less than 2%

A laboratory should include 2 control material ( high and Low% )

Samples with HBA1c results below lower limits of the reference interval or greater than
15 % HBA1c should Be verified by repeat testing.

HbA1c values that are inconsistent with the clinical presentation should be
investigated further.

The repeat measurement should be performed with a method based on

analytical principal that is Different from the initial assay

If the results are confirmed the variant should be considered.

28
 14-08-2020

Removal of labile GHB

Schiff's base pre JBA1c

( Labile HBA1C)

It interferes with HBA1c assay method

Most recently available methods either removes the labile labile pre HBA1c or do not measure them.

When NGSP certified method is used each 1% change in the mean plasma glucose concentration
Of approximately 30mg%.

eAG = 28.7 ×HBA1C – 46.7

Small changes in HBA1c (0.3%) over time may reflect assay imprecision rather a true change
in glycaemic status.

29
14-08-2020

30
14-08-2020

31
 14-08-2020

GENETIC MARKERS

32
 14-08-2020

Autoimmune diabetes is strongly associated with HLADR and HLA DQ

HLADQA1 and DQB1 genotyping is useful to indicate absolute risk in T2DM

Some haplotype induce susceptibility however others provide delay or even protection.

33
 14-08-2020

Non HLA genetic factors include:

INS ( Insulin )

PTPN22( protein tyrosine phosphatase non receptor type 22

CTLA4 ( Cytotoxic T lymphocyte associated protein 4

Early diagnosis may prevent hospitalisation for ketoacidosis

and preserve residual beta cells

AUTOANTIBODIES

34
14-08-2020

35
14-08-2020

36
 14-08-2020

ALBUMINURIA

37
14-08-2020

38
14-08-2020

39
14-08-2020

40
 14-08-2020

INSULIN

PROINSULIN

C PEPTIDE

41
 14-08-2020

THANK YOU AND BEST WISHES

42