The document discusses the diagnosis of diabetes mellitus. It outlines the evolution of diagnostic guidelines and tests over time, from initial diagnosis based on sugar in the urine to current use of HbA1c, fasting plasma glucose, and oral glucose tolerance tests. Key tests discussed include insulin, C-peptide, proinsulin, glucagon, and autoantibodies which can help distinguish types of diabetes.

1. DIAGNOSIS OF
DIABETES MELLITUS
Presented by:
Dipesh Tamrakar
MSc. Clin. Biochemistry

2. OVERVIEW
• History
• Diagnostic guidelines for DM
• Evolution of diagnosis of DM
• Various diagnostic and supportive tests

3. HISTORY on diagnosis of DM
• 1674: physician Thomas Willis of Oxford notes
sweet flavor of urine in patients with diabetes
• Initial diagnosis centered around presence of sugar
in the urine
• 1917: Oral glucose tolerance test introduced by
Jacobsen (Jacobsen, Aa. Th.B. (1917). The Blood Sugar in Normal
Persons and Diabetic Patients (in Danish).Gyldenal, Copenhagen)
• 2 hour OGTT value was generally diagnostic test of
choice before 1979

4. 1979: National Diabetes Data Group
• Diabetes:
• FPG  140 mg/dl or
• OGTT 2 hr glucose  200 mg/dl or
• Classic symptoms present
• Impaired glucose tolerance:
• FPG  140 mg/dl (Expert Committee on the Diagnosis and Classification
of Diabetes Mellitus.)
• 2 hrs OGTT glucose 140-200 mg/dl (Report of the expert
committee on the diagnosis and classification of diabetes mellitus.
Diabetes Care2003;26Suppl 1:S5–20.2).
• 1997: ADA expert committee
• FPG criteria  126 mg/dl

5. • The oral glucose tolerance test (OGTT) should be retained as a
diagnostic test for the following reasons:
1. Fasting plasma glucose alone fails to diagnose approximately
30% of cases of previously undiagnosed diabetes,
2. OGTT is the only means of identifying people with IGT,
3. OGTT is frequently needed to confirm or exclude an
abnormality of glucose tolerance in asymptomatic people.
4. An OGTT should be used in individuals with fasting plasma
glucose 6.1 – 6.9 mmol/l (110 – 125 mg/dl) to determine
glucose tolerance status.

6. • In November 2005 a joint WHO and International Diabetes
Federation (IDF) Technical Advisory Group met in Geneva to
review and update the current WHO guidelines.
• Recommendations:
1. Fasting plasma glucose ≥ 7.0mmol/l (126mg/dl) or 2–h
plasma glucose ≥ 11.1mmol/l (200mg/dl).
2. the term ‘normoglycaemia’ should be used for glucose
levels associated with low risk of developing diabetes or
cardiovascular disease
3. The current WHO definition for Impaired Glucose Tolerance
(IGT) should be maintained for the present
4. Impaired Fasting Glucose (IFG) should remain at 6.1mmol/l.
5. Venous plasma glucose should be the standard method for
measuring and reporting glucose concentrations in blood
6. The oral glucose tolerance test (OGTT) should be retained
as a diagnostic test
7. Table below:

10. 2009: HbA1c as new diagnostic criteria
• HbA1c had been considered in 1997 but not used because of lack
of standardization of assay
• Advantages of A1c over FPG or OGTT
• Better index of overall glycemic exposure
• Less biologic variability
• Stable samples
• No need for fasting or time samples
• Relatively unaffected by acute stressors
• Used to guide management and adjust therapy
(Nathan D. International Expert Committee report on the role of the A1C
assay in the diagnosis of diabetes. Diabetes Care 2009)

12. Evolution of testing for diabetes

14. Type 1 DM
• Most patients have antibodies that identify an autoimmune
process
• Approximately 75% acquire the disease before the age of 18, but
onset in the remainder may occur at any age.
• Age at presentation is not a criterion for classification.

16. Insulin
• The primary clinical application of insulin measurement is in the
evaluation of patients with fasting hypoglycemia
• Measurement of circulating insulin could be helpful in evaluating
insulin resistance and insulin secretion.
• Reference range: 12 to 150 pmol/L (2 to 25 μIU/mL)

17. Proinsulin
• High proinsulin concentrations are usually noted in patients
with benign or malignant β-cell tumors of the pancreas
• Patients with type 2 diabetes have increased proportions of
proinsulin and proinsulin conversion intermediates
• Similarly, women with GDM have higher concentrations of
proinsulin
• An increased ratio of proinsulin-like molecules to insulin-like
molecules at screening may be a better predictor of GDM than
age, obesity, or hyperglycemia
• Increased proinsulin concentrations may also be detected in
patients with chronic renal failure, cirrhosis, or
hyperthyroidism.
• Reference : 1.1 to 6.9 pmol/L to 2.1 to 12.6 pmol/L

18. • Accurate measurement of proinsulin has been difficult for
several reasons:
• the blood concentrations are low; antibody production is
difficult;
• most antisera cross-react with insulin and C-peptide, which
are present in much higher concentrations;
• the assays measure intermediate cleavage forms of
proinsulin; and
• reference preparations of pure proinsulin are not readily
available.
• However, RIA method with biosynthetic human C-peptide
coupled with agarose (to eliminate cross-reactivity with C-
peptide.)
• The detection limit is 0.25 pmol/L.

19. C-peptide
• Measurement of C-peptide has a number of advantages over
insulin measurement.
• Because hepatic metabolism is negligible, C-peptide
concentrations are better indicators of β-cell function than is
peripheral insulin concentration.
• Furthermore, C-peptide assays do not measure exogenous
insulin and do not cross-react with insulin antibodies, which
interfere with the insulin immunoassay

20. Fasting Hypoglycemia
• The primary indication for measuring C-peptide is for the
evaluation of fasting hypoglycemia.
• Some patients with insulin-producing β-cell tumors,
particularly if hyperinsulinism is intermittent, may exhibit
increased C-peptide concentrations with normal insulin
concentrations.
• When hypoglycemia is due to surreptitious insulin injection,
insulin concentrations will be high but C-peptide values will be
low; this occurs because C-peptide is not found in commercial
insulin preparations and exogenous insulin suppresses β-cell
function.
C-peptide

21. Insulin Secretion
• C-peptide concentrations provides estimates of a patient’s insulin
secretory capacity and rate.
• In rare cases, this strategy may be helpful before discontinuation
of insulin treatment (e.g., in an obese adolescent).
• Urinary and fasting serum C-peptide concentrations appear to
be of some value in differentiating patients with type 1 diabetes
from those with type 2 diabetes.
• In addition, patients who have type 1 diabetes but who have no
C-peptide response are usually more labile than those with some
residual β-cell function.
• Despite these observations, C-peptide measurement has a
negligible role in the routine management of patients with
diabetes.
• A relatively new indication for C-peptide analysis is the recent
requirement that Medicare patients in the United States must
have low C-peptide concentrations to be eligible for coverage of
insulin pumps.
C-peptide

22. Monitoring Therapy
• Measurement of C-peptide is used to monitor patients’ response
to pancreatic surgery.
• In addition, a stable C-peptide concentration is used as an
endpoint in immunomodulatory trials for the prevention of type
1 diabetes
• Measurements of urine C-peptide are useful when continuous
assessment of β-cell function is desired, or when frequent blood
sampling is not practical.
• The 24-hour urine C-peptide content (in the absence of renal
failure, which produces increased concentrations) correlates
well with fasting serum C-peptide concentration
C-peptide

23. • However, the fraction of secreted C-peptide that is excreted in
the urine exhibits high intersubject and intrasubject variability,
limiting the value of urine C-peptide as a measure of insulin
secretion
• Fasting serum concentrations: 0.78 to 1.89 ng/mL (0.25 to 0.6
nmol/L).
• After stimulation with glucose or glucagon, values range from
2.73 to 5.64 ng/mL (0.9 to 1.87 nmol/L), or 3 – 5 times the
prestimulation value.
• Urinary C-peptide is usually in the range of 74  26 μg/L (25 
8.8 μmol/L)
• C-peptide is excreted primarily by the kidney, and concentrations
in the serum are increased in renal disease.
C-peptide

24. Glucagon
• Very high concentrations of glucagon are seen in patients with
α-cell tumors of the pancreas called glucagonomas.
• Patients with this tumor frequently have weight loss, necrolytic
migratory erythema, diabetes mellitus, stomatitis, and
Diarrhea.
• Low glucagon concentrations are associated with chronic
pancreatitis and long-term sulfonylurea therapy
• Fasting plasma concentrations of glucagon: 70 - 180 ng/L (20 -
52 pmol/L).
• Values up to 500 times the upper reference limit may be found
in patients with autonomously secreting α-cell neoplasms

25. Insulin antibodies
• Assays for insulin antibodies fall into three categories:
1. Quantitative radioimmunoelectrophoresis, which measures
the binding of immunoglobulin (Ig)G antibody to radiolabeled
insulin by rocket immunoelectrophoresis into anti–IgG-
containing agarose;
2. RIAs with separation of bound and free insulin by
precipitation with PEG or a second antibody; and
3. Solid-phase immobilization of insulin to test tubes or
Sepharose.

26. Antibodies
• The most practical markers of β-cell autoimmunity are circulating
antibodies, which have been detected in the serum years before
the on set of hyperglycemia.
1. Islet cell cytoplasmic antibodies (ICAs) react with a
sialoglycoconjugate antigen present in the cytoplasm of all
endocrine cells of the pancreatic islets.
• These antibodies are detected in the serum of 0.5% of normal
subjects and 75 to 85% of patients with newly diagnosed type
1 diabetes.
• The antibodies are detected by immunofluorescence
microscopy on frozen sections of human pancreatic tails.
• expressed in Juvenile Diabetes Foundation (JDF) units.
• Although not universal, many laboratories use 10 JDF units on
two separate occasions or a single result of greater than or
equal to 20 JDF units as a significant titer.

27. 2. Insulin autoantibodies (IAAs) are present in more than 90% of
children who develop type 1 diabetes before age 5, but in less
than 40% of individuals who develop diabetes after age 12
• Results are positive when concentrations exceed the 99th
percentile or the mean + 2 (or 3) standard deviations (SDs) in
healthy controls
3. Antibodies to the 65 kDa isoform of glutamic acid
decarboxylase (GAD65) have been found up to 10 years before the
onset of clinical type 1 diabetes and are present in ≈60% of
patients with newly diagnosed diabetes.
• GAD65 antibodies may be used to identify patients with
apparent type 2 diabetes who will subsequently progress to
type 1 diabetes.
• Several different assay formats have been used for the
measurement of anti-GAD65 antibodies, including enzymatic
immunoprecipitation assay, radiobinding assay, ELISA,
immunofluorescence, and Western blotting

28. • In type 1 diabetes, a number of autoantibodies are thought to
circulate including those which target glutamic acid
decarboxylase.
• Presence of these autoantibodies suggests type 1 diabetes.
• The test is particularly useful for adults over 30 who get
diabetes where a type 2 diabetes diagnosis is in doubt - such as
if the patient is not overweight.
• The test may also be used to determine whether gestational
diabetes (diabetes within pregnancy) may be type 1 diabetes.
• The test can also be used to measure the progression of type 1
diabetes or indicate a risk of type 1 diabetes or LADA.

29. 4. Insulinoma-associated antigens (IA-2A and IA-2βA), directed
against two tyrosine phosphatases, have been detected in more
than 50% of newly diagnosed type 1 diabetes patients.
• A widely used method to measure IA-2A uses 35S-labeled
recombinant IA-2 in a dual micro-method and RIA.
5. Zinc transporter ZnT8 was identified recently as a major
autoantigen in type 1 diabetes.
• Initial analysis identified ZnT8 in 60 to 80% of patients with
new-onset type 1 diabetes compared with less than 2% of
controls and less than 3% of individuals with type 2
diabetes.

30. • The Centers for Disease Control and Prevention (CDC) and the
Immunology of Diabetes Society have developed the Diabetes
Autoantibody Standardization Program (DASP).
• The major goals of DASP are
1. to assist laboratories in improving methods,
2. to organize workshops for harmonization of antibody testing
for type 1 diabetes, and
3. To provide reference materials. ASP provides serum from 50
patients with newly diagnosed type 1 diabetes and from 50
to 100 control patients.
• A WHO standard for GAD65 and IA-2A has been established and
allows laboratories to express results in common units

31. • Autoantibody markers of immune destruction are present in 85
to 90% of individuals with immune-mediated diabetes when
fasting hyperglycemia is initially detected.
• Approximately 5 to 10% of white adult patients who have the
type 2 diabetes phenotype also have islet cell autoantibodies,
particularly to GAD65. This condition has been termed Latent
Autoimmune Diabetes Of Adulthood (LADA).
• Up to 1 to 2% of healthy individuals have a single autoantibody
and are at low risk of developing immune-mediated diabetes.
• The presence of multiple islet autoantibodies (IAA, GAD65, and
IA-2A and/or IA-2βA) is associated with a greater than 90% risk
of immune-mediated diabetes.

32. New strategies
• In type 1 diabetes: focus on immunosuppressive therapy to
attenuate the autoimmune response, replacement of insulin-
producing β-cells by transplantation, or restoration of insulin with
gene therapy
• Hyperglycemia in diabetic mice was completely reversed by gene
therapy that induced the development of β-cells of the pancreas
in the liver.
• Although type 1 diabetes can be prevented in animal models, the
possible role of immune intervention in humans remains
unresolved.

33. Type 2 DM
• Insulin concentrations may be normal, decreased, or increased,
and most people with this form of diabetes have impaired
insulin action.
• insulin resistance and is thought by many to be the primary
underlying pathologic process. The other is β-cell dysfunction
• type 2 diabetes mellitus is an extremely heterogeneous disease,
and no single cause is adequate to explain the progression from
normal glucose tolerance to diabetes.
• The fundamental molecular defects in insulin resistance and
insulin secretion result from a combination of environmental
and genetic factors.
• Obesity is commonly associated, and weight loss alone usually
improves hyperglycemia in these persons.

34. Insulin Resistance
• Insulin resistance is defined as “a decreased biological response
to normal concentrations of circulating insulin”;
• it is found in obese, nondiabetic individuals and in patients with
type 2 diabetes.
• Measurement of insulin resistance in a routine clinical setting is
difficult, and surrogate measures, namely, fasting insulin
concentration are used to provide an indirect assessment of
insulin function.
• Homeostatic model assessment (HOMA) is a method for
assessing β-cell function and insulin resistance (IR) from basal
(fasting) glucose and insulin or C-peptide concentrations.

35. a. Optimal insulin sensitivity: < 1
b. Early insulin resistance: > 1.9
c. Significant insulin resistance: > 2.9
• The use of HOMA to quantify insulin sensitivity and β-cell
function can be helpful in normal populations as it allows
1. comparisons of β-cell function and insulin sensitivity
to be made with subjects with abnormal glucose
tolerance and
2. the collection of longitudinal data in subjects who go
on to develop abnormal glucose tolerance

36. Impaired Glucose Tolerance
• Impaired glucose tolerance (IGT) is diagnosed in people who
have fasting blood glucose concentrations less than those
required for a diagnosis of diabetes mellitus, but have a
plasma glucose response during the OGTT between normal
and diabetic states
• The 2 hour postload plasma glucose following an OGTT is 140
to 199 mg/dL for this classification. An OGTT is required to
assign a patient to this class.

37. Impaired Fasting Glucose
• This category is analogous to IGT, but it is diagnosed by a fasting
glucose value between those of normal and diabetic individuals,
namely, fasting plasma glucose (FPG) between 100 and 125
mg/dL.
• It is a metabolic stage between normal glucose homeostasis and
diabetes.
• As with IGT, persons with impaired fasting glucose (IFG) are at
increased risk for the development of diabetes and
cardiovascular disease.
• IFG and IGT are not clinical entities, but rather are risk factors
for diabetes and cardiovascular disease.

38. • The following conditions should be met before an OGTT is
performed:
1. discontinue, when possible, medications known to affect
glucose tolerance;
2. perform in the morning after 3 days of unrestricted diet
(containing at least 150 g of carbohydrate per day) and activity;
and
3. perform the test after a 10 to 16 hour fast only in ambulatory
outpatients (bed rest impairs glucose tolerance), who should
remain seated during the test without smoking cigarettes.
• Glucose tolerance testing should not be performed on
hospitalized, acutely ill, or inactive patients.

39. • The test should begin between 7 am and 9 am. Venous
plasma glucose should be measured fasting, then 2 hours
after an oral glucose load.
• For nonpregnant adults, the recommended load is 75 g,
which may not be a maximum stimulus;
• for children, 1.75 g/kg, up to 75 g maximum is given. The
glucose should be dissolved in 300 mL of water and ingested
over 5 minutes.

41. Other causes
• This subclass includes uncommon patients in whom
hyperglycemia is due to a specific underlying disorder, such as:
1. genetic defects of β-cell function;
2. genetic defects in insulin action;
3. disease of the exocrine pancreas;
4. endocrinopathies (e.g., Cushing’s syndrome, acromegaly,
glucagonoma);
5. administration of hormones or drugs known to induce β-
cell dysfunction (e.g., dilantin, pentamidine) or to impair
insulin action (e.g., glucocorticoids, thiazides, β-
adrenergics);
6. infection;
7. uncommon forms of immune-mediated diabetes; or
other genetic conditions (e.g., Down syndrome,
Klinefelter syndrome)

42. GESTATIONAL DIABETES MELLITUS
• Normal pregnancy is associated with increased insulin
resistance, especially in the late second and third trimesters
• Recommendations for screening and diagnosis were
formulated in 1984 at the Second International Workshop-
Conference on Gestational Diabetes Mellitus,
• they were refined at the Third, Fourth, and Fifth International
Workshop-Conferences in 1990, 1998, and 2007,157
respectively.
• Based on the Fourth Workshop- Conference, the ADA
modified its recommendations for laboratory diagnosis of
GDM by adopting 5 to 10% lower glucose value

43. These modified guidelines are as follows:
1. Low-risk patients require no testing. Low-risk status is limited to
women who meet all of the following:
• Age younger than 25 years
• Weight normal before pregnancy
• Member of an ethnic group with a low prevalence of diabetes
• No known diabetes in first-degree relatives
• No history of abnormal glucose tolerance
• No history of poor obstetric outcome
2. Average-risk patients (all patients who fall between low and high
risk) should be tested at 24 to 28 weeks’ gestation
3. Very high-risk patients should undergo immediate testing. They
are defined as having any of the following:
• Severe obesity
• Prior history of GDM or delivery of large-for-gestational age
infant
• Glycosuria
• Strong family history of type 2 diabetes
• Diagnosis of polycystic ovarian syndrome

44. • Average- and high-risk patients receive a glucose challenge test
given by one of two methods :
1. One step: Perform a diagnostic 100 g OGTT without prior
plasma or serum glucose screening.
A 75 g OGTT is deemed acceptable by some, but it is not as well
validated
2. Two step: The first step is a 50 g oral glucose load (the patient
does not need to be fasting), followed by a plasma or serum
glucose determination at 1 hour.
A plasma glucose value greater than or equal to 140 mg/dL
(7.7 mmol/L) indicates the necessity for definitive testing.
Approximately 15% of patients have a 1 hour venous plasma
glucose concentration of 140 mg/dL (7.7 mmol/L) or
greater, and require a full diagnostic glucose tolerance test
as the 100 g test.

49. Comprehensive Medical Evaluation and Assessment
of Comorbidities
1. Influenza
2. Pneumococcal pneumonia
3. Hepatitis B
4. Cancer
5. Cognitive
impairment/Dementia
6. Hypoglycemia
7. Hypoglycemia
8. Nutrition
9. Fatty liver disease
10.Statins
11.Fractures
12.HIV
13.Hearing impairment
14.Low testosterone in Men
15.Obstructive sleep apnea
16.Periodontal disease
17.Anxiety disorders
18.Depression
19.Disordered eating behavior
20.Serious mental illness

51. ASSESSMENT OF GLYCEMIC
CONTROL