Diabetes Mellitus, Diagnosis and Tests - PowerPoint Presentation

Slide1

Diabetes Mellitus, Diagnosis and Tests

By

Zohreh

Rahimi

Prof. of Clinical Biochemistry

Slide2

The concentration of glucose in blood is normally controlled within narrow limits by many hormones, the most significant of which,

insulin

,

is produced by the endocrine pancreas

. Diabetes mellitus is the most common disease of carbohydrate metabolism.

Most individuals with diabetes have either type 1 (beta cell destruction with absolute insulin deficiency) or type 2 (insulin resistance and defective insulin secretion).

FUNCTION

OF THE ENDOCRINE PANCREAS

The pancreas functions as both an endocrine and an exocrine organ

in the

control

of carbohydrate metabolism.

As an exocrine gland

, it produces and

secretes

amylase responsible for the breakdown of ingested complex

carbohydrates

.

Further digestion leads to the production of monosaccharides.

Once

absorbed,

the monosaccharides signal the endocrine pancreas, which

regulates

hormones involved in energy homeostasis.

Enteroendocrine

cells

in

the gastrointestinal tract are also stimulated by nutrients to secrete

incretins

, peptide hormones that affect pancreatic function, gastric

emptying

, appetite, and intestinal motility

.

Slide3

The endocrine pancreas secretes four hormones from different cells

residing

in the islets of Langerhans.

Insulin is produced by the beta cells,

glucagon

by the alpha cells, somatostatin by the delta cells, and pancreatic

polypeptide

(PP) by the PP cells.

In insulin-sensitive tissues such as

skeletal

muscle, fat, and liver, insulin stimulates glucose uptake and the

formation

of glycogen and inhibits glucose production

.

Glucagon

acts primarily

in

the liver

, where it stimulates glucose production and, over time, keto-genesis.

Somatostatin

, on the other hand,

inhibits insulin and glucagon

secretion

, as well as the secretion of several other hormones.

Nutrient

ingestion

, through vagal-cholinergic stimulation, increases PP secretion.

The

PP family also includes peptide YY and neuropeptide Y, which

are

found in the gastrointestinal tract and nervous system and affect

gastrointestinal

function, food intake, and satiety.

Slide4

Insulin

Insulin is secreted from beta cells of pancreas as pro insulin. After

processing of pro insulin one mole insulin and one mole connection peptide (C-peptide) is secreted from beta cells.

The biological activity of pro insulin is

10%

to

15% of the biologic activity of insulin

.

In tumors of pancreas the production of pro insulin increases.

C-peptide is used for diagnosis of endogenous from exogenous insulin, since in exogenous insulin there is no C-peptide.

In adults, small amounts of intact proinsulin and these metabolically active conversion intermediates,

especially

des-31,32 split proinsulin, are

cosecreted

with insulin.

Slide5

Slide6

Proinsulin and its metabolites may cross-react

with

insulin in some insulin

radioimmunoassays

.

This can be significant,

especially

because the half-life of proinsulin is at least three times as long

as

that of insulin

.

Elevated

proinsulin levels (intact and partially processed proinsulin)

and

high proinsulin : insulin ratios have been found in

type 2 diabetes. This

is

associated with reduced

proprotein

convertases (PC1/PC3)

activity and decreased ability of the

beta

cells to secrete insulin.

Less common conditions associated with high proinsulin levels

include

insulinomas

.

Approximately 50% of insulin is rapidly removed by its initial

passage

through the liver,

but hepatic extraction of C-peptide is negligible.

In

cirrhosis, hyperinsulinemia is observed as the result of decreased hepatic

insulin

clearance.

In healthy individuals, the half-life of both C-peptide

and

proinsulin is approximately 30 minutes, whereas it is only 4 to 9

minutes

for insulin. There is evidence that

C-peptide has significant

biologic activity.

Slide7

Sera

from

insulinoma

patients

have

high insulin and C-peptide levels, whereas hypoglycemia from

injected

or exogenous insulin is characterized by high insulin levels and

low

C-peptide levels.

Commercially available insulin preparations are free

of

C-peptide and proinsulin. Because

C-peptide is less stable than insulin,

serum

samples should be separated quickly and frozen

.

In people with diabetes mellitus, C-peptide and glucose can be mea-

sured

after an overnight 8-hour fast and after stimulation by an oral

mixed

meal or after glucagon stimulation to provide information con-

cerning

beta cell secretory capacity.

Urine C-peptide

: creatinine

has also

been

used to assess beta cell function

.

Low C-peptide

levels

are characteristic of the absolute insulin deficiency of type 1

diabetes

.

Slide8

C-peptide

measurement can also be useful in follow-up evaluations after pancreatectomy and

postpancreatic

transplantation.

Unlike insulin, both C-peptide and proinsulin are primarily degraded in the kidneys, so levels are elevated in renal failure.

Serum insulin measurements may be falsely low in the presence of hemolysis. An insulin-degrading enzyme found in red blood cells

, as well as in other tissues, is responsible for this problem.

C-peptide and proinsulin measurements appear to be less affected by hemolysis

.

The pancreatic beta cell secretes a 37 amino acid protein called islet amyloid polypeptide (IAPP), or amylin.

First discovered in 1987,

amylin is

colocalized

and

cosecreted

with insulin in response to stimulation with nutrients.

Slide9

Amylin slows gastric emptying and inhibits postprandial glucagon secretion. The overall result is a lowering of postprandial hyperglycemia.

High

levels

of

amylin have been observed in

hyperinsulinemic

, insulin-resistant states,

such

as impaired glucose tolerance and early type 2 diabetes

, and pancreatic cancer

and low levels

are

seen in type 1 diabetes and insulin-requiring type 2 diabetes.

Slide10

Insulin measurement

Sample:

Serum

or

EDTA treated

whole blood (plasma)

without hemolysis

Need to fasting

Reference method: RIA

Preference method

:

Chemiluminescence

Interference:

Insulin autoantibodies especially in RIA method, intake food and obesity increase insulin, Drugs (corticosteroids, levodopa, OCP), complete cross reaction with recombinant human insulin (

Novolin R and Novolin

N)

Icteric, lipemic and hemolysis in serum and high biotin have interference.

Slide11

GLUCAGON

Proglucagon

is synthesized in the pancreatic alpha cells and the L cells of

the

distal small bowel.

Through

differential processing

, the glucagon

family

of gene products is formed. This includes the production of

glucagon

in the pancreatic alpha cells

and the incretin glucagon-like peptide 1

(

GLP-1) in the L cells of the intestines

.

In type 2 diabetes, inappropriately

elevated

postprandial levels of glucagon are observed

.

Serum glucagon concentrations are rarely measured in clinical

prac

-tice.

Glucagonomas

are rare islet cell tumors that produce excessive

glucagon

.

Clinically,

glucagonomas

present with a characteristic necrotizing

migratory

erythematous rash, stomatitis, glossitis, weight loss, anemia,

and

mild diabetes mellitus.

Slide12

GLUCOSE MEASUREMENTS

SPECIMEN 

CONSIDERATIONS

The standard clinical laboratory analysis of glucose is performed on

plasma

or serum derived from a phlebotomy specimen.

Glycolysis causes

plasma

glucose to decline over time while the plasma is in contact with

cells

. The decline can be several milligrams per deciliter

per hour,

depending

on cell counts and other factors.

A specimen is appropriate for glucose

analysis

if serum or plasma is separated from the cells within 30 minutes.

If plasma is in contact with cells for much longer than 30 minutes, a

preservative

such as sodium fluoride that inhibits glycolysis should be added

.

However, even with the use

of

fluoride, plasma glucose will decline in the first hour after blood

collection

.

Slide13

GLUCOSE MEASUREMENT 

METHODS

Most measurements of glucose employ enzymatic methods

. These methods

provide

specificity and can be packaged to furnish point-of-care

determinations

.

Enzyme systems that have been commonly used to measure

glucose

are glucose dehydrogenase, glucose oxidase, and hexokinase.

These reactions produce an electrical current that is proportional to the

initial

glucose concentration, or a product that measured

spectrophotometrically

is proportional to the initial glucose concentration. The assays

can

be initial rate-of-change assays, where the velocity of the reaction is

dependent

on the initial glucose, or end-point assays

.

The most accurate method is isotope dilution gas

chromatography/mass

spectrometry.

The YSI glucose analyzer, which measures glucose in

plasma

, serum, or blood using an immobilized enzyme electrode,

produces

more accurate results than the point-of-care home blood glucose

monitoring

devices.

Slide14

Slide15

Glucose

oxidase, a

flavoenzyme

, catalyzes the reactions shown in

Equation

16-3. The peroxidase reaction can be measured

spectrophoto

-metrically and can be inhibited by high concentrations of uric acid,

ascorbic

acid, bilirubin, glutathione, creatinine, l-cysteine,

l-dopa

, dopamine,

methyldopa

, and citric

acid.

In addition, the glucose

oxidase

reaction can be coupled to the

ferricyanide

/

ferricyanide

couple to

produce

an electrical current, as shown in Equation 16-4.

This system is

dependent

on the partial pressure of

O2

because

oxygen will compete in

the

reaction to form hydrogen peroxide, so the higher the partial pressure

of

O2, the lower the electrically measured

glucose. Glucose

oxidase can be used in another electrical system, as shown in

Equation

16-5.

Slide16

Slide17

In

the hexokinase system assay, the glucose concentration is

proportional

to the rate of production of reduced nicotinamide adenine

dinucleotide

phosphate (NADPH), which is followed spectrophotometrically

(

Equation 16-6).

Depending on the source of the glucose-6-phosphate

dehydrogenase

, the enzyme can require specificity for NADP, or from

some

sources, it can use NAD as well.

Hemolyzed

samples can be

problematic

in that contents released from the erythrocytes may interfere

with

the stoichiometric relationship between glucose and NAD(P)H

accumulation

.

Slide18

Slide19

Whole Blood Glucose

Whole blood

glucose

specimens

, analyzed with point-of-care monitoring

devices

, are used in the home, in the physician’s office, or at the bedside

in

the hospital to monitor for hypoglycemia and hyperglycemia.

Whole

blood tends to give

approximately

10% to 15% lower glucose readings than plasma, but the

percentage

varies on the basis of hematocrit, analysis technique, and

sample

timing (fasting vs.

Post

-

glucose

load).

(Because erythrocytes make up a

significant fraction of blood volume, their removal by centrifugation leaves a

supernatant fluid, the plasma, containing the “blood glucose” in a smaller

volume.

To convert blood glucose to plasma glucose concentration, multiply

the blood glucose level by 1.14.)

Slide20

Capillary

blood is the source for most of these whole blood glucose measuring devices. Capillary blood glucose is similar to arterial glucose but can vary markedly from venous samples, depending on timing relative to food ingestion.

For example, a postprandial specimen is higher in the capillary sample than in the venous sample.

Capillary glucose tests, using point-of-care devices, should not be used to diagnose diabetes or hypoglycemic disorders. To establish these diagnoses, confirmation with laboratory measurements of plasma glucose is essential because of their greater accuracy.

Slide21

Home

blood glucose monitoring devices help people with diabetes to

better

self-manage their disease. A wide variety of devices are available for

home

measurements.

Proper training of patients in the use of individual

meters

is critical in avoiding operator errors, which, in one study, were

reported

in 12% of

users.

Errors that may contribute

to

inaccurate readings in certain devices include the application of an

insufficient

volume of blood, milking the finger to acquire sufficient blood,

use

of outdated test strips, use of alternative sites, environmental factors

(

humidity, heat, altitude), use of a malfunctioning meter, use of a dirty

meter

, hypertriglyceridemia, hypotension, measurements outside of the

hematocrit

or temperature range, and other

factors.

The accuracy of point-of-care home blood glucose monitoring devices

varies

, especially at very high and low glucose concentrations.

Slide22

Interstitial Glucose

Interstitial glucose measuring

devices have

been developed for

continuous

monitoring of glucose levels in people with diabetes. Most of these

devices

use electrochemical methods to automatically and frequently

measure

glucose levels in the interstitial fluid of dermis or subcutaneous

fat

tissue

and require repeated calibration to plasma or whole blood glucose

levels

.

Currently available continuous glucose monitoring systems use a

subcutaneous

tissue sensor that provides glucose measurements every 1 to

5

minutes. They are used independently or connected to insulin pumps.

The sensor is changed every 6 days. Interstitial glucose is in slow (5-30 min)

equilibrium

with capillary blood glucose and therefore is not equal to

blood

glucose

,

except in stable

systems Particularly

during times when glucose levels are rapidly changing, such

as

after meal ingestion or recovery from hypoglycemia, interstitial fluid

readings

will lag behind

finger stick

glucose levels.

Although the precision

and

accuracy of currently available portable continuous glucose monitors

are

improving, they are not as high as for home blood glucose monitoring

devices

. They are therefore recommended to supplement but not replace

conventional

home blood glucose monitoring.

Slide23

Hypoglycemia

can occur at low or high

HbA1c levels

and

can

be discovered through continuous glucose monitoring

.

There is currently an FDA-approved insulin

pump/continuous

glucose monitoring system that has incorporated a low–glucose

threshold

suspend feature. Insulin infusion is temporarily stopped when

glucose

levels fall below the threshold suspend level, reducing time in

hypoglycemia

.

Early

studies of a bionic pancreas

(

an automated wearable insulin and glucagon subcutaneous infusion

system

that communicates with a continuous glucose monitor and uses an

automated

adaptive algorithm) are

promising.

Slide24

Diabetes mellitus

is a group of diseases in which blood glucose levels are

elevated.

A

fasting

plasma glucose level of 126 mg/

dL

(7.0

mmol

/L) or higher on at

least

two occasions is diagnostic of diabetes

(Table 16-1). The fasting

glucose

level should be obtained

after an 8-hour fast

.

Symptoms of

hyperglycemia

(e.g., polyuria, polydipsia, polyphagia, unexplained weight loss)

with

a casual plasma glucose level of 200 mg/

dL

(11.1

mmol

/L) or higher

or

hemoglobin A1c(HbA1c) of 6.5% or higher on two different days is

sufficient

to diagnose diabetes

.

Prediabetes designates conditions in which glucose homeostasis is

abnormal

, but serum glucose levels are not high enough to be classified as

diabetes

.

This group includes individuals with impaired fasting glucose and

impaired

glucose

tolerance.

They are also at increased risk

for

cardiovascular and cerebrovascular diseases.

Slide25

Slide26

Fasting Blood

Suger

(FBS

),

Fasting Plasma glucose

,Fasting

  blood

glucose

Sample:

Serum or EDTA treated whole blood (plasma), at least 8 hours fasting, not more than 16 hours (because increases glucose falsely)

Not use Insulin or hypoglycemic drugs before test

Increased FBS

: DM, response to severe stress (surgery, infection), Cushing syndrome, Acromegaly,

Glucagunoma

, Renal failure

Decreased FBS

: Hypothyroidism, Hypopituitarism,

Insulinoma

, Addison

disease,liver

disease, fasting, high dose Insulin

Interference

: severe hemolysis of sample, smoking, stress,

Drugs Increases FBS

: Drugs such as antidepressant drugs, corticosteroids, beta blockers, dextrose, lithium, diuretics, epinephrine, glucagon

Drugs Decreases FBS

: Acetaminophen, anabolic steroids, Insulin,

pentamidine

, propranolol

Reference method

: Hexokinase

Preference method

: Glucose oxidase

Slide27

Oral

glucose tolerance

test (OGTT):

Before an oral glucose tolerance test is performed, individuals should

ingest

at least 150 g/day of carbohydrates for the 3 days preceding the test

without

limitation in physical activity, and the test should be performed

after

an overnight 8- to 14-hour fast. The individual should not eat food;

drink

tea, coffee, or alcohol; or smoke cigarettes during the test, and he

or

she should be seated. Venous glucose samples are preferably collected

in

gray-top tubes containing fluoride and an anticoagulant.

Slide28

Formal

oral glucose tolerance tests are not generally recommended for

routine

clinical use in the diagnosis of diabetes.

If used, the procedure

described

by the World Health Organization (1985)utilizing a 75-gram

glucose load should be followed.

For children, 1.75 grams glucose/kg up

to

75 grams is recommended.

The exception is for the diagnosis of diabetes

during

pregnancy.

Pregnant women with risk factors for type 2 diabetes

should

be screened using an oral glucose tolerance test at their initial

prenatal

visit. Other women should be screened between 24 and 28 weeks’

gestation

.

Slide29

Slide30

Slide31

TYPE 1 DIABETES

Type 1 diabetes mellitus represents approximately

10%

of all cases of

diabetes

. There usually is an

autoimmune destruction of insulin-producing

beta

cells

in the islets of the pancreas, causing an

absolute deficiency in

insulin

production

. The genetic susceptibility to develop type 1 diabetes

is

related, at least in part, to the inheritance of specific immune response

genes

associated with HLA-DR/DQ

on chromosome 6, as well as other genes and genetic markers.

Antibody markers of beta cell destruction are commonly present before

and

at the time of onset of diabetes

and can be useful for prediction and

diagnosis

and for research on the treatment and prevention of type 1

diabetes

. These include antibodies to antigens for which recombinant

autoantibody

assays are available:

antibodies to the 65

kDa

isoform of

glutamic

acid decarboxylase (GAD65)

,

insulin autoantibodies (IAAs), auto-antibodies to

insulinoma

-associated protein 2 (IA-2)

, and

autoantibodies

to

zinc transporter 8 (ZnT8

).

IA-2 is a tyrosine phosphatase–related

protein

.

Slide32

Those individuals at greatest risk of developing type 1 diabetes have

high

titers of multiple autoantibodies.

In family as well as in population

studies

, the detection of at least two autoantibodies is associated with

increased

risk of developing type 1 diabetes

.

GAD65 has the highest sensitivity (91%) as a

single

screening marker for detecting multiple antibody-positive

individuals.

IAAs are more common in young children who

develop

type 1 diabetes,

whereas GAD65 is more common in adults.

The “prediabetes” period of

gradual

and progressive beta cell destruction can last for months, years, or

decades

. During this period, the acute insulin response to intravenous

glucose

, called the first-phase insulin release, becomes depressed or absent.

Eventually

, in most people with

type 1 diabetes

, most or all of

the

beta cells are destroyed, resulting in inadequate or absent insulin

secretion

.

C-peptide levels and endogenous insulin levels therefore are very low

or

undetectable

.

People with untreated type 1 diabetes develop diabetic

ketoacidosis

. Insulin therapy is required for all patients with type 1

diabetes

.

Slide33

Diabetes Mellitus

Autoantibodies (

DMA

) in Serum

Or

Insulin

Autoantibodies

Sample : 2ml

No need to fasting, only

serum

Hemolysis,

lipidemic

, and Icteric samples are not acceptable.

Preference method: RIA

Other methods: ELIZA

, Semi-Quantitative Indirect Fluorescent

Antibody

Clinical applications:

Differentiate T1DM from T2DM, Detection high risk T1DM, care of patients receiving pancreas islet cells transplantation

Slide34

Normal levels

Anti IA

2

: < 7.5 U/ml 

Anti-GAD

65

: < 5 U/ml

Insulin Ab: < 10 U/ml

ICA Ratio Value: < 0.95

ZnT8 Ab: < 15 U/ml

Slide35

Type 2 diabetes

is the most common type of diabetes, affecting

approximately

90% of Americans with diabetes.

This disease is familial, but

the

underlying genetic defects for most of those affected have yet to be

determined

.

Risk factors include overweight (BMI ≥25

kg/m2),

sedentary

lifestyle

, family history of diabetes, advanced age (≥45 years), ethnicity

(

African Americans, Latinos, Native Americans, Asian Americans, and

Pacific

Islanders),

and polycystic ovary disease, as well as history of

gestational

diabetes

or delivery before diabetes of a baby weighing more

than

9 pounds,

hypertension, vascular disease or dyslipidemia

(

HDL-cholesterol ≤35 mg/

dL

[0.90

mmol

/L]

and/or triglyceride level ≥250

mg/

dL

[2.82

mmol

/L]),

HbA1c

of

5.7% or greater, impaired fasting glucose or

impaired

glucose tolerance,

and other conditions associated with insulin

resistance

(i.e., acanthosis

nigricans

).

This is not an autoimmune disease

,

so

antibody testing is not worthwhile.

C-peptide levels are measurable in

type

2 diabetes, with a reduction in beta cell mass over time.

Unlike

undiagnosed

type 1 diabetes, in which patients are usually symptomatic, people with new-onset type 2 diabetes can be free of symptoms.

Slide36

Testing

for diabetes should be considered in

any

overweight or obese adult.

In general, it is recommended that adults

ages

45 and older be screened for diabetes every 3 years

, but screening

should

be performed earlier and more frequently if the individual is at high

risk

. The preferred test is a fasting plasma glucose or

HbA1c level

.

If a

random

plasma glucose level is 160 mg/

dL

(8.9

mmol

/L) or higher, a

FBS, HbA1c

, or 2-hour 75-g OGTT should be

performed.

Slide37

The

American Diabetes Association (ADA, 2010)

recommends

screening children and adolescents, beginning at age 10 or

at

onset of puberty, who are overweight (BMI >85th percentile or weight

>

120% of ideal)

with two of the following risk factors: family history (type

2

diabetes in first- and second-degree relatives), high-risk race/ethnicity

(

Native Americans, African Americans, Hispanic Americans, Asians/South

Pacific

Islanders),

signs of insulin resistance

(acanthosis

nigricans

,

hypertension

, dyslipidemia, polycystic ovary

syndrome),

or maternal history of diabetes or gestational diabetes

during

the child’s gestation.

Slide38

Most people with

type 2 diabetes

are insulin resistant, obese, and have

a

relative or absolute deficiency in insulin secretion.

Inappropriately high

hepatic

glucose production occurs, along with impaired glucose utilization

peripherally

.

Decreased glucose transport can be demonstrated in muscle

and

adipose tissue. For glucose tolerance to remain normal, the pancreas

must

secrete enough insulin. If the pancreas is unable to secrete sufficient

insulin

, impaired glucose tolerance or type 2 diabetes results.

Hyperglycemia

is toxic to beta cell function and further impairs insulin secretion. Over

time

, beta cell failure is usually progressive, and the beta cells produce

lesser

amounts of insulin, contributing to increasing insulin deficiency.

Although

many people with type 2 diabetes can be effectively treated with

diet

, exercise, and oral glycemic control agents, others require insulin

therapy.

Slide39

HbA1c is

defined by the International Federation of Clinical Chemistry

Working

Group on

HbA1c as

the hemoglobin A that is irreversibly

glycosylated

at one or both N-terminal valines of the β-chains of the tetrameric

hemoglobin

molecule, including hemoglobin

that may also (but not solely)

be

glycosylated on lysine residues.

HbA1c testing

provides an index of

average

blood glucose levels over the past 2 to 4 months

.

It has been established that improved glycemic control is associated with

preventing

or delaying the progression of microvascular complications in

diabetes

.

The

Diabetes Control and Complications Trial (DCCT)

demonstrated

that lowering glucose levels in patients with type 1 diabetes

slows

or

prevents the development of retinopathy, neuropathy, and

nephropathy.

A

50% to 75% decrease in complications

was

observed in the intensively treated group, in which an

HbA1c of

7.2%

was

achieved (compared with 9.0% in the conventionally treated group).

Slide40

MEASURES OF GLYCEMIC 

CONTROL

It has been established that improved glycemic control is associated with

preventing

or delaying the progression of microvascular complications in

diabetes

.

The Diabetes Control and Complications Trial (DCCT)

demonstrated

that lowering glucose levels in patients with

type 1 diabetes

slows

or

prevents the development of retinopathy, neuropathy, and

nephropathy.

A 50% to 75% decrease in complications

was

observed in the intensively treated group, in which an HbA1cof 7.2%

was

achieved (compared with 9.0% in the conventionally treated group

).

Reduction

in microvascular complications in type 2 diabetes was reported in the

United

Kingdom Prospective Diabetes Study (UKPDS), as well as in a

smaller

Japanese

study

.

In the UKPDS, microvascular

complications

were decreased by 25% in intensively treated patients by

lowering the

HbA1cfrom

7.9% to 7.0%.

Slide41

Glycosylated hemoglobin (

GHb

) is formed

nonenzymatically

by the

two-step

reaction shown in Figure 16-3.

The first reaction is rapid,

reversible

, and dependent on the ambient glucose concentration

, and it produces

a

labile

aldimine

or Schiff base

. Over time, the

aldimine

slowly undergoes

Amadori

rearrangement and is converted to a stable

ketoamine

,

glycosylated

hemoglobin.

Most HbA1cassays measure this stable

ketoamine

,

not

the

labile product, which is more prone to be influenced by recent dietary

intake.

HbA1c is

defined by the International Federation of Clinical Chemistry

Working

Group on HbA1cas

the hemoglobin A that is irreversibly

glycosylated

at one or both N-terminal valines of the β-chains of the tetrameric

hemoglobin

molecule, including hemoglobin that may also (but not solely)

be

glycosylated on lysine residues.

HbA1ctesting provides an index of

average

blood glucose levels over the past 2 to 4 months.

Slide42

Several

types of certified

methods

are available for measuring hemoglobin A1c: immunoassay, ion-exchange HPLC, electrophoresis,

boronate

affinity HPLC, and enzyme

methods

. Reliable benchtop point-of-care analyzers, such as the one that

uses

a cassette-based immunoassay method, also are available. Most U.S.

laboratories

use a certified method and the College of American

Pathologists

’ proficiency testing program, which utilizes whole blood and

lyophilized samples.

To obtain and retain a “Certificate of Traceability to the DCCT Refer-

ence

Method” in the NGSP, the laboratory must annually satisfy precision

criteria

(CV ≤5%; ≤3% for Level 1 laboratories

).

Level

1

laboratories are usually large and involved in research studies.

Slide43

Slide44

HbA1cassays

vary in reliability in the presence of a variety of factors.

Interference by

carbamylated

hemoglobin can occur with uremia, hyper-

triglyceridemia

, and hyperbilirubinemia, and salicylates can cause

interference

by acetylated species.

Hemoglobinopathies

(

HbSS

,

HbSC

,

HbCC

) associated with high red blood cell turnover and the need for transfusions will adversely affect accuracy, as will chronic alcohol or opiate use, iron deficiency, and lead poisoning.

Vitamins C and E can falsely lower levels by inhibiting glycosylation, but vitamin C can also increase levels for some assays

.

Sample storage effects may occur. Conditions

associated

with shortened red blood cell survival or lower mean red blood

cell

age, such as hemolysis, recovery from acute blood loss, transfusions,

or

splenectomy, will lower the HbA1clevel as the result of reduced exposure

to

plasma glucose.

Slide45

Hyperglycemia

has been associated with a decrease in

erythrocyte

survival, suggesting that

HbA1c levels

in poorly controlled

patients

may underestimate their mean plasma glucose

concentration.

The correlation between HbA1cand estimated average plasma glucose

levels

is shown in Table

16-5.

This is based on data

from

the International A1c Derived Average Glucose trial,

but there is

uncertainty

about the accuracy of this correlation in children and African

Americans

.

There is evidence that the relationship between A1c and mean

blood

glucose may vary in

different racial and ethnic groups due to

differences in hemoglobin

glycation and/or other factors that are not well

understood.

Slide46

For

patients in whom A1c/

eAG

and measured blood glucose appears

to

be discordant, clinicians should consider the possibility of interfering

factors

discussed above.

In the presence of abnormal red cell turnover, such

as

with pregnancy, recent blood loss, or transfusions, only blood glucose

criteria

should be used to diagnose diabetes.

For patients with an abnormal

hemoglobin

but normal red cell turnover,

such as sickle cell trait, an

HbA1c assay

without interference from abnormal hemoglobin should be used.

Slide47

Slide48

The turnover time of serum proteins, primarily albumin, is much shorter

than

that of erythrocytes (14-20 days), so their glycosylation reflects

glycemic

control over narrower periods of time.

The

nonenzymatic

glycation

of

these serum proteins occurs similarly to that of hemoglobin, with

the

formation

of

ketoamine

-linked glucose protein

.

Several methods are

available

for measuring glycosylated proteins or glycosylated albumin, including

affinity

chromatography, immunoassays, and enzymatic methods.

Fructosamine

and glycated albumin

assays may be useful in patients for whom

HbA1c assays

are inaccurate, such as those with

hemoglobinopathies

,

advanced

chronic kidney disease, and hemolytic anemias, but unlike

HbA1c levels

, their clinical utility has not been firmly established

.

The relationship between A1C and

estimated average glucose (

eAG

)

is described by the formula 28.7

⨯

A1C

– 46.7 =

eAG

.

Slide49

Fructosamine

is a compound that is formed by the non-enzymatic reaction between fructose and ammonia or an amine, with a molecule of water being released.

Fructosamines

are also formed when the carbonyl group of glucose reacts with an amino group of a protein. When

fructosamines

are formed from blood proteins such as albumin, they are known as Glycated Serum Protein or Glycated Albumin.

Since albumin has a much shorter half-life than hemoglobin, serum

fructosamine

generally reflects the state of glycemic control for only the preceding 2 weeks

.

The test for serum

fructosamine

is simpler and less costly than that for hemoglobin A1C, but at present is less frequently used. The level of

fructosamine

correlates well with fasting glucose and with hemoglobin A1C

levels.

For several years, there was a home

fructosamine

meter that allowed patients to monitor their own

fructosamine

weekly, but it was taken off the market by the manufacturer because of inaccurate readings.

Slide50

The American Diabetes Association (ADA) recognizes the utility of both tests, and says that

fructosamine

may be

a better choice

when A1C cannot be reliably measured. These situations include:

1)

The evaluation of changes in diabetic treatment

, since the effects of adjustment can be evaluated after a couple of weeks rather

than months

.

2)

In pregnancy, since the glucose and insulin needs of the mother and fetus change rapidly during gestation.

3)

Any condition that affects the average age of red blood cells, such as hemolytic anemia, sickle cell anemia, or blood loss.

Fructosamine

is not affected by such conditions, and may be a better choice for monitoring glucose control.

Slide51

When

is

fructosamine

not a useful test?

Measured

fructosamine

may be falsely low in the setting of decreased protein levels, such as

nephrotic syndrome or hepatic disease

. Further,

because of lack of standardization and concern with reproducibility,

fructosamine

is not recommended for routine use, or as a replacement or supplement for A1C

when the A1C appears to be providing an accurate representation of glycemic control.

Slide52

HbA1C

Specimen: 3 ml EDTA or heparin treated, washed RBC or

hemolysate

Did not need to fasting

, the clot sample is not acceptable

Stable in 4ºC for 7 days and in RT for 24 hours

Reference method:

HPLC

Preferred method:

Ion exchange chromatography (cationic)

Increased level

in Diabetes, Gestational diabetes and non diabetic hyperglycemia (acute stress, Cushing syndrome,

glucagonema

, acromegaly, treatment with corticosteroids).

Decreased level

in hemolytic anemia, chronic bleeding, chronic renal failure

Interference:

Hemoglobinopathies

, changes in life span of RBC, carbamoyl

Hb

in uremia, morphine and propranolol and

HbF

A reasonable A

1C

goal for many

nonpregnant

adults is below 7% (53

mmol

/

mol

)

Slide53

Pancreatic transplantation for patients with type 1 DM

is

a possibility in some referral centers. It is performed most commonly with simultaneous kidney transplantation for end-stage renal disease (ESRD

).

Laboratory Studies

Plasma glucose

Patients with type 1 diabetes mellitus (DM) typically present with symptoms of uncontrolled hyperglycemia (

eg

, polyuria, polydipsia, polyphagia). In such cases, the diagnosis of DM can be confirmed with a random (

nonfasting

)

plasma glucose

concentration of 200 mg/

dL

or a fasting plasma glucose concentration of 126 mg/

dL

(6.99

mmol

/L) or higher.

Slide54

The reference range for nondiabetic people is 6% in most laboratories

.

American Diabetes Association (ADA) guidelines recommend measuring

HbA

1c

at least every 6 months in patients with diabetes who are meeting treatment goals and who have stable glycemic control

. For patients whose therapy has changed or

who are not meeting glycemic goals, the guidelines recommend HbA

1c

testing every 3 months.

HbA

1c

cannot be used as an indicator of glycemic control

in patients with neonatal diabetes mellitus (NDM) because of the high levels of fetal hemoglobin (

HbF

) remaining in the blood

.

Moreover, the overall efficacy of HbA

1c

testing in diabetes diagnosis remains uncertain. A study presented in 2019, using data derived from 9000 adults, reported diabetes diagnosis with the HbA

1c

blood test to be unreliable. The investigators found

evidence that in comparison with the oral glucose tolerance test, HbA

1c

testing would lead to a 42%

overdiagnosis

of glucose tolerance and a 73%

underdiagnosis

of diabetes, in adults.

Slide55

Other laboratory studies

Fructosamine

levels also test for glucose levels.

Fructosamine

is formed by a chemical reaction of glucose with plasma protein and reflects glucose control in the previous 1-3 weeks. This assay, therefore, may show a change in control before HbA

1c

and often is helpful when applying intensive treatment and in short-term clinical trials.

Urine

ketones are not reliable for diagnosing or monitoring diabetic ketoacidosis (DKA), although they may be useful in screening to see whether a hyperglycemic individual may have some degree of

ketonemia

. The plasma acetone level—specifically, the

beta-

hydroxybutyrate

level—is a more reliable indicator of DKA, along with measurement of

plasma bicarbonate or arterial pH as clinically required

Slide56

C-Peptide

Measures for detection the function of pancreas and differentiate T1DM from T2DM, detection the cause of hypoglycemia and detection of

insulinemia

tumor

Measurement of C-peptide in blood needs to at least 8 hours fasting

Increased C-peptide: in Insulin resistance and Cushing syndrome,

Insulinemia

and using high dose of glyburide

Decreased C-peptide in liver disease and severe infection, Addison disease

Interference:

1. Using Insulin or sulfonyl urea drugs, 2. Renal failure (increase C-peptide due to the route of excretion), 3. overweight due to high production of insulin and C-peptide

Slide57

Fructosamine

assays

are

the most widely used to assess short-term

(

3- to 6-week) glycemic control because the average half-life of the

proteins

is 2 to 3 weeks.

These assays have the advantage of using serum

samples

and automated equipment, so they are simple to perform and low

in

cost.

They are more reliable than other glycosylated protein assays

but

can be affected by alterations in serum protein levels that are present

during

acute illnesses and liver disease.

Whether

fructosamine

values

should

be corrected for serum protein or albumin concentrations is

controversial

. The assay should not be performed if the serum albumin level

is

3.0 mg/

dL

or lower.

High uric acid, triglyceride, and bilirubin levels and

the

presence of heparin or hemolysis can also affect the assay

.

Another

approach

under investigation for the assessment of glycemic status is the

measurement

of glycated protein in saliva.

If successful, this may provide

a

truly noninvasive test with vast clinical implications. Neither

HbA1c nor

fructosamine

values capture the magnitude of glycemic

exursions

.

Slide58

One

such

test is

1,5-anhydroglucitol (1,5-AG),

the results of which reflect

postprandial

glycemia

and short-term (

1- to 2-week

) hyperglycemia.

1,5-anhydroglucitol

, a dietary monosaccharide, is filtered in the

glomerulus

and competes with glucose for reabsorption in the renal tubules.

Hyperglycemia decreases 1,5-AG blood concentrations

. Several small

studies

have suggested an association between 1,5-AG and microvascular

complications

.

Abnormal renal function will interfere with the accuracy of

this

test

Slide59

KETONE TESTING

The ketone bodies

β-

hydroxybutyric

acid, acetoacetic acid, and acetone

are

products of fatty acid degradation.

β-

hydroxybutyric

acid and

acetoacetic

acid are normally present in a 1 : 1 ratio at concentrations of 0.5 to

1.0

mmol

/L each.

Ketone testing, using urine or blood, is particularly

important

for individuals with type 1 diabetes mellitus to detect ketosis.

Diabetic ketoacidosis (DKA) is a serious and potentially fatal

hyperglycemic

condition requiring urgent treatment

. It is frequently associated with

nausea

, vomiting, abdominal pain, electrolyte disturbances, and severe

dehydration

.

Type 2 diabetes patients who are poorly controlled,

particularly

in the presence of extreme stress or severe acute illness, can also

develop

DKA. Ketone testing may be useful in pregnancy and in

determining

the cause of hypoglycemic disorders.

Slide60

The

ratio of β-

hydroxybutyric

acid to acetoacetic acid is greatly

increased

in DKA as a result of the altered redox state and elevated levels

of

NADH in the hepatic mitochondria.

The most commonly used strips

and

tablets use sodium nitroprusside (sodium nitroferricyanide) and turn

purple

in the presence of elevated levels of acetoacetic acid

.

Acetone is

detected

in the presence of glycine

.

False-negative results can occur with

old

strips and with strips that have had excessive contact with air and

after

ingestion of large amounts of vitamin C.

False-positive results have

been

observed with the use of sulfhydryl-containing medications such as

captopril

(but not other angiotensin-converting enzyme inhibitors not

containing

the sulfhydryl group), acetylcysteine,

penicillamine

, and

mesna

.

Slide61

β-

hydroxybutyric

acid is not detected by these methods. Because

β-

hydroxybutyric

acid levels fall and acetoacetic acid and acetone levels

rise

during the treatment of DKA, these tests are not useful for the

monitoring

of therapy.

β-

hydroxybutyric

acid can be measured in serum by enzymatic,

electrochemical

, chromatographic, electrophoretic, and colorimetric methods.

Reference intervals of β-

hydroxybutyrate

vary among assay methods, but

concentrations

in healthy individuals who have fasted overnight are

generally

less than 0.5

mmol

/L.

Patients with well-documented DKA (i.e., HCO3 <17, arterial pH <7.3, plasma glucose >250 mg/dl) generally have

β-

hydroxybutyrate

of over 2.0

mmol

/L.

Slide62

For

the monitoring of recovery from DKA in the hospital setting

,

serial

measurements

of serum electrolytes, including bicarbonate with

calculation

of the anion gap, are used commonly.

Serial measurement of

β-

hydroxybutyric

acid provides an additional tool to monitor improvement

in

DKA. This can be especially useful in patients with DKA in the presence

of

a concomitant additional acid-base abnormality interfering with proper

interpretation

of bicarbonate concentrations and the anion gap, such as

seen

in renal failure.

Assessment of ketone levels by breath testing is also

under investigation.