tests to diagnose diabetes mellitus

Question 1

Tests used to diagnose/ monitor diabetes mellitus

Answer 1

fasting plasma glucose ( FPG)
oral glucose tolerance test ( OGTT)
HbA1c
symptoms & family history

Question 2

adults hemoglobin fractions and Hb A subdivisions

Answer 2

Hb A …………97%
Hb A2………. 2.5%
Hb F……………0.5%

Hb A can be subdivided into:
HbA1a
HbA1b
HbA1c

HbA1c is the biggest fraction of the ( 3-6% of total Hb)

Question 3

glycohemoglobins

Answer 3

formed when glucose reacts non-enzymatically with an amino group of hemoglobin 
other names :
HbA1c
glycosylated Hb
glycated Hb 
glycohemoglobin 
Fast Hb

Question 4

HbA1c formation

Answer 4

Hb A has 2 alpha & 2 Beta chains
glucose attaches to each of the beta chains forming HbA1c
this attachment is unstable
after undergoing an Amadori Rearrangement it becomes a stable ketamine

Glucose = N-terminal amino group Aldimine ( Schiff base)

Aldimine ( Schiff base) Ketoamine

Question 5

Amadori rearrangement

Answer 5

the -H from the -OH group next to the C=N moves to the N leaving a stable ketone

Question 6

HbA1c

Answer 6

forms of the lifespan of an RBC
is directly proportional to the glucose concentration in the blood

the amount formed depends on :

• the average concentration of blood glucose
• the RBC lifespan

reflects the blood glucose levels over the previous 2-3 months

used to monitor control of diabetes mellitus

interpretation is based on normal RBC lifespan
- hemolytic disease = reduced HbA1c values

Question 7

Interpretation of HbA1c values

Answer 7

Good test Q

Falsely decreased values in :

Hemolytic disease ( shortened RBC lifespan ) 
-compare values to patients previous values , not reference range 

Recent significant blood loss
- higher fraction of young RBC

falsely increased values in:

Iron- deficiency anemia

Question 8

Interpretation HbA1c sources of error

Answer 8

Hemoglobins variants 
HbF
HbS
HbC
Results may be falsely increased or decreased depending on the method used 

Carbamylated hemoglobin

• formed by the attachment of urea
• large amounts in renal failure ( common in diabetics )

Question 9

HbA1c testing recommendations

Answer 9

Testing should be performed twice a year for patients who are meeting treatment goals & have a stable glycemic control

Testing should be performed quarterly when there has been a change in therapy or when patients are not meeting treatment goals

Target HbA1c value for non- pregnant patients < 7%
Target for pediatrics < 7.5%

Reference range : 4-6 % ***

Question 10

HbA1c in diagnosis & monitoring of diabetes

Answer 10

values > 6.5% are used for diagnosis ***

concentrations between 5.7-6.4% indicate high risk of developing diabetes

good measure for determining the risk pof developing microvascular complications, retinopathy & nephropathy

levels are directly related to the risk of cardiovascular disease in non-diabetic patients as well

Question 11

methods of glycated hemoglobin measurement

Answer 11

Hemolytic reagent is mixed with a small amount of EDTA whole blood to lysed cells and release hemoglobin

2 approaches :

1. Based on charge differences between glycosylated & non- glycosylated hemoglobin
   - cation- exchanged chromatography
   - electrophoresis
   - isoelectric focusing
2. Based on structural characteristics of glycogroups on hemoglobin
   - affinity chromatography
   - immunoassay

Question 12

preferred method for glycated hemoglobin measurement

Answer 12

Affinity Chromatography

• gel columns separate the glycated hemoglobin from non-glycated fraction
• A1c attaches to resin & is eluted from the column using a buffer ( sorbitol)
• absorbance is measured at 415nm

Advantages:

• no interference from non-glycated hemoglobin
• not affected by vacations in temperature
• relatively good precision
• hemoglobin variants produce little affect

Question 13

cation-exchange chromatography - hemoglobin measurement

Answer 13

negatively charged hemoglobins attach to positively charged resin bed

A1c is eluted using a buffer of a specific pH

DISADVANTAGES

• highly temp dependent
• affected by hemoglobinopathies ; Hb F causes false increase in results, Hb S & C causes false decrease in results

Question 14

high performance liquid chromatography

Answer 14

hemoglobin fractions are separated using cation-exchange chromatography

fingerstick sample ( 5 microL)

hemolysis reagent containing borate

incubated at 37 degrees for 30 mins to remove schiff base

sample introduced into auto sampler

3 phosphate buffers of increasing ionic strength are passed through column & detection is performed at 415nm & 690 nm

Question 15

immunoassay

Answer 15

antibodies against amadori product of glucose

measurement by inhibition of latex agglutination

agglutination produces light scattering which is measured as an increase in absorbance

a decrease in light scattering is seen when HbA1c in patients sample competes for antibody on the latex; inhibiting agglutination

Question 16

POCT for HbA1c

Answer 16

based on latex ummunoagglutonation inhibition

total Hb & HbA1c measured
- concentration of total Hb reported as %

glycated Hb F >10% causes false decrease in results

Question 17

capillary electrophoresis ( used in NL)

Answer 17

charged particles are separated by their electrophoretic mobility in an alkaline buffer ( pH 9.4)

hemoglobin fractions are detected by cathodic end of the capillary by absorption spectroscopy

ADVANTAGES
high resolving ability
small sample volume

Question 18

HbA1c specimen collection & storage

Answer 18

no fasting required

EDTA, oxalate or fluoride

whole blood stable at
4 degrees for 1 week ( 7 days in fridge )
-70 degrees for 18 months

type 1 & 2 diabetics who are meeting treatment goals should be monitored at least every 6 months

Reference intervals
4-6% *****
increase with age
slighter higher in African Americans & Hispanics

Values >15% & <4% should be investigated for presence of variant hemoglobin *******

Question 19

Glycated serum protein

Answer 19

nonenzymatic attachment of glucose to amino groups of proteins other than hemoglobin to form ketoamines

Fructosamine - stable ketoamine ****
-used in patients with hemoglobin variants associated with decreased RBC lifespan

• plays no role in diagnosis of diabetes ( can be used for monitoring )
• similar to HbA1c test but measures average blood glucose levels over 2-3 weeks ( vs 2-3 months)

Question 20

Other testing to investigate / monitor diabetes

Answer 20

ketones
microalbumin
c-peptide

these can be tested on both serum & urine samples

Question 21

ketones

Answer 21

3 ketone bodies present in low amounts in the body
acetone (2%)
acetoacetic acid ( 20%)
ß-hydrocybutyric acid ( 78%)

produced in the liver through metabolism of fatty acids

ketone levels increase with carbohydrate levels are low ( ex. diabetes, starvation/fasting, high-fat diets, prolonged vomiting, or glycogen storage disease)

Question 22

ketone bodies in uncontrolled diabetes

Answer 22

low insulin levels lead to breakdown of fat & decreased reesterification
- results in increased free fatty acids in plasma

increased counterregulatory hormones also increase the breakdown of fats & production of ketones
- leads to acetoacetate accumulation in the blood

in healthy person all=most all serum ketones consist of ß- hydroxybutrate & acetoacetate

in uncontrolled diabetes, high NADH concentration favours ß- hydroxybutrate production causing elevated levels in serum

Question 23

conditions causing ketones

specimen

Answer 23

ketonemia - accumulation of ketones in the blood
ketonuria- accumulation of ketones in the urine

excessive production of ketones occurs when:
decreased availability of carbohydrates
- starvation, frequent vomiting

decreased use of carbohydrates
- diabetes mellitus, glycogen storage disease, alkalosis

specimen
fresh serum or urine
tightly stoppered & analyzed immediately

Question 24

measuring ketones in blood vs urine

when should type 1 diabetics test for ketones

Answer 24

blood ketones in the blood is more accurate than the urine, urine is used to monitor type1 due to convenience

type 1 diabetics should test fro ketones when:
acute stress or illness
consistently high blood glucose levels ( >16.7mmol/L)
pregnancy
symptoms of ketoacidosis

Question 25

measurement of ketones

Answer 25

Nitroprusside method - actoacetic acid - used with urine reagent stir tests & acetest tablets enzymatic method - ß-hydrocybutrate - used in automated instruments

Question 26

microalbumin/albuminuria

Answer 26

can aid in early diagnosis of diabetic renal nephropathy ``` over time (7-10+ yrs) increased glomerular capillary permeability allows small amounts of albumin to pass in urine - if detected early enough kidney failure can be prevented ``` an albumin- creatinine ration ( ACR) is done at least yearly on diabetic patients. Persistent elevations are indicator of diabetic kidney disease

Question 27

albuminuria

Answer 27

renal damage is common in patents with diabetes mellitus ~1/3 of type 1 patients will develop end-stage renal disease patients with persistent proteinuria have overt nephropathy - albumin excretion rate (AER) of >300mg/24hr - usually on ly seen with long standing disease - real function deteriorates rapidly - treatment can slow progression but not stop or reverse damage before nephropathy occurs, there is a period of increased AER in the range of 30-300 mg/24 hrs that will not be picked up by routine dipstick termed MICROALBUMINURIA

Question 28

albuminuria - specimen collection

Answer 28

albumin to creatine ration ( ACR) can be measured to correct the variation in urine flow rate - albumin excretion rate ( AER) is increase by things like : exercise within 24hrs , posture, infection, fever, hypertension ``` Acceptable specimens - 24hr collection -overnight (8-12 hrs, timed) -1-2 hour timed specimen first morning*****( best, less within -person variation) ``` at least 3 specimens collected on different fays - high within-subject biological variation - diurinal variation ( 50-100% ) higher during the day specimen stood at 4 degrees untreated urine is stable for 1 week at 4 degrees & 5 months at -80 degrees

Question 29

albuminuria- semiquantitative analysis

Answer 29

test strips used to determine if albumin is positive using a predetermined concentration recommended for screening false negs may occur with dilute urine specimens should reach at least 10 degrees before performing analysis

Question 30

albuniuria reference ranges

Answer 30

albumin creatinine ratio-what we use in lab (mg/mmol) normal : <3.5 high: 3.5-30 very high/ overt nephropathy >30 the albumin excretion rate (mg/24hrs) ranges are x10 each of these values

Question 31

C-peptide

Answer 31

a short chain of amino acids that are released into the roof during the formation of insulin by the pancreas mainly analyzed to evaluate th because of hypoglycaemia healthy individual : 0.25-0.6 nmol/L

Question 32

self monitoring blood glucose

Answer 32

blood glucose is the preferred method of assessing glycemic control ( not urine) portable glucose meters are used : at patient bedside in hospital in physicians offices by patients ( or caregivers) at their own home patients modify their insulin doses based in the results type 1 diabetics - 1-3 times daily urine ketone testing -type 1 & gestational diabetes

Question 33

glucose meters

Answer 33

use glucose oxidase or glucose dehydrogenase enzyme catalyzed reaction test strip is instead into meter, drop of blood added from finger stick results appear on digital display screen in 5-45 seconds methods of analysis - reflectance photometry - electrochemistry

Question 34

Glucose meters- methods of analysis

Answer 34

reflectance photometry - measures amount of light reflected from the test pad containing reagent electrochemical ( accuchek) - an electrode is incorporated into test strip - glucose dehydrogenase enzymatic reaction produces a flow of electrons - current produces is directly proportional to the amount of glucose in sample - current converted to digital readout whole blood is ~10-12% low than plasma/serum conc - some meters are calibrated to report plasma glucose values using a factor of 1.11x

Question 35

disadvantages of glucose meters

Answer 35

operator errors have been minimized by - system is sorted if testing volume is too small - simplified quality control - increased memory to store glucose readings factors affecting accuracy & precision - used variability - hematocrit - anemia ( false increase) - polycythemia ( false decrease) - defective regent strips or instrument malfunction - changes in altitude, temp & humidity - hypotension - hypoxia - high triglyceride levels blood glucose meters are unreliable at very high & very low glucose concentrations : <3.3mmol/L & >28mmol/L **** patients who are dehydrated will have increased blood viscosity, causing inaccurately low blood glucose level

Question 36

alternatives to blood glucose meters

Answer 36

glucose meters can be painful & inconvenient implanted biosensors that are enzyme based, electrodes or fluorescence can be used -most widely studied method is the subcutaneous implant of an electrochemical sensor glucose oxidase is used to measure glucose every 1- 5 mins with results being sent to monitor sensors need to be calibrated when implanted & at least twice a day - new sensors have been developed to eliminate calibration by used benefits : - improved long term glycemic control through continuous monitoring - reduced rate of hypoglycaemia - improved HbA1c

Question 37

diagnosis of diabetes mellitus in the lab

Answer 37

screening test such as immunologic markers, HLA typing & insulin secretion have been developed they are only used for research purposes diagnosis is made through - blood glucose - HbA1c other tests analyzed in the clinical lab : - OGTT - Ketones - C-peptide - insulin analysis

Question 38

tests for management for diabetes mellitus

Answer 38

acute conditions : diabetic ketosis hypersmolar nonketotic coma ( type 2 patients) hypoglycaemia lab tests conducted : glucose ( blood & urine) ketones ( blood & urine ) acid-base status ( pH) - checks in in ketoacidosis lactate electrolytes, osmolality ( abnormal during dehydration )

Question 39

tests to detect & monitor long term complications of diabetes

Answer 39

glucose ( fasting& random ) HbA1c Albumin urine protein ( shouldn't be protein in urine ) Creatinine( something otg w/ kidneys), cholesterol , triglycerides C-peptides, insulin ( determine success of pancreas transplant)

Question 40

Hypoglycaemia

Answer 40

``` decrease in blood glucose when levels drop between 2.8-3.1mmol/L we see the following - hunger -sweating - nausea / vomiting -dizziness -nervousness/ shaking -blurred speech / vision -mental confusion ```

Question 41

Hypoglycaemia casuses

Answer 41

hormonal : excess insulin ( ß-cell tumor- insulinoma) - decreased growth hormone -decreased ACTH - decreased adrenal steroids ( cortisone, cortisol) Hepatic : decreased liver glycogen - fasting, starvation, liver damage, drug toxicity Glycogen Storage diseases - von gierke's ( deficiency of glucose- 6- phosphatase enzyme)

Question 42

Insulinoma

Answer 42

pancreatic ß-cell tumor ( inc. insulin= dec. glucose ) - decreased plasma glucose - extremely elevated insulin levels

Question 43

carbohydrate metabolism defects

Answer 43

genetic defects in carbohydrate metabolism occur die to deficiency of a necessary enzyme examples: Von Gierke Disease Galactosemia/Galactosuria Fructosuria

Question 44

Von Gierke Disease

Answer 44

most common congenital ( from birth ) glycogen storage disease also called glucose- 6-phosphotase deficiency type 1 glycogen is unable to be converted back to glucose & accumulates in the liver - causes hepatomegaly managed by controlling blood glucose levels so that they don't go into the hypoglycaemic range

Question 45

Galactosemia / Galactosuria

Answer 45

deficiency of 1 of 3 enzymes involved in galactose metabolism - most common is Galactose- 1-phosphate uridyltransferase ** ``` galactose accumulates in the serum ( galactosemia ) & in the urine ( galactosuria) effects: hepatomegaly splenomegaly cirrhosis of liver cataracts mental defects ``` managed by removing galactose from diet to prevent irreversible complications urine testing - urine dipstick - negative for glucose - clinitest - positive for reducing sugar ( galactose )

Question 46

Fructosuria

Answer 46

deficiency of fructose-1 - phosphate - aldolase ***** fructose- 1-phosphate accumulates in the serum & urine ``` effects : gastropathies enteropathies neuropathies mental defects ``` urine testing - urine dipstick - negative for glucose - clinitest -postive ( fructose )