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chemistry > tests to diagnose diabetes mellitus > Flashcards

tests to diagnose diabetes mellitus Flashcards

1
Q

Tests used to diagnose/ monitor diabetes mellitus

A

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

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2
Q

adults hemoglobin fractions and Hb A subdivisions

A

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)

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3
Q

glycohemoglobins

A 
formed when glucose reacts non-enzymatically with an amino group of hemoglobin 
other names :
HbA1c
glycosylated Hb
glycated Hb 
glycohemoglobin 
Fast Hb
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4
Q

HbA1c formation

A

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

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5
Q

Amadori rearrangement

A

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

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6
Q

HbA1c

A

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

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7
Q

Interpretation of HbA1c values

A

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

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8
Q

Interpretation HbA1c sources of error

A 
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 )
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9
Q

HbA1c testing recommendations

A

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 % ***

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10
Q

HbA1c in diagnosis & monitoring of diabetes

A

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

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11
Q

methods of glycated hemoglobin measurement

A

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
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12
Q

preferred method for glycated hemoglobin measurement

A

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
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13
Q

cation-exchange chromatography - hemoglobin measurement

A

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
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14
Q

high performance liquid chromatography

A

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

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15
Q

immunoassay

A

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

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16
Q

POCT for HbA1c

A

based on latex ummunoagglutonation inhibition

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

glycated Hb F >10% causes false decrease in results

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17
Q

capillary electrophoresis ( used in NL)

A

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

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18
Q

HbA1c specimen collection & storage

A

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 *******

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19
Q

Glycated serum protein

A

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)
20
Q

Other testing to investigate / monitor diabetes

A

ketones
microalbumin
c-peptide

these can be tested on both serum & urine samples

21
Q

ketones

A

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)

22
Q

ketone bodies in uncontrolled diabetes

A

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

23
Q

conditions causing ketones

specimen

A

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

24
Q

measuring ketones in blood vs urine

when should type 1 diabetics test for ketones

A

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

25
Q

measurement of ketones

A

Nitroprusside method

  • actoacetic acid
  • used with urine reagent stir tests & acetest tablets

enzymatic method

  • ß-hydrocybutrate
  • used in automated instruments
26
Q

microalbumin/albuminuria

A

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

27
Q

albuminuria

A

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

28
Q

albuminuria - specimen collection

A

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

29
Q

albuminuria- semiquantitative analysis

A

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

30
Q

albuniuria reference ranges

A

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

31
Q

C-peptide

A

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

32
Q

self monitoring blood glucose

A

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

33
Q

glucose meters

A

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
34
Q

Glucose meters- methods of analysis

A

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

35
Q

disadvantages of glucose meters

A

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

36
Q

alternatives to blood glucose meters

A

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
37
Q

diagnosis of diabetes mellitus in the lab

A

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
38
Q

tests for management for diabetes mellitus

A

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 )

39
Q

tests to detect & monitor long term complications of diabetes

A

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)

40
Q

Hypoglycaemia

A 
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
41
Q

Hypoglycaemia casuses

A

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)

42
Q

Insulinoma

A

pancreatic ß-cell tumor ( inc. insulin= dec. glucose )

  • decreased plasma glucose
  • extremely elevated insulin levels
43
Q

carbohydrate metabolism defects

A

genetic defects in carbohydrate metabolism occur die to deficiency of a necessary enzyme

examples:
Von Gierke Disease
Galactosemia/Galactosuria
Fructosuria

44
Q

Von Gierke Disease

A

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

45
Q

Galactosemia / Galactosuria

A

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 )
46
Q

Fructosuria

A

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 )

chemistry (13 decks)

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