tests to diagnose diabetes mellitus Flashcards
Tests used to diagnose/ monitor diabetes mellitus
fasting plasma glucose ( FPG)
oral glucose tolerance test ( OGTT)
HbA1c
symptoms & family history
adults hemoglobin fractions and Hb A subdivisions
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)
glycohemoglobins
formed when glucose reacts non-enzymatically with an amino group of hemoglobin other names : HbA1c glycosylated Hb glycated Hb glycohemoglobin Fast Hb
HbA1c formation
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
Amadori rearrangement
the -H from the -OH group next to the C=N moves to the N leaving a stable ketone
HbA1c
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
Interpretation of HbA1c values
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
Interpretation HbA1c sources of error
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 )
HbA1c testing recommendations
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 % ***
HbA1c in diagnosis & monitoring of diabetes
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
methods of glycated hemoglobin measurement
Hemolytic reagent is mixed with a small amount of EDTA whole blood to lysed cells and release hemoglobin
2 approaches :
- Based on charge differences between glycosylated & non- glycosylated hemoglobin
- cation- exchanged chromatography
- electrophoresis
- isoelectric focusing - Based on structural characteristics of glycogroups on hemoglobin
- affinity chromatography
- immunoassay
preferred method for glycated hemoglobin measurement
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
cation-exchange chromatography - hemoglobin measurement
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
high performance liquid chromatography
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
immunoassay
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
POCT for HbA1c
based on latex ummunoagglutonation inhibition
total Hb & HbA1c measured
- concentration of total Hb reported as %
glycated Hb F >10% causes false decrease in results
capillary electrophoresis ( used in NL)
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
HbA1c specimen collection & storage
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 *******
Glycated serum protein
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)
Other testing to investigate / monitor diabetes
ketones
microalbumin
c-peptide
these can be tested on both serum & urine samples
ketones
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)
ketone bodies in uncontrolled diabetes
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
conditions causing ketones
specimen
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
measuring ketones in blood vs urine
when should type 1 diabetics test for ketones
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
measurement of ketones
Nitroprusside method
- actoacetic acid
- used with urine reagent stir tests & acetest tablets
enzymatic method
- ß-hydrocybutrate
- used in automated instruments
microalbumin/albuminuria
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
albuminuria
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
albuminuria - specimen collection
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
albuminuria- semiquantitative analysis
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
albuniuria reference ranges
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
C-peptide
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
self monitoring blood glucose
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
glucose meters
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
Glucose meters- methods of analysis
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
disadvantages of glucose meters
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
alternatives to blood glucose meters
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
diagnosis of diabetes mellitus in the lab
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
tests for management for diabetes mellitus
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 )
tests to detect & monitor long term complications of diabetes
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)
Hypoglycaemia
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
Hypoglycaemia casuses
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)
Insulinoma
pancreatic ß-cell tumor ( inc. insulin= dec. glucose )
- decreased plasma glucose
- extremely elevated insulin levels
carbohydrate metabolism defects
genetic defects in carbohydrate metabolism occur die to deficiency of a necessary enzyme
examples:
Von Gierke Disease
Galactosemia/Galactosuria
Fructosuria
Von Gierke Disease
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
Galactosemia / Galactosuria
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 )
Fructosuria
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 )
