MAACB Prep Flashcards
Name 3 types of potentiometric electrode
1- redox
2- ion selective membrane ( glass and polymer)
3- PCO2
Define precision , repeatability and reproducibility
Precision is the closeness of agreement of independent results , measured under stipulated conditions
Repeatability is within run precision.
Reproducibility is under changed conditions eg time, operator , calibrators
Name 3 enzymes that can be used to measure glucose
Hexokinase
Glucose dehydrogenase
Glucose oxidase
What is the most widely used glucose method with laboratories
Hexokinase coupled with glucose dehydrogenase , measuring NADH
What is the equation for the glucose Hexokinase method
Glucose + MgATP —HK—->G6P + MgADP
G6P + NAD —G6PD—-> 6-phosophogluconolactone + NADH
NADH measured at 340nm
How many umol of glucose is consumed for every umol NADH?
1
How many calibration points and what calibrator is used for the Hexokinase glucose method
3 point cal
Blank plus MCC1 and MCC 2
What is the LOD, LOQ for serum glucose
LOD= 0.14mmol/L with 95%probability LOQ= 0.28mmol/L with CV 20%
What is the LOD and LOQ for urine/csf glucose
LOD= 0.056mmol/L 95%propbability LOQ= 0.056mmol/L 20%CV
What is the linearity of glucose HK Abbott method
0.28 - 44.40mmol/L
What kind of interference does haemolysis cause Glucose HK spectro method ?
Haem concentrations >5g/L causes negative interference
What kind of interference does ict and lip cause glucose HK method ?
Positive interference >55mmol/L
Fasting glucose reference range ?
Adult- 2.8-5.6 mol/L
Describe the glucose oxidase reaction ?
Glucose + O2 —GO—-> gluconlactone + H2O2
H2O2 + chromogenic oxygen acceptor (e.g o-diasidine ) —peroxidase—> chromogen colour + H2O
What is mutarotase used for?
In kits to convert alpha D-glucose to beta D-glucose for use in glucose oxidase polarography reaction - measuring oxygen consumption
Where is albumin synthesised ?
Parenchymal cells of the liver
+ fall in oncotic pressure
- IL6
What is the function of albumin
Non-specific transport protein eg FFA, ca2+,Zn2+, unconjugated bilirubin and many drugs.
Contributes to oncotic pressure
Minor buffer of H+ ions
Possible causes of low albumin by decreased synthesis?
Inadequate nitrogen intake
Malabsorption
Chronic liver disease
Possible cause of low albumin by increased catabolism?
Sepsis
Possible cause of low albumin by redistribution?
Ascites
Oedema
Sepsis
Possible cause of low albumin by increased loss?
Protein-losing enteropathy
Nephrotic syndrome
Loss of plasma eg.burns
How do you convert mg/dl to mol/L?
Mol/L= (mg/dL X 10) / Mw
List 4 hormones increased by stress
Glucocorticoids eg cortisol
Catecholamines eg epinephrine
Growth hormone
Prolactin
List causes of elevated urate
Gout Renal failure Leukaemia Multiple myeloma Toxiemia of pregnancy
List causes of raises bilirubin
Increased hepatocellular damage
Infra and extra hepatic biliary tree obstruction
Haemolytic disease
Neonatal physiological jaundice
List causes of in vitro haemolysis
Incorrect needle size
Incorrect tube size selection (difficult bleed)
Prolonged tourniquet
Vigorous mixing of tubes
List biochemical analytes that change with dehydration
Urea Creatinine Sodium HB Protein ADH
What is the ref method for lactate ?
There isn’t one
What is the reaction for the lactate oxidase method?
L-lactate + O2 —-LOD—-> Pyruvate + H2O2
H2O2 + 4-AA+ H+ ——Peroxidase—> chromogen + 2H2O
548nm
Fluoride oxalate sample
What is precision?
Closeness of agreement between independent results obtained under stipulated conditions.
What is repeatability ?
Closeness of agreement between results of successive measurements under the same conditions (within run precision )
What is reproducibility?
Closeness of agreement between results of measurements performed under changes conditions ( time , operators , calibrators )
What is drift?
Instrument or reagent instability over time
What is accuracy?
Closeness of the agreement between the result of a measurement and the true concentration of an analyte.
What is the limit of blank?
Highest apparent concentration in analyte-free material. 20 rots of sample with no analyte (0 calibrator)
LOB= mean(blank) + 1.645(SD blank)
What is LOD?
Lowest concentration confidently separated from blank 20 rots if verifying /60 if establishing)
LOD = LOB + 1.64( SD low conc sample)
What is LOQ?
Aka functional sensitivity-limit of reporting . Lowest concentration with acceptable reproducibility CV <20%
What is analytical sensitivity?
The smallest quantities that the assay measures
LOB, LOD, LOQ
Define positive likelihood ratio, how is it calculated and how does it influence clinical decision making?
LR+ is the probability that a person with the disease test positive (TP) divided by the probability that a person without the disease test positive (FP)
LR+= sensitivity/1-specificity
LR- = 1-sensitivity/specificity
Applying likelihood ratios can change the probability of a diagnosis eg low enough to rule out disease or high enough to start treatment
Name 4 methods for urinary free cortisol
LC-MS/MS
Beckman coulter -EIA(comp chemilum) 30mins
Roche - ECLIA ( comp electro chemilum IA) 18mins NB:biotin
Abbott -CMIA( comp chemilum Microparticles IA) 30 mins
What do the corticotroph cells of the anterior pituitary gland synthesize and secrete and to which receptor do they bind?
ACTH
Binds to the melanocortin receptor 2. Needs presence of MC2 accessory protein to respond to ACTH.
ACTH acts through the formation of cAMP which facilitates the transfer of cholesterol into the mitochondrial inner membrane for the synthesis of adrenal steroids
What are the series of reactions used for alkaline phosphatase measurement ?
4-nitrophenylphosphate(colourless) + H2O ——Alp , Mg2+—> 4-nitrophenoxide (benzenoid form) + HPO4 ——pH10.3–> 4-nitrophenoxide( yellow , quinod form)
List causes of an elevated serum cholesterol
Familial hypercholesterolemia Cushing’s syndrome Hypothyroidism Pregnancy 2ndary to obstructive liver disease
List sources of alkaline phosphatase and how to differentiate them.
Liver and bone chromosome 1
Intestinal & placental chromosome 2
Alkaline phosphatase isoenzyme electrophoresis - separates different isoforms
Placental is heat labile and may need to be treated to differentiate
List causes of hypovolaemic hyponatraemia with low serum osmolality
Urine Na >20 & UOsm> 450 = renal loss: Addisons , salt losing nephritis, cerebral salt loss
Urine Ana <20 & UOsm >450 = GIT loss, sweat
List causes of euvolaemic hyponatraemia with low serum Osm
UNa >20 & UOsm >100 = SIADH, hypothyroidism, cortisol def
UNa variable & UOsm <100 = severe polydipsia , beer potomania, excess IV fluids
List causes of hypervolaemic hyponatraemia (low serum osmolality)
UNa>20 ARF of CRF
UNa <20 nephrotic cirrhosis CCF
After a water deprivation test what urine osmolality would indicate central (neurogenic ) diabetes insipidis?
<300 after fluid deprivation
>800 after desmopressin
After water deprivatation test what urine osmolality would indicate nephrogenic DI
After Fluid deprivation UOsm <300
After desmopressin UOsm <300
What UOsm results after water deprivation test would indicate primary polydipsia ?
After Fluid deprivation UOsm >800
After desmopressin UOsm >800
After water deprivation test what UOsm results would indicate partial DI or polydipsia?
After Fluid deprivation UOsm 300-800
After desmopressin UOsm <800
What is functionally sensitivity?
Lowest concentration of analyte that can be measured at a specified precision
What is a heterogeneous immunoassay?
Immunoassay formate that uses 2 phases , usually liquid and solid to separate reacted from unreacted components
What is a heterophilic antibody?
Artefact seen in sandwich immunoassays, in which patients endogenous antibody to reagent antibodies links labeled antibody to capture antibody in the absence of antigen. Results in false elevations of analyte.
What is high dose hook effect?
Artefact seen in sandwich immunoassay in which labeled antibody bound in the sandwich decreases in the presence of excess antigen. Results in falsely low assay results
What is prozone phenomenon ?
Apparently lower reactivity or nonreactivity caused by a relative antigen excess
What is osmolality ?
The measurement of the number of moles of particles per kilogram of water
What is osmolarity?
An alternative measure of colligative property that is clinically equivalent to osmolality;
Measurement of the number of moles of particles per litre of water.
How do you calculate osmolarity(mOsm/L)?
Osmolarity(mOsm/L) = 2X Na + glucose + urea
Name 4 measurements that depend on colligative properties
Colloid osmotic pressure(oncotic pressure)
Boiling point elevation
Freezing point depression
Vapor-pressure depression
Why is boiling point elevation measurement not used to determine osmolality?
Because proteins will coagulate, causing gross changes in the sample composition
What kind of thermometer is used to measure freezing point depression ?
A thermistor ( thermal resistor) a semiconductor made from a mixture of oxides of transition metals such as manganese, cobalt, and nickel. Thermistors become better conductors as temperature rises. The conductance or resistance of metals can be related to the temperature and hence to the osmolality
Describe how freezing point depression is measured.
1- sample cooled rapidly by cold block to -5’C
2- vigorous agitation induces the crystallisation process. Once crystals begin to form , additional water molecules are added rapidly to the ice crystals. Heat is released in the freezing process , this raises the Temperature of the sample until freezing stops and equilibrium temperature establishes.
This temp is inversely related to the osmolality of the sample
What is the osmolar gap?
Serum osmolality gap= measured - calculated
The average gap is near zero
What is the ideal osmolality gap for mannitol therapy?
Mannitol is used to treat patients with Edema , especially cerebral edema.
Aim for osmolal gap of 10 - 50 mOsm/L
How do you calculate stool osmolal gap
Stool osmolal gap= measured osmolality (stool) - 2([Na+] + [K+]) stool
Why is stool osmolal gap calculated?
To differentiate causes of chronic diarrhoea
Gap <50mOsm/L = secretion all diarrhoea
>50mOsm/L = Unabsorbed material eg food. Excessive use of laxatives
Measured osm should be similar to plasma . A hypo-osm< 280mOsm/L may be factitious diarrhoea (munchausen)
What is vitamin D2 also known as where is it obtained?
Ergocalciferol
Obtained through diet or supplements
Metabolises to 25(OH) vitaminD2
What is vitamin D3 also known as and where is it obtained?
Cholecalciferol
Mainly produced in skin through UV B exposure
Metabolised to 25(OH) vitamin D3
What are some advantages / challenges of using the enzymatic method for HbA1c
Advantages: no analytical interference from sub variants
Challenge: unable to detect Hb variants
What is the principle of the enzymatic HbA1c method?
Uses an enzyme which specifically cleaves the N-terminal valine.
Lysed RBCs undergo proteolytic digestion to release substrate fructosyl valine
Fructosyl valine + O2 —-FVO+ FADH2–> H2O2 + valine + glucosone
H2O2 + leuco dye —-HPO—> colorimetric result 670nm
What is the principle of the immunoassay for HbA1c?
Uses Abs which target the first 4-10a.a at the N-terminal of the beta chain
List advantage/challenges of using immunoassay for HbA1c
Advantages: no analytical interference from most common Hb variants using newer gen assays
Challenge: unable to detect Hb variants, still susceptible to rare Hb variants
What is the Principle of boron are affinity HPLC for HbA1c?
The structural difference between HbA0 & HbA1c resulted from the presence of the glucose group constitutes the basis for this method. Glycohaemoglobin binds affinity resin while nonglycated Hb passes through the column. M-aminophenyboronic acid binds glucose moiety to affinity resin
Name advantage/challenges of boron are affinity HPLC for HbA1c
Advantages: considered to have the least analytical interference from Hb variants.
Challenge: measures all glycated Hb and cannot detect Hb variants
What is the principle of ion-exchange HPLC for HbA1c measurement?
Uses buffers of increasing ionic strength to separate HbA1c from other Hb species by differences in charge. Concentration of each fraction is quantified by calculating the area under each peak
Name advantages/challenges to using ion-exchange HPLC for HbA1c measurement
Advantage :ability to detect most common Hb variants can give presumptive ID
Challenges: can have interference from Hb variant that co-elite with peaks of interest.
Schiff
Labour intensive
TAT
What is the principle of capillary electrophoresis for HbA1c testing?
Separates by charge and mass. Charged molecules are highly resolved by electrophoretic mobility and their separation depends on electrolyte pH and electro-osmotic flow.
List advantages/challenges to CE for HbA1c
Advantages: high chromatic resolution and resulting ability to detect many Hb variants.
Challenge: throughput
List four methods of HbA1c measurement
Enzymatic Immunoassay Capillary electrophoresis HPLC- ion- exchange HPLC- boronate affinity
Define diagnostic sensitivity
How good a test is at identifying those with disease. True positives
Sensitivity = TP/TP + FN
Define diagnostic specificity
How good at identifying those without disease. True negatives
Spec= TN/TN+FP
List causes of in Vitro haemolysis
Collection into syringe-drawing up blood under pressure
Collection with small needle large tube -vacuum through small hole
Transfer of sample from syringe to tube
Alcohol contamination with capillary collection
Freezing sample
Name reasons for in vivio haemolysis
Hereditary - sickle cell
Acquired (auto immune)
Transfusion reactions , heart valves
What is the reference method for measurement of haemolysis?
Cyanomethaemoglobin
List the mechanisms of haemolysis interference
Addiitive - released from cells( K, LD, AST, Phos, Fe, folate , ammonia)
Spectral - method wavelength around Hb peak (415, 540, 570nm) eg GGT, ALP
Chemical - cross -reactivity of cell analytes and method eg adenylte kinase in RBCs cross reacts with CK, proteolytic enzymes can destroy small peptides eg insulin, glucagon, calcitonin, PTH, ACTH and TnT
Dilutional - gross haemolysis (Na, Cl etc)
List mechanisms of interference for lipids
Spectrophotometric - methods using lower wavelength
Volume displacement - indirect ISE methods dilute the sample enhancing solution effect
Physical - block Ag-Ab complex formation, bind lipophilic compounds eg drugs
What can give a falsely high lipaemic index?
IgM paraproteins
What are the mechanisms of interference for Icteria?
Spectral - interference with Jaffe creatinine method
Chemical - can react with H2O2 - chol, trig, urate, Fe and enzymatic creatinine method
What can be added to remove bilirubin (ictera ) interference?
Ferrycyanide solution destroys bilirubin.
Or alternative method eg chromatography
What areas need to be checked when testing for carryover?
Probe wash process
Curette wash process
Tube components
How do heterophile antibodies give false positives in sandwich immunoassays ?
By forming a bridge between two antibodies as the analyte would.
What is a HAMA?
Human anti-mouse antibody
Name a protein that can behave like a heterophile antibody
Rheumatoid factor
At what protein levels should a direct ISE method be used for na
<40 (pseudo hyper state)
>100g/L ( pseudo hypo state)
What is Rayleigh scattering?
When particle size is significantly smaller than the wavelength. Light is scattered symmetrically in the direction of the incoming light as well as against incoming light.
What is Raleigh-Debye scattering?
When particle size is only a little smaller than , or is equal to the wavelength , asymmetric forward scattering .eg IgM ,chylomicrons
What is Mie scattering?
Asymmetric forward scattering increases significantly when particle size is larger than the wavelength eg RBC, bacteria
Name examples of Atomic absorption spectrometry
Flame atomic absorption spectroscopy
Graphite furnace atomic absorption spectroscopy
Name types of atomic emission spectrometry
Flame atomic emission spectroscopy
ICP -OED ( inductively coupled plasma - optical emission spectroscopy
What kind of sample prep is required for ICPMS?
Blood , urine or plasma - dilute and shoot. Acid -2% nitric acid, base - 1% ammonium , 0.1% EDTA, 0.05% triton x-100
Tissue sample , hair, nail - chemical digestion - concentrated acid HCL, or base TMAH
Name possible sources of error in ICPMS
Memory effects Contamination Matrix effects Isobaric elements Double charged ions Polyatomic ions
