Term
| What are the two most common assays for measurement of total calcium? |
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Definition
Both are photometric assays:
o-Cresolphthalein assay- o-Cresolphthalein reacts with Ca+2 to form a red complex.
Arsenazo III dye- bound Ca is liberated from anions and then reacts with Arsenazo dye to produce a colored complex. If the sample is exposed to air and CO2 evaporates, the pH increases and the measured [Ca] increases. |
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Term
| What methodology is most commonly employed for measurement of free (ionized) calcium? |
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Definition
| calcium selective electrodes via potentiometry |
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Term
| What is the most common assay for measurement of Phosphorus? |
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Definition
Ammonium molybdate reacts with PO4 to form a colored complex that is measured by photometry
**the strongly acidic reagent can precipitate immunoglobulins resulting in positive interference in animals with prominent monoclonal gammopathies |
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Term
| What is the most common clinical assay for measurement of total magnesium? |
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Definition
Metallochromic indicators or dyes change color when they selectively bind to Mg and are measured photometrically.
**atomic absorption spectrometry is the gold standard reference method but not routinely performed. fMg can be measured with ion specific electrodes (like fCa) however the clinical utility of fMg is not well established currently |
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Term
| What are the two most common assays for measurement of urea nitrogen? Any interferances? |
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Definition
Reflectance spectrophotometry (vitros dry reagent slide)- urea is hydrolyzed (by urease) to form NH3 and CO2, NH3 interacts with an indicator to generate a colored dye. Hemolysis causes positive interference.
Kinetic spectrophotometry (Roche wet reagents)- same reaction as vitros slide but NH4 is coupled to another reaction that consumes NADH resulting in decreased absorbance (inverse relationship between BUN and absorbance)
*Flouride inhibits ureas activity to fluoride containing anticoagulants (NaF) cause negative interference for both assays |
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Term
| What are the two most common assays for measurement of creatinine? Interferences? |
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Definition
Jaffe's reaction- creatinine reacts with picric acid to form a colored complex, the rate of this complex formation is measured via spectrophotometer. ***Effect of non-creatinine chromagens is minimized by using a kinetic assay which is the standard method; however, doesn't entirely ameliorate the effect of proteins, so a correction factor assuming normoproteinemia is applied to some assays to account for this positive interference***
enzymatic method with reflectance spectrophotometry (Vitros dry reagent slide)- Creatine is enzymatically hydrolyzed to creatinine resulting in production of H2O2 that generates a colored dye. *Negative bias compared to Jaffe reaction* |
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Term
| What is the gold standard assay for cortisol? |
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Definition
RIA
*ELISAs and chemiliminescent assays are also available |
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Term
| Describe the two major methods for detecting proteinuria? Which protein can be best detected by both methods? Interferences? |
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Definition
reagent pad- has a pH indicator that changes color when binding negatively charged amino groups on proteins
SSA turbidity- proteins are denatured in acid to form a precipitate
**both methods detect albumin better than globulins. Alkaline urine affects test result (proportional positive interference for reagent pad and negative interference for SSA turbidity) |
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Term
| What methods are available to detect bence jones proteins (immunoglobulin light chains- kappa or lambda) in urine? |
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Definition
Preferred method is immunoelectrophoresis (human antibodies cross react with light chain proteins of domestic animals) but can also do regular electrophoresis on urine (bence jones proteins migrate to beta-2 globulin fraction)
Heat test: urine is acidified to 4.9 and heated to 56C (bence jones proteins precipitate) if positive urine is then placed in boiling water bath and observed for dissolution of precipitate. Finally urine is filtered and placed into a new tube and observed for precipitate formation at 60C and then dissolution at 40C.
**also bence jones proteins are detected better on SAA than reagent pad so discordant results on routine UA could suggest their presence |
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Term
| What is the reagent pad method for detection of glucosuria? Other tests? |
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Definition
reagent pad: glucose oxidase converts glucose to gluconic acid and H2O2, H2O2 reacts with an indicator to produce a color change
Positive reactions can be confirmed with copper reduction method: glucose (or other sugars) reduce copper to form cuprous oxide and cuprous hydroxide, cuprous hydroxide interacts with an indicator to produce a color change ***more accurate than reagent pad but less sensitive (higher detection limit)*** |
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Term
| Describe the methodology use to detect ketonuria? |
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Definition
Reagent pad and Acetest tablet use the same method: acetoacetate (mostly) and acetone (less reactive) form colored complexes with nitroprusside.
*Tablet method has a lower detection limit so can be used to confirm trace results on reagent pad. Can also use tablet in blood, plasma and milk (in addition to urine)
***beta-hydroxybutyrate is NOT DETECTED by either method |
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Term
| What methodology is used to detect hemoglobinuria? |
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Definition
reagent pad and hematest tablet use same methodology:
peroxidase activity of heme catalyzes oxidation of either
o-toludine or another chromogen
**methemoglobin and myoglobin also cause a positive reaction. The reaction is more sensitive than the protien method so small amounts of blood will cause marked heme reactions and negative protein reactions |
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Term
| Describe the methodology used to detect bilirubinuria? |
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Definition
Reagent pad and Ictotest use the same method: In a strongly acidic medium bilirubin couples with a diazonium salt to produce a color change
*the ictotest has a lower detection limit and can be used to confirm trace positives on the reagent pad. UV light degrade bilirubin to biliverdin which is not detected by either method so samples should be expediently processed or protected from UV light during storage. |
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Term
| Describe the reagent pad methodology used to detect urobilinogen in urine? |
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Definition
modified Ehrlich's aldehyde reaction (urobilinogen reacts with p-dimethylaminobenzaldehyde to form a red pigment)
*can only detect increased urobilinogen. UV light also degrades urobilinogen (in addition to degrading bilirubin) |
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Term
| What is the methodology used to detect nitrite in urine? |
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Definition
Reagent pad: nitrite reacts with p-arsanilic acid to form a diazonium compound that couples with N-1-naphthyl ethylenediamine to form a pink product
*technically increased with gram negative bacterial infections (can reduce nitrate to nitrite) but significant bacteriuria may not be detected so the test is not very useful |
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Term
| What are the limitations of the leukocyte esterase reagent pad for use in dog and cat urinalysis? |
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Definition
false negatives in dogs and false positives in cats
*Just ignore it |
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Term
| Describe three possible methods for quantitative urine total protein analysis, how do they differ from the reagent pad and SAA turbidity tests for semiquantitative protein evaluation of urine? |
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Definition
In general the quantitative methods are better at identifying the presence of globulins compared to reagent pad and SSA turbidity test.
Trichloroacetic acid method- proteins precipitate in acid Benzethonium chloride
Coomassie brilliant blue- amount of dye binding to amine groups is proportional to the quantity of protein
Species specific immunoassays (detect small amounts of albumin)- ELISA or nephelometric assays for dogs and cats |
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Term
| What is the only method for determining fT4 in dogs? |
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Definition
equilibrium dialysis *non-dialysis methods underestimate fT4 and are unreliable in dogs
**RIA, chemiluminescent immunoassays and ELISA can all be used to measure total T4, T4AA can result in falsely increased TT4 with the RIA |
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Term
| What is the major assay for measurement of Na? Limitations/interferences? |
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Definition
Potentiometry via ion selective electrode is the major assay, can be direct (undiluted) or indirect (highly diluted). The [Na] is measured within the water portion of the sample so the presence of dissolved substances (e.g. proteins, lipids) can result in a negative interference with indirect potentiometry.
*flame photometry has the same issue as indirect potentiometry and is no longer routinely used. |
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Term
| What type of interferences can be observed with respect to chloride specific electrodes (potentiometry)? |
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Definition
Halides (e.g. bromide, iodine) can cause marked positive interference
organic anions (e.g. lactate, ketones) can cause a very mild positive interference
**if using indirect potentiometry lipemia and hyperproteinemia can cause a negative interference due to excessive dilution of H20 portion of the sample |
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Term
| What are the two major assays for determination of total CO2 (as an estimate of HCO3- on routine chemistry)? Any interferences? |
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Definition
Vitros (hitachi and olympus)- sample is placed in an alkaline environment --> most CO2 is converted into HCO3- which reacts with phosphoenolpyruvate and other reactions that ultimately consume NADH. Amount of NADH is measured with reflectance spectrophotometry and corresponds to tCO2.
Beckman- sample is placed in an acidic environment --> most tCO2 converted to gaseous CO2 which diffuses into a HCO3- solution, change in pH of the solution is measured via electrode and corresponds with tCO2 **Lactate dehydrogenase (from muscle if rhabdomyolysis, or RBC if hemolysis) can consume NADH and cause positive intererence. Oxamate can be added to reactions to consume LD and reduce interference |
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Term
What is the major methodology used by automated assays to determine cholesterol concentrations in serum?
Interferences? |
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Definition
enzymatic methods that hydrolyze cholesterol esters, followed by oxidation via cholesterol oxidase to generate hydrogen peroxide which reacts with an indicator dye.
*high bilirubin can cause negative interference |
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Term
What is the major methodology used by automated assays to determine triglyceride concentrations in serum?
Interference? |
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Definition
enzymatic reaction utilizing lipase to liberate glycerol from TG followed by additional reactions resulting in products detected by spectrophotometry.
Centrifugation of a sample can separate out some of the lipid component falsely lowering TG concentration, however, increased turbidity of samples with high TG can also interfere with spectrophotometric assays. |
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Term
Describe the methodology for measurement of nonesterified free fatty acids in serum or EDTA plasma?
Interferences? |
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Definition
enzymatic method, fatty acids are acetylated and then oxidized to form hydrogen peroxide which is linked to a reaction that produces a colored product.
Hemolysis can cause positive interference. Heparinized plasma is not appropriate (heparin stimulates LPL) |
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Term
| What three enzymes are most commonly used for photometric glucose assays? |
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Definition
glucose oxidase (inhibited by NaF tubes)
glucose dehydrogenase
hexokinase
*icterus, hemolysis, and lipemia can all interfere with light transmission |
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Term
| What is the methodology used for nonphotometric glucose assays (fewer photometric and chemical interferences)? |
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Definition
glucose oxidase results in cosumption of O2 and production of H2O2. Some assays measure rate of O2 consumption and others rate of H2O2 production via specific electrodes
**can still be inhibited by NaF anticoagulant, promise also causes a negative interference |
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Term
| What is the assay methodology is most accurate for measurement of fructosamine? |
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Definition
Fructosyl lysine oxidase enzymatic assay- specific for fructosyl lysine, a specific glycosylated amino acid
nitroblue tetrazolium assay- in an alkaline medium fructosamine acts as a reducing agent to generate formazine, which is detected spectrophotometrically. Nonspecific reduction occurs in the first 10 minutes, so measurements are taken after this time period. Results are 3-4x higher than the enzymatic assay, also more affected by hemolysis and lipemia. |
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Term
| What methodology is typically used to measure [cobalamin]? |
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Definition
competitive binding assays (bioassays are much more technically challenging)
*if using a human assay with gastric derived intrinsic factor, the presence of contaminating R protein (binds ingested cobalamin in acid environment of the stomach) can bind inactive cobalamin metabolites resulting in falsely increased result |
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Term
| What is the competitive binding agent in most folate assays? Does this reflect the amount of functional folate metabolite? |
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Definition
beta-lactoglobin (milk folate binder)
**binds primarily N5-methyltetrahydrofolate (so even if there is a functional folate deficiency (e.g. deficiency in tetrahydrofolate due to decreased methionine synthase activity secondary to cobalamin deficiency) serum [folate] may be normal |
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Term
| For serum enzyme assays what methodology is most commonly used? What is the difference between end-point and kinetic assays? |
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Definition
Most are spectrophotometric assays and most are kinetic.
end point- reaction is stopped at a specific time and either quantity of substrate used or quantity of product formed is measured
kinetic- multiple readings are taken during a specified time frame and the rate of the reaction is determined |
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Term
| Which of the ALP isoforms is resistant to levamisole inhibition? |
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Definition
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Term
| Which of the ALP isoforms is heat stable? |
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Definition
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Term
| Which of the ALP isoforms is not precipitated by wheat germ lecithin? |
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Definition
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Term
| What is the preferred method for measurement of amylase? |
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Definition
amyloclastic assay- a dye binds to available starch, the amount of dye binding is inversely proportional to the amylase activity (not affected by normal glucoamylase activity of serum but hard to automate)
Saccharogenic assay (AMS cleaves a starch into glucose and maltose; can be falsely increased due to normal glucoamylase) and chromogenic assays (AMS catalyzes cleavage of a dye bound to a substrate) are also available |
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Term
| How is delta bilirubin measured? |
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Definition
dry chemical method involving modified diazo reaction
**most bilirubin assays utilize conventional spectrophotometry (wet reagent methods) |
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Term
| What methodology is used to measure bile acids most commonly? |
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Definition
spectrophotometric assay that useless 3alpha-hydroxysteroid dehydrogenase.
*RIA and ELISAs are also available and may be more economical in reference lab setting |
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Term
| Describe the general enzymatic method for detection of NH4 |
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Definition
NH4+ alpha-ketoglutarate +NADPH --> glutamate + NADP
**there are also dye binding methods and ion specific electrodes |
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Term
| When measuring L-lactate, which enzyme is used for reference automated assays vs. POC/in-clinic assays? interferences? |
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Definition
Reference assays are usually spectrophotometric and utilize lactate dehydrogenase
POC methods utilize L-lactate oxidase *bromide can cause a negative interference in some assays using lactate oxidase, free Hgb can cause negative interference in these assays (peroxidase activity of heme removes H2O2 generated by L-lactate oxidase reaction) |
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Term
| What are the two most common assays to measure ketone bodies? |
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Definition
quantitative spectrophotometric assays: use betahydroxybutyrase dehydrogenase to measure [BHB] by catalyzing conversion of BHB to acetoacetate
Nitroprusside methods are common and can be used for whole blood, serum, urine, and milk but only detect acetoacetate and acetone (BHB doesn't react)
*there are less common semiquantitative or qualitative POC tests for BHB in urine and milk |
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Term
| What type of vWD may be less accurately assessed via functional vWF assays (botrocetin cofactor assay and vWF collagen binding activity assay)? |
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Definition
| Type 2 vWD (associated with disproportionally decreased large vWF multimers, which are more functional than small multimers) the functional vWF assays will underestimate amount of vWF in animals with type II vWD (german pointers, horses) |
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Term
| What is the main use of immunoelectrophoresis with regards to vWD? |
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Definition
to distinguish between type 1 (all multimers decreased by >50%) and type 2 (disproportionately decreased large multimers) vWD
**otherwise the quantitative ELISA is a better overall test for quantification of vWF |
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