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RCPA Part 1 > Analytical > Flashcards

Analytical Flashcards

1
Q

What is the gold standard method for separating MacroPrl and Prolactin?

A

Gel filtration chromatography

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2
Q

What are the mechanisms by which lipaemia causes interference? (name 4)

A
  1. Spectrophotometric interference (light absorption and light scattering)
  2. Volume depletion effect (e.g. pseudohyponatreamia)
  3. Partitioning of the sample
  4. Physicochemical mechanisms (e.g., disturbance of the electrophoretic pattern).
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3
Q

Methods for removing lipids from a lipaemic sample?

A
  • Ultracentrifugation or high speed centrifugation
  • Lipid clearing agents such as Lipoclear
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4
Q

Reaction catalysed by CK used in enzymatic method?

A

Creatine phosphate + ADP –> Creatine + ATP

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5
Q

In the enzymatic spectrophotometric method of bicarbonate measurement, PEPC catalyses the reaction of HCO3- + PEP to form ___ ?

A

Oxaloacetic acid

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6
Q

The enzyme PEPC is involved in the spectrophotometric method of bicarbonate measurement. What does PEPC stand for?

A

Phosphoenolpyruvate carboxylase

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7
Q

Approximately how much CO2 is lost from uncapped tubes per hour?

A

4mmol/L

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8
Q

What can interfere with the paracetamol enzymatic assay?

A

N-acetylcysteine
Salicyclic acid

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9
Q

What is the method used for paracetamol measurement?

A

Enzymatic/colourimetric using aryl acylamidiase

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10
Q

What issue can occur with creatinine measurement in the setting of paracetamol OD?

A

Therapeutic concentrations of NAC can interfere with the Cr enzymatic method

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11
Q

Method for ethanol measurement?

A

Enzymatic method using alcohol dehydrogenase

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12
Q

In scanning spectroscopy in investigating for CSF bilirubin, at what wavelengths do the peaks for oxyhaemoglobin and bilirubin occur?

A

Oxyhaemoglobin peak 415nm
bilirubin peak 476nm

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13
Q

What are the different fractions of bilirubin

A

Unconjugated (alpha)
Conjugated: mono (beta) and di (gamma)
Delta bilirubin: covalently bound to albumin

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14
Q

Reference method for bilirubin measurement

A

Modified Jendrassik and Grof
- Caffeine-benzoate-acetate accelerator

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15
Q

Name 6 methods for bilirubin analysis

A
  • Chemical (diazo reaction)
  • Direct spectrophotometry
  • Enzymatic (oxidation)
  • HPLC
  • Scanning spectrophotometry (CSF)
  • Reflectance (transcutaenous bilirubin)
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16
Q

What can cause interference in Diazo reaction for bilirubin measurement

A

Haemolysis
Lipaemia
Paraproteinaemia

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17
Q

What is the pKa of bicarbonate?

A

6.1

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18
Q

What will happen to the pH of sample if the specimen is left uncapped?

A

pH will increase
Loss of CO2 from sample into room air

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19
Q

What is the difference between actual and standard bicarbonate measurements on blood gas machine?

A

Actual bicarbonate is a derived measurement calculated from pH and pCO2 on anaerobically collected sample.
Standard bicarbonate indicates bicarbonate level at 37C and pCO2 of 40mmHg

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20
Q

Osmolality is a _______ property of solutions that depends on the number of dissolved particles present in the solution

A

Colligative

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21
Q

As the number of dissolved particles in a solution increases, the freezing point and vapour pressure of a solution will ________

A

decrease

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22
Q

Formula for calculating urine osmolality

A

= 2 x (Na + K) + urea

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23
Q

Interferences in osmolality measurement

A

Citrate can increased measured osmolality
Any particular matter (e.g. microclots in serum/plasma)

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24
Q

Interferences in analytical measurement of iron?

A

Chelators (e.g. EDTA, citrate)

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25
Q

What is osmolality?

A

The concentration of a solution expressed as the total number of solute particles per litre.

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26
Q

What are colligative properties?

A

The physical changes that result from adding solute to a solvent.

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27
Q

The Jaffe method involves Creatining reacting with ________ in alkaline medium to produce an orange-red complex?

A

Picrate

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28
Q

Sources of interference in the Kinetic Jaffe method?

A

Ketoacids
Cephalosporins
Bilirubin - negative interference

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29
Q

The kinetic Jaffe method measures creatinine-picrate at what wavelength?

A

500nm

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30
Q

What enzymes are involved in the enzymatic method for Creatinine (Abbott Alinity)?

A
  1. Creatininase
  2. Creatinase
  3. Sarcosine oxidase
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31
Q

In the enzymatic method for creatinine, detection of ______ is measured at 548nm?

A

H2O2

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32
Q

Causes of positive inteference in enzymatic assay for Creatinine?

A

Lidocaine
Creatine supplements

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33
Q

Causes of false low results of Creatinine with Alinity enzymatic assay?

A

N-acetyl-L-cysteine
Alpha-methyldopa

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34
Q

Interferences in Jaffe method?

A

Positive bias from:
alpha-ketoacids
Cephalosporins
Ascorbic acid
Glucose
Glutathione
Uric acids

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35
Q

Reference method for Creatinine

A

Isotope Dilution Mass Spectometry

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36
Q

What is Stokes shift? (Fluorometry)

A

The difference between the maximum wavelength of the excitation light and the maximum wavelength of the emitted fluorescence

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37
Q

Do faecal elastase assays detect the porcine elastase used in supplements?

A

No

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38
Q

What is the type of test used for FOBT?

A

Immunochemical test for human globin
(iFOB uses turbidimetry)

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39
Q

Components of a Fluorometer

A
  1. Excitation source
  2. An excitation monochromator
  3. Cuvet
  4. Emission monochomator
  5. Detector
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40
Q

Excitation sources that can be used in Fluorometers

A

Xenon lamp
Lasers
Light-emitting diode (LEDs)

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41
Q

The absorption spectra of most fluorescent compounds of interest are in the spectral region of…

A

300-700nm

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42
Q

Types of photodetectors used in Fluorometry

A

Photomultiplier tubes (PMT)
Charge coupled detectors (CCD)

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43
Q

Patient preparation for a Hydrogen/Methane test?

A
  • No antibiotics for 4 weeks prior
  • Avoid fermentable carbohydrates day prior
  • Overnight fast
  • No smoking
  • Oral hygiene (if poor may influence results)
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44
Q

How are Hydrogen and Methane analysed following a breath test?

A

chromatographic separation of gases on
molecular sieve column

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45
Q

Gold standard test for determining Small Intestinal Bacterial Overgrowth (SIBO)

A

Culture of intestinal fluid

Breath tests are surrogate markers

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46
Q

Causes of false negatives in a urea breath test for H. pylori

A

PPIs (past 2 weeks)
Antibiotics (past 4 weeks)
Bismuth therapy (past 4 weeks)
Active peptic ulcer bleeding

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47
Q

Recommended method for metanephrine measurement

A

LCMS

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48
Q

Pre-analytical factors influencing metanephrine results

A
  • Position (lower reading with supine)
  • Recent intake catecholamine rich foods (banana, pineapple, walnuts) - increase in 3MT
  • coffee - increase normet
  • exercise - can increase normet, met
  • medications e.g. antidepressants, sympathomimetics
  • stress, critical illness
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49
Q

Method used in NIPT

A

Next generation sequencing

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50
Q

Difference between Rayleigh and Raman light scattering

A

Rayleigh scattering occurs with no change in wavelength
Raman scattering occurs with lengthening of a wavelength

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51
Q

How is phosphorescence different to fluorescence?

A
  • results from relaxatin of molecules in an excited triplet electronic state
  • decay time is longer
  • shows a larger shift in emission light wavelength
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52
Q

What does MLPA stand for

A

Multiplex Ligation dependent Probe Amplification

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53
Q

6 steps of MLPA

A
  1. Denaturation
  2. Hybridisation
  3. Ligation
  4. Amplification by PCR
  5. Fragment separation
  6. Data analysis
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54
Q

Genetic analysis technique commonly used for imprinting syndromes (e.g. Angelman, Prader-willi)

A

MS-MLPA

Methylation specific MLPA

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55
Q

A key difference between 2nd and 3rd generation PTH assays is that 2nd gen assays are affected by cross reactivity with … ?

A

C-terminal fragments

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56
Q

Components of Atomic Absorption Spectrophotometer (flame)

A
  1. Hollow cathode lamp
  2. Chopper
  3. Flame
  4. Monochromator
  5. Detector
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57
Q

Principle of AAS

A

Each element absorbs light of a specific (unique) wavelength

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58
Q

What is HbA1c

A

The fraction of haemoglobin where a glucose has been nonenzymatically attached to the N-terminal valine of the beta chain

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59
Q

In agarose gel electrophoresis, most proteins migrate towards the …

A

+ve anode

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60
Q

In capillary zone electrophoresis, proteins migrate towards the…

A

cathode (-ve)

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61
Q

Order of detection of proteins in CZE

A

gamma; B-2; B-1; a2; a1; albumin

opposite to gel electrophoresis

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62
Q

Conditions other than myeloma/MGUS that can produce monoclonal band on EPP?

A
  • Plasmacytoma
  • Lymphoma (including Waldenström’s
    macroglobulinaemia)
  • Amyloidosis
  • Cryoglobulinaemia
  • Chronic lymphocytic leukaemia
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63
Q

Changes seen on electrophoresis with nephrotic syndrome?

A

Hypoalbuminaemia
Lipoproteins
Alpha2 macroglobulin
Hypogamma

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64
Q

How does glucose concentration differ in whole blood, plasma and serum?

A

Whole blood <plasma < Serum

(serum 2-5% higher than plasma due to fluid shifts from erythrocytes to plasma because of anticoagulants)

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65
Q

Requirements (definition) of a high sensitivity troponin assay

A
  • CV <10% at 99th percentile URL
  • detectable cTN in at least 50% of “normal” male and female patient populations
  • LoQ <20% CV

Rerpoted in ng/L

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66
Q

What does FTIR stand for?

A

Fourier Transform Infra-Red Spectrometer

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67
Q

Components of HPLC

A
  • Solvents
  • Pump
  • Column
  • Sample injector
  • Detector
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68
Q

Reverse phase chromatography has a stationary phase that is …

A

Non polar

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69
Q

Commonly used solvents in reverse phase chromatography

A

Water
Methanol
Acetonitrile

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70
Q

What is used as the stationary phase in ion exchange chromatography

A

Resin

Mobile phase: aqueous soln of salt + buffer

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71
Q

What methods are used in the lab to purify water?

A
  • Filtration
  • Reverse osmosis
  • Deionisation
  • UV oxidation
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72
Q

Water that is fit for most laboratory purposes is termed

A

Clinical Laboratory Reagent Water (CLRW)

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73
Q

Specifications of CLRW

A
  • Microbiological content <10 conlony forming units/mL
  • Resistance at 25degC >10 MOhm
  • Particulate matter (water passed through 0.2 um filter)
  • Total organic content **<500 ng/g
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74
Q

In the enzymatic method of total bile acids, the rate of formation of __ ? __ is determined by measuring the change of absorbance at 405nm as bile acids are oxidised by 3-a-HSD.

A

Thio-NADH

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75
Q

Interferences in the enzymatic method for bile acids?

A
  • Haemolysis
  • Lipaemia
  • Ursodeoxycholic acid (UDCA) – patients on this therapy are not suitable for analysis.
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76
Q

The direct method for bilirubin measures which fraction of bilirubin?

A

Conjugated

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77
Q

In the direct diazo method, bilirubin couples with a diazonium salt in the presence of saulfamic acid to form the coloured compound…

A

Azobilirubin

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78
Q

The diazo method for bilirubin measures the increase in absorbance due to azobilirubin at what wavelength?

A

548nm

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79
Q

What is used as the accelerator in the Jendrassik-Grof method for bilirubin measurement?

A

Caffeine

Modification of this method includes the addition of surfactant

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80
Q

Reference method for ALP uses what as the substrate?

A

4-nitrophenyl phosphate

Kinetic spectrophotometry
Buffer = AMP at 37C

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81
Q

Most heat stable form of ALP

A

Placental ALP

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82
Q

Methods of measuring bone specific ALP

A
  • Colourimetric assays + pre treatment to select BALP
  • Immunoassays
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83
Q

Misleading causes of NAGMA

A
  • Bias and imprecision in Na+, K+, Cl-, HCO3-
  • low albumin
  • high cations e.g. Ca2+, monoclonal proteins
  • Interference with chloride electrode - e.g. bromide
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84
Q

Measurement issues with urine bicarbonate

A
  • rapidly lost to the air in the form of CO2
  • concentration too low to measure on enzymatic automated analysers
  • frequently done on blood gas analysers - ?validation
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85
Q

The Friedewald equation is inaccurate when:

A
  • when TG are high
  • when LDL is low
  • in patients with type 3 hyperlipoproteinaemia
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86
Q

In a K+ ISE, potassium selective liquid membrane uses…

A

Valinomycin

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87
Q

The calcium ISE is a liquid membrane electrode. It uses an ion-selective carrier such as..

A

Dioctylphenyl phosphonate

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88
Q

Reference electrode commonly used in pH electrode

A

Ag/AgCl immersed in KCl

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89
Q

Ion selective electrodes can be classified into 4 main types:

Based on selective membrane used

A
  • Glass
  • Solid state/crystalline
  • Liquid ion-exchange
  • Enzyme
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90
Q

In a pO2 electrode, the anode consists of..

A

Ag rod coated with AgCl

Oxidatio of Ag occurs

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91
Q

In a pO2 electrode, the cathode consists of…

A

Platinum wire encased in glass

Reduction of O2 occurs here

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92
Q

What is the role of the platinum black catalyst in the pO2 electrode?

A

Conversion of H2O2 to O2 for reduction at the cathode

H2O2 is produced from O2 not completely reduced.

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93
Q

In blood gas measurement of glucose/lactate, what occurs in the middle enzyme layer?

A

Production of H2O2

Glucose + O2 -> Gluconic acid + H2O2
Lactate + O2 -> Pyruvate + H2O2

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94
Q

Lipaemic index is measured at what wavelengths

A

660/700nm

At which there is only a small influence by the haemolysis + icterus

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95
Q

Haemolysis index is measured at what wavelengths

A

570/600nm

To repress the influence of the icteric content as much as possible

96
Q

Icteric content is measured at what wavelength

A

480 / 550 nm

97
Q

Analytes which are higher in serum than plasma

A
  • Lactate
  • Troponin
  • K+
  • P
  • Glucose
  • ALP
  • Albumin
98
Q

Analytes which are higher in plasma than serum

A
  • LDH
  • Protein (due to Fibrinogen)
99
Q

Lipaemic samples can result in falsely low values due to increased turbidity. Which analytes?

A

ceruloplasmin, prealbumin, transferrin

measured by immunoturbidimetry

100
Q

Steps of solid phase extraction

A
  1. Conditioning - Solvent is added to cartrigde containing silica
    2.** Load **sample
  2. **Wash **- potential interferences remove
  3. Selective elution of analyte for further work up/analysis
101
Q

In the routine method for bilirubin, bilirubin couples with a ________

A

Diazo reagent

102
Q

Interferences in Diazo method for Bilirubin

A
  • Haemolysis
  • Lipaemia
  • Indicans (present with high concentrations of ureaemia)
  • Indocyanine green
103
Q

How does haemolysis cause interference in Diazo method for bilirubin?

A
  • Spectral interference and
  • Free Hb inhibits the reaction
104
Q

Possible acclerators in Diazo reaction

A

Methanol
Caffeine (Jendrassik-Grof)
Dimethyl Sulfoxide
Surfactant

105
Q

Enzyme reactions used in ALT method

A
  1. Alanine + alpha-ketoglutarate -> Pyruvate + Glutamate
  2. Pyruvate + NADH -> Lactate + NAD+

Measurement at 34onm

106
Q

Enzyme reactions in AST method

A
  1. Asparate + alpha ketoglutarate -> Oxaloacetate + Glutamate
  2. Oxaloacetate + NADH -> Malate + NAD+

Malate dehydrogenase catalyses 2nd reaction

107
Q

Method for LDH

A

Lactate + NAD+ -> Pyruvate + NADH + H+

IFCC recommended forward reaction

108
Q

Preferred speciment for LDH

A

Serum

Plasma may be contaminated with platelets, high conc of LDH

109
Q

Enzyme reactions used in measurement of CK

A
  1. Creatine phosphate + ADP -> Creatine + ATP
  2. ATP + glucose – (Hexokinase)–> Glucose-6-phosphate + ADP
  3. G-6-P + NADP –(G6PD)–>6-P-G + NADPH + H

These reactions occur in presence of NAC (Enzyme reactivator)

110
Q

On a CSF xanthochromia scan, net bilirubin absorbance and net oxyhaemoglobin absorbance are measured at what wavelengths?

A

Net Bilirubin Absorbance: 476 nm
Net OxyHaemoglobin Absorbance: 415nm

111
Q

A CSF xanthochromia scan result is consistent with SAH if net bilirubin absorbance (NBA) is…

A

> 0.007 AU

(not the only criteria. Also NOA >0.02 with vis peak or CSF protein <1)

112
Q

What is the significance of a CSF total protein >1g/L in interpreting a xanthochromia scan?

A

Interpret with caution as ↑ CSF protein → ↑ CSF bilirubin

CSF bilirubin `correction’ NOT recommended if protein CSF > 1 g/L

113
Q

What is an ionophore?

A

Chemical species that reversibly binds ions

Used in ion selective electrodes

114
Q

How do activity coefficients of ions change with concentration?

A

 Diluted samples e.g calibrators activity coefficient ≈ 1 (more stable)
 Concentrated specimens e.g. Blood, serum - Activity coefficient
varies and usually <0.7

115
Q

Analytical method for Beta-hydroxybutyrate

A

Beta hydroxybutyrate dehydrogenase +/- electron mediator

116
Q

Routine method for total cholesterol - what are the 3 enzymes used?

A
  1. Cholesterol esterase
  2. Cholesterol Oxidase
  3. Peroxidase

H2O2 reacts to form chromophore (quinoneimine dye) - measured at 500 nm

117
Q

Interferences in Total Cholesterol enzymatic method

A
  • N-Acetyl-L-Cysteine - may lead to falsely low results
  • Bilirubin – negative interference
  • Ascorbic acid - negative interference
118
Q

What are the enzymes used in the measurement of triglycerides?

A
  1. Lipase (+protease)
  2. Glycerol kinase
  3. Glycerol phosphate oxidase

H2O2 formed - reacts to form a coloured dye

119
Q

High glycerol levels in blood can occur with

A
  • increased endogenous glycerol in liver disease, diabetes mellitus
  • glycerol-coated stopper used in vacutainer phlebotomy tubes
  • glycerol containing IV medications
    -mannitol infusion
120
Q

Reference method for LDL cholesterol

A

Ultracentrifugation with beta quantification

121
Q

Method for calcium measurement

A

Arsenazo dye reacts with calcium in an acid solution to form a blue-purple complex.
Measured at 660nm

122
Q

Electroenzymatic reaction used in glucometer?

A

Glucose + O2 –(Glucose oxidase)–> Gluconic acid + H2O2

Current generated in the test strip is proportional to glucose conc

123
Q

Light sources used in spectrophotometry

A
  • Incandescent tungsten or tungsten-iodide lamp (visible and near IR)
  • Deuterium (for UV)
  • Mercury lamps (UV to mid visible)
124
Q

Monochromators used in Spectrophotometry

A
  • Coloured glass filters
  • interference filters
  • simple glass prism
  • Diffraction gratings
125
Q

Types of photodetectors used in spectrophotometry

A
  • Photocell (no amplification)
  • Phototube (outside voltage applied)
  • Photomultiplier tube (series of anodes with increasing voltage - 200x more sensitive than phototube)
  • Photodiode array (excellent linearity and speed)
126
Q

Background correction is much more necessary for which type of AAS?

A

Electrothermal techniques

More than for flame AAS

127
Q

What is the Zeeman correction

A

Correction of background absorbance for flameless AAS which utilises a magnetic field.
Magnetic fiels splits atomic energy levels into 2 components: polarised parallel and perpendicular

Absorption measurements at different polariser settings

128
Q

Interferences of AAS

A
  • Spectral - closely absorbing atomic species
  • Non-spectral , non specific. Matrix (may affect nebulisation; viscosity, surface tension, density; sample flow rate)
  • Non-spectral, specific. e.g. phosphate may interfere with calcium due to formation of calcium phosphate (overcome by adding cation e.g. strontium)
129
Q

Advantages of AAS (Flame and Graphite)

A
  • Low set up cost
  • Simple sample preparation
  • Few interferences
130
Q

Disadvantages of AAS (Flame + graphie)

A
  • Single element technique
  • limited analytical range
131
Q

Which form of AAS has a higher detection limit?

A

Flame AAS has higher detection limit than graphite furnace

i.e. less sensitive technique

132
Q

How is chemiluminescence different to fluorescence

A
  • no excitation radiation is required
  • no monochromators are needed as CI arises from one species
133
Q

Chemiluminescence reactions are oxidation reactions of …

A
  • Luminol
  • Acridium esters
  • Dioxetanes
134
Q

Advantages of Chemiluminescence

A
  • subpicomolar detection limits
  • speed
  • ease of use
  • simple instrumentation
135
Q

Disadvantages of chemiluminescence

A

Impurities may cause a B/G signal –> reduce sensitivity, specificity

136
Q

What is electroendosmosis

A

Movement of buffer ions and solvent relative to the fixed support

137
Q

Ionisation technique used in GCMS

A

Electron ionisation - Electric potential applied which bombards molecules in source with high energy electrons

138
Q

Ionisation techniques used in LC MS

A
  • Electrospray
  • Atmospheric pressure chemical ionisation
  • MALDI
139
Q

What does MALDI stand for

A

Matrix Assisted Laser Desorption Ionisation

140
Q

When should a sweat test be delayed?

A
  • Patient <2 weeks of age or <3kg
  • Unwell
  • dehydration or oedema
  • corticosteroid treatment
141
Q

How does neuraminidase affect ALP isoenzyme separation by electrophoresis?

A

Removes sialic acid residues.
Separation of bone and liver isoenzymes.
Bone is slowed down more than liver

142
Q

What is the coloured molecule formed in the ALP enzymatic method?

A

p-nitrophenol

Yellow colour

143
Q

The rate of absorbance increase of p-nitrophenol is measured at what wavelength?

A

404nm

144
Q

Causes of increased osmolar gap

A

ethanol
methanol
ethylene glycol
polyethylene glycol (IV lorazepam)
propylene glycol (IV lorazepam, diazepam and phenytoin)
glycine (TURP syndrome)
maltose (IV IG – Intragram)
mannitol
sorbitol

145
Q

Method for renin measurement

A

Sandwich immunoassay

146
Q

Method for plasma renin activity involves measuring the amount of…

A

Angiotensin I
Generated after incubation of plasma at 37C, buffered to pH6 for optimal activity.

Endogenous angiotensiongen is utilised

147
Q

Causes of decreased renin:

A
  • Medications: beta blockers, methyldopa, clonidine, NSAIDs, OCP
  • sodium loading
  • renal impairment
  • luteal phase
148
Q

Conditions associated with a higher concentration of HbF

A
  • Pregnancy
  • Beta thalassaemia
  • Sickle cell anaemia
  • Leukaemia
  • Insulin therapy
149
Q

Non glucose adducts the Hb beta chain can occur via..

A
  • Carbamylation (increased urea in RF)
  • Acetylation (aspirin, alcohol, vit C)
150
Q

Reference method for HbA1c

A

HPLC + MS
or HPLC + capillary electrophoresis

151
Q

How does FTIR differ from simple IR spectroscopy

A
  • it uses an interferometer - causes the IR radiation to interfere with itself.
  • Computer software uses data from the interference of waves with different frequencies to determine the frequencies of IR that were absorbed by the sample.
152
Q

What are the disadvantages of wet chemical analysis for renal calculi analysis?

A
  • requires at least 10-15mg of sample
  • only identifies ions present not the specific compounds
  • can miss rare material
153
Q

What is the main disadvantage of using Nitroprusside to measure ketones?

A

Beta-hydroxybutarate does not react with nitroprusside

154
Q

Nitroprusside is most sensitive at measuring which type of ketone

A

Acetoacetate

Nitroprusside + Acetoacetate (or acetone) -> Red colouration

155
Q

The enzymatic method for ketone analysis uses which enzyme

A

Beta hydroxybutarate dehydrogenase

Beta HB + NAD –> Acetoacetate + NADH. Change in absorbance at 340nm

156
Q

Point of care device for Beta hydroxybutarate uses …

A

Electrochemical sensor.
Enzyme: Beta HB dehydrogenase
Generated NADH reoxidised to NAD- and current is measured

157
Q

Glucose oxidase method for glucose:
Glucose + O2 –>

A

Gluconic acid + H2O2

158
Q

Hexokinase method for glucose:
1) Glucose + ATP –> __________ + ADP
2) _________________ + NADP –> 6-phosphogluconate + NADPH + H+

A

Glucose- 6 Phosphate

159
Q

Point of care device using glucose oxidase involves measurement of what coloured chromogen

A

Oxidised ortho-dianisidine

Formed by reaction with H2O2

160
Q

Disadvantages of POC glucose

A
  • No reference method for whole blood glucose
  • Affected by medications, oxygen therapy, anaemia, haematocrit
  • Not easily connected to LIS
  • no Medicare rebate
  • errors due to lack of expertised and insufficient QC
161
Q

What does ELISA stand for

A

Enzyme linked immunosorbent assay

162
Q

Method for chromagranin A

A

ELISA
Sandwich immunoassay incorporating 2 monoclonal Abs targeting different epitopes

163
Q

Newborn screening for CF uses

A

Immunoreactive trypsinogen

164
Q

In the enzymatic method of ammonia, what is the enzyme used?

A

Glutamate dehyrodgenase (GLDH)

Change in absorbance measured at 340nm

165
Q

Why does plasma ammonia concentration increase after collection?

A

Deamination of plasma proteins and amino acids by enzymes

Increases with higher GGT concentrations

166
Q

Interferences in enzymatic assay for Lactate

A
  • Haemolysis
  • Elevated bilirubin
  • Glycolic acid
  • N-acetyl cysteine
167
Q

What is the effect of excess heparin in the blood gas tube when measuring HCO3?

A

Decreases the pCO2 and calculated HCO3-

168
Q

Method for bone ALP

A

2 site immunoassay

169
Q

Reference method for ALP

A

IFCC kinetic spectrophotometry

Substrate = 4-nitrophenyl phosphate

170
Q

Types of tissue non-specific ALP

A
  • Bone
  • Liver
  • Kidney
171
Q

Buffer used in IFCC reaction for ALP measurement

A

AMP (2- amino-2-methyl-1-propanol)

Transphosphorylating buffer (accepts PO4) and increases reaction rate

172
Q

In measuring ALP isoenzymes, addition of neuraminidase separates which forms?

A

Bone and liver

173
Q

How does neuraminidase separate bone and liver isoenzymes?

A

Removes sialic acid. Bone contains more sialic acid than liver –> greater reduction in mobility

174
Q

Reference method for glucose

A

Isotope dilution LC-MS

175
Q

Albumin is measured at what wavelength?

A

604nm

176
Q

Reference method for albumin

A

IFCC Optimized immunoturbidimetry/nephelometry

177
Q

Reference method for total protein

A

INSTAND spectrophotometric method based on biuret reaction

178
Q

Interferences in biuret method for total protein

A
  • Haemolysis, Icterus, Lipaemia
  • Dextran
  • Contamination with ammonium ion
179
Q

Reference method for ALT and AST

A

IFCC kinetic spectrophotometry with P5P

180
Q

Method used for Cystatin C

A

Immunonephelometry or immunoturbidimetry

181
Q

What is the dye formed in the Trinder reaction

A

quinoneimine dye

182
Q

Reference method for Total cholesterol

A

Isotope Dilution MS

183
Q

Reference method for Triglycerides

A

Isotope Dilution MS

184
Q

U/L of enzyme = ________ of substrate consumed per minute

A

micromol of substrate

185
Q

Formula for correcting calcium for albumin

A

Corrected calcium = Measured calcium – ((Albumin-41) x 0.02)

186
Q

Reference method for total calcium

A

ICP-MS

187
Q

When does total calcium not reflect free/ionised calcium?

A
  • abnormal protein concentration
  • when patient has acid-based disturbance (higher H+ -> higher free Ca2+)
188
Q

The Payne formula for Calcium correction was derived using which method for albumin measurement

A

BCG

If a lab uses BCP, this can affect the performance of BCG-equations

189
Q

Enzyme that conjugates bilirubin

A

UDP Glucuronosyltransferase 1A1 (UGT1A1)

190
Q

What does NIST stand for

A

National Institute of Standards and Technology

191
Q

What is a diazo group

A

An organic moiety consisting of 2 linked Nitrogen atoms (azo) at the terminal position

192
Q

Interferences in Diazo method

A
  • Haemoglobin
  • Lipaemia
  • Paraproteins
  • Ascorbic acid
  • Indican
  • Indocyanine green
193
Q

Reaction for enzymatic measurement of bilirubin

A

Vanadate OR Bilirubin Oxidase - oxidises
Bilrubin –> Biliverdin

Monitor decrease in absorbance

194
Q

Most common method for Lithium measurement

A

Colourimetric
(Abbott - substituted porphyrin reacts with Lithium)

Alkaline pH required

195
Q

Reference method for Lithium

A

ID-ICP-MS

196
Q

What is heparin

A

A mucoitin polysulfuric acid

Accelarates action of antithrombin III

197
Q

What is Daratumumab?

A

Human monoclonal IgG1 anti-CD38
Used in treatment of multiple myeloma

198
Q

Reasons for derivitisation

A
  • improve performance in chromatographic environment
  • thermal stability
  • volatility
  • improve separation
  • improve sensitivity of detection
199
Q

Identification of CSF by beta trace protein can be limited in what situations

A
  • Bloody discharge - low levels may be found in blood
  • Meningitis - may reduce beta trace protein levels
200
Q

Analytical issues with serum free light chains - name 5

A
  • Interference from turbidity/lipaemia
  • Hook effect
  • Non-linearity
  • Reagent lot to lot variation
  • Results can differ between methods (polyclonal vs monoclonal Abs)

In CKD, different K/L ratio RI may be needed depending on assay

201
Q

Method for plasma free metanephrines

A

LCMS-MS

202
Q

Impact of OCP on AMH levels

A

Decrease by approx 10%

203
Q

In a chemiluminescence reaction (acridium ester),
Dye system + H202 –> ??

A

2H20 + oxidised dye + LIGHT

Light is emitted when the electron returns to the ground state

204
Q

Assays (Abbott) affected by High Bilirubin

A
  • Cholesterol - decrease
  • Trigs - increased
  • Amylase - increased
205
Q

Medications that increase 5-HIAA measurements

A
  • Paracetamol
  • Phenacetin
  • Glyceryl guaiacolate (found in many cough syrups)
206
Q

Properties of a good internal standard

A
  • similar mass of the analyte of interest
  • similar ionisation potential
  • not present in clinical samples
  • not affected by spectroscopic interferences
  • not a source of analyte contamination
207
Q

Common internal standards used in ICP-MS

A
  • Scandium
  • Germanium
  • Rhodium
  • Yttrium
208
Q

Anti-hypertensive that do not affect ARR

A
  • verapamil
  • prazosin
  • hydralazine
  • moxonidine
209
Q

Definition of Km (Michaelis-Menten constant)

A

Concentration at half the maximum velocity of the enzymatic reaction

210
Q

Difference between enzyme activators and coenzymes

A
  • Enzyme activators: usually ions that increase the activity by facilitating more stable form of enzyme e.g. Mg++ for CK
  • Coenzymes usually more complex that activators but smaller than enzymes themselves e.g. NAD, NADP, P-5-P
211
Q

How can a Propofol infusion affect lipid results?

A

Propofol infusions (used in ICU as sedative) contain glycerol
Can result in falsely high triglyceride results

212
Q

Types of detectors used for measuring radioactivity

A
  1. Gas filled
  2. Scintillation - more common
    - absorption of radiation produces a flash of light
    - liquid or crystal
213
Q

Enzymatic method of HbA1c uses which enzyme

A

FPOX (Fructosyl peptide oxidase)

214
Q

What formula is used (by Sonic) to calculate free testosterone?

A

Vermuelen formula
accounts for both SHBG and albumin

215
Q

Name 4 equations for calculating free testosterone and the components they include

A
  • Vermuelen - T, SHBG, albumin
  • Sodergard - T, SHBG, albumin
  • Nanjee-Wheeler - T, SHBG
  • Ly-Handelsman - T, SHBG
216
Q

What does PETIA stand for?

A

Particle Enhanced Turbidimetric Immunoassay

217
Q

What does PETINIA stand for?

A

Particle Enhanced Turbidimetric Inhibition Immunoassay

218
Q

What does CEDIA stand for

A

Cloned Enzyme Donor Immunoassays

219
Q

What does ELISA stand for

A

Enzyme Linked Immunosorbent Assays

220
Q

Cause of falsely low phosphate result

A

IV Mannitol (e.g. for high ICP)

Interferes with reaction of phosphate with molybdate

221
Q

Preferred specimen for LD?

A

Serum

Plasma has higher LD; released from platelets when they activate

222
Q

Conditions of standard gel electrophoresis

A
  • Agarose gel medium (typically 0.5-2% concentration); low endosmosis effect
  • buffer pH 8.6 (constant) - most proteins negative charged -> anode
223
Q

What does EDTA stand for?

A

Ethylene-diamine-tetraacetic acid

224
Q

Cause of clear green appearing serum with positive lipaemic index

A

Patent blue dye

Used in sentinal lymph node biopsy

225
Q

Types of analysers available for Mass Spec

A
  • Quadrupole
  • Ion trap
  • Tandem mass spec
  • TOF
  • Orbitrap

TOP and Orbitrap are types of “high resolution MS”

226
Q

Most common type of detector used in MS

A

Electron multiplier

227
Q

Ionisation types used in LC/MS

A
  • Electrospray
  • Atmospheric pressure chemical ionisation (APCI)
  • MALDI
228
Q

Types of CK isoenzymes

A
  1. CK-BB - brain, lung, intestine
  2. CK-MB - cardiac muscle
  3. CK-MM - skeletal muscle

Above located in cytosol
CK-mitochondria - can occur w necrosis

229
Q

Types of Macro CK

A
  • MacroCK 1 - typically CK-BB with IgG. Females >50 years
  • MacroCK 2 - complexes of mitochondrial CK
230
Q

What is derivitisation and why is it used?

A

Chemical modification of the analyte (e.g. alkylation, Silylation).
Used to improve:
- volatility and thermal stability in gas chromatography
- retention and resolution from interferences
- detection (e.g fluorescence detection)

231
Q

Formula for theoretical plates

A

N = 16 (t / w) ^2
where t is retention time
and w is width of peak

232
Q

Formula for Height of theoretical plates (HETP) in chromatography

A

HETP = column length / N
where N is number of theoretical plates

233
Q

How to increase number of theoretical plates in chromatography

A
  • longer column
  • smaller average packing particle size
  • optimising flow rates
  • higher mobile phase viscosity
  • smaller sample volume
234
Q

Buffer used in SPIFE method for serum protein electrophoresis

A

Tris buffer

235
Q

Indicator dye used on urine dipstick for protein detection

A

Bromphenol blue

236
Q

Difference between isoenzyme and isoform

A

Both catalyse the same reaction
* Isoenzyme - different genetic loci
* Isoform - same genetic loci but different post translational modifications

RCPA Part 1 (6 decks)

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