Methods

Question 1

Compare/contrast lactose tolerance breath test vs blood test

Answer 1

Larger dose of lactose given with blood test, which can cause more side effects from the test. More needle-sticks and need to remain in collection centre, while breath test can be performed at home. Lower false negative rate with breath than blood test and can be avoided by measuring methane in addtion to hydrogen.
Breath tests can be contaminated by room air or dead space air if there is improper sample collection. Correction for alveolar CO2 is included to compensate for any dilution by room air.
Breath testing is a manual process but there is minimal system maintenance required.
Patient preparation is more complex for breath test (longer fasting period 12 vs 10 hours, more foods to be avoided for a longer period of time).

Question 2

HbA1c methods

Answer 2

Based on charge differences:
- ion exchange HPLC*
- electrophoresis - gel (rare) or capillary
- isoelectric focusing (rare)

Based on structural differences:
- affinity chromatography
- immunoassay*

Chemical analysis
- enzymatic
- photometry (rare)
- spectrophotometry

*most common

Question 3

Considerations when selecting a method for HbA1c?

Answer 3

Sample volume
Patient population
Cost
Clinician preferences
Recommended to choose only HbA1c assays certified by the NGSP (National Glycohemoglobin Standardization Program) as these are traceable to the DCCT reference

Question 4

Advantages and disadvantages of HPLC for HbA1c

Answer 4

Advantages:
-Automation possible
-Small sample volumes required, even fingerstick possible
-Labile A1c may be separated chromatographically (improving speed of assay)
-HPLC was used for analysis of all DCCT patient samples

Disadvantages:
- If the charge on Hb is altered by the attachment of noncarbohydrate moieties, this may affect accuracy (eg uraemia, alcoholism, lead poisoning, chronic treatment with large doses of aspirin)
- Hb variants may be detected
- Hb variants may also be glycated
- If modified Hb or its glycated derivative cannot be separated from HbA or HbA1c spuriously increased or decreased results will be obtained: variants that elute with HbA1c will increase the result; variants that elute with HbA will decrease the result

Question 5

Advantages and disadvantages of immunoassay for HbA1c

Answer 5

Advantages:
- Labile intermediates and other glycated haemoglobins do not interfere due to specificity of antibody for both ketoamine linkage and specific amino acid sequences
- Similarly, Hb variants are not detected

Disadvantages:
- Prone to similar interferences as other immunoassays

Question 6

Roche blood gas method vs Radiometer ABL 800flex for chloride?

Answer 6

Quaternary ammonium ion exchanger in Roche method unaffected by nitrates, whereas ABL800flex has a polyamide ionophore which is affected by nitrates

Question 7

Methods for diagnosing AAT deficiency

Answer 7

Phenotyping by electrophoresis or MS. Genotyping.

Question 8

Methods for determining AAT

Answer 8

Immunoturbidimetry or immunonephelometry

Question 9

List 7 total protein methods

Answer 9

1. Kjeldahl
2. Biuret
3. Direct optical
4. Lowry/Folin-Ciocalteu
5. Dye-binding
6. Refractometry
7. Light scattering