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Question 1

What conditions are cryoglobulins associated with?

Answer 1

Malignant B cell diseases (myeloma, Waldenstroms)
Connective tissue disorders (RA, SLE, immune complexes)
Acute and chronic infections eg syphilis, CMV
Other (sarcoid, cirrhosis)

Question 2

How do cryoglobulins cause disease?

Answer 2

Vascular occlusion, immune complex nephritis/vasculitis, secondary release of histamine enhancing ischaemic symptoms

Question 3

Specimens required for cryoglobulins and specific handling requirements

Answer 3

EDTA plasma AND clot tube kept at 37deg C until separated

Question 4

Difference between cryoglobulin and cryofibrinogen

Answer 4

Cryofibrinogen precipitates out of plasma but not serum

Question 5

Significance of cryofibrinogen

Answer 5

May be seen in healthy individuals but also associated with malignancy, infection, connective tissue and autoimmune disorders

Question 6

Cause of brown serum

Answer 6

myoglobin, methemoglobin, methemalbumin

Question 7

Where is AFP produced in the fetus?

Answer 7

Fetal yolk sac (small quantities)
Fetal liver (as the yolk sac degenerates)
HCC

Question 8

Methods for AFP determination

Answer 8

Immunoenzymometric or chemiluminescent immunoassay
Same methods for amniotic fluid and serum, however, amniotic fluid specis must be diluted

Question 9

Difference in sample processing of amniotic fluid and serum for AFP

Answer 9

While AFP is stable in serum, it is degraded in amniotic fluid if left at room temperature, necessitating refrigerated transport of amniotic fluid samples

Question 10

Cause of spuriously elevated amniotic fluid AFP and what to do about it

Answer 10

Fetal blood contamination will cause an increased amniotic fluid AFP. If AFP > 2.0 or 2.5 MoM, lab should test for presence of fetal blood.

Question 11

Indications for urate/uric acid measurement

Answer 11

1. Diagnosis and management of gout (monitoring urate-lowering treatment)
2. High urate a feature of pre-eclampsia
3. high urate a feature of tumour lysis syndrome
4. Ethambutol and pyrizinamide reduce renal urate excretion causing hyperuricaemia
5. Urinary urate in pts with renal caluli
6. Urate crystals in synovial fluid using microscopy with polarised light - negative birefringence

Question 12

what is urate/uric acid?

Answer 12

A heterocyclic nitrogenous compound. The acid has 4 dissociable H+ ions and pKa is 5.8, hence in blood, uric acid exists as urate ions. It is the end product of purin breakdown (dietary and endogenous) and excreted by kidney (2/3) and large intestine (1/3)

Question 13

Methods for urate measurement

Answer 13

1. Colorimetric
2. Enzymatic
3. HPLC (either ion exchange or reverse phase columns)

Question 14

Colorimetric method for uric acid?

Answer 14

Phosphotungstic acid reduced by urate in alkaline solution to Tungsten blue, measured at 650-700nm. Many interferents limiting the use of this method

Question 15

Enzymatic method for uric acid?

Answer 15

Uricase oxidises urate to allantoin, hydrogen peroxide and carbon dioxide. Decrease in absorbance at 293 nm could be measured, but requires a narrow bandwidth spectrophotometer which cannot be automated. Utilising Trinder reaction generates a chromogen that can be detected with automated systems. Has been adapted for dry chemistry systems. Interfering compounds can accumulate in renal failure (bilirubin, ascorbate, phenolic compounds

Question 16

Reference and definitive methods for uric acid

Answer 16

Proposed definitive method - isotope dilution MS
Reference method - HPLC

Question 17

Causes of hyperuricaemia

Answer 17

1. Primary - renal retention due to increased tubular resorption, inherited metabolic diseases eg Lesch-Nyhan syndrome
2. Secondary - alcohol, obesity, high purine intake, drugs (cytotoxics, diuretics, ciclosporin), tumour lysis, lead poisoning, obesity, renal impairment

Question 18

Investigation of hyperuricaemia

Answer 18

1. Look for secondary causes
2. Renal function
3. Assess for other features of metabolic syndrome

Question 19

Pre-analytical factors when collecting uric acid for tumour lysis syndrome?

Answer 19

Collect on ice and analyse ASAP - this is to inhibit the action of rasburicase to convert uric acid through to allantoin, which will cause falsely low uric acid results

Question 20

What is alpha1 antitrypsin?

Answer 20

A serine protease inhibitor (serpin). Inhibits elastase, trypsin, chymotrypsin and thrombin.

Question 21

How is liver damage caused in AAT deficiency?

Answer 21

Accumulation of misfolded ptn

Question 22

What is the cause of lung disease in AAT deficiency?

Answer 22

Unchecked elastase activity

Question 23

Causes of increased AAT

Answer 23

Inflammation
Malignancy
Trauma
Pregnancy/ oestrogens
Neonates

Question 24

AAT method in your lab

Answer 24

Immunoturbidimetry. Sample incubated with PEG and anti-alpha1-antitrypsin. Formation of antibody-antigen complex results in increased turbidity measured as the amount of light absorbed at 340/596nm

Question 25

Limitations of AAT method

Answer 25

High dose hook effect. Designed to have less than <10% interference from HIL.

Question 26

Threshold concentration for determining normal PiMM allele of AAT?

Answer 26

20umol/L

Question 27

Protective threhold concentration of AAT (prevention of emphysema)?

Answer 27

11 umol/L

Question 28

Diagnosis of AAT deficiency?

Answer 28

AAT concentration < 11 umol/L plus genotyping of SERPINA gene showing alleles associated with severe deficiency (Z, S, I) OR phenotyping with iso-electric focusing.

Question 29

Utility of faecal alpha-1-antitrypsin

Answer 29

Aid diagnosis of protein-losing enteropathy. Estimate clearance of alpha-1-antitrypsin from plasma into faeces (an estimate of intestinal plasma protein loss).

Question 30

What other test should accompany faecal alpha-1-antitrypsin?

Answer 30

FOBT - if there is blood in the faeces, then an increased AAT may be due to this, rather than enteropathy. Plasma AAT - if this is low due to deficiency, faecal AAT will also be low, potentially falsely low.

Question 31

Gold standard for protein losing enteropathy?

Answer 31

Methods utilising administration of radioactive albumin or dextran

Question 32

How is alpha-1-antitrypsin genotyping performed?

Answer 32

RT PCR for Z and S allele variants; Sanger sequencing if required

Question 33

Gene encoding AAT?

Answer 33

SERPINA1

Question 34

Wild-type alpha-1-antitrypysin allele?

Answer 34

M

Question 35

AAT allele associated with severe deficiency?

Answer 35

Z

Question 36

Effect of acute inflammation on interpretation of AAT?

Answer 36

AAT is an acute phase reactant. In Z/M and S/Z heterozygotes, an acute inflammatory illness may increase AAT into the normal range, but is unlikely to mask ZZ homozygotes.

Question 37

Is there a role for iso-electric focusing for AAT deficiency diagnosis?

Answer 37

It is still part of the diagnostic criteria. Genotyping for all alleles associated with AATD (F, I, S, Z) is not widely available and in individuals with severe deficiency whose genotype does not match, iso-electirc focusing can be used to detect rarer variants such as F and I. FZ heterozygotes can have a severe phenotype and be mis-genotyped as MZ.

Question 38

Challenges faced when developing vitamin D assays

Answer 38

1. Highly lipophillic 2. Multiple cross-reacting vit D metabolites 3. High affinity of vit D to its binding protein

Question 39

Vitamin D methods

Answer 39

1. Manual IAs 2. Automated IAs 3. HPLC with UV detection 4. LCMS