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1
Q

What conditions are cryoglobulins associated with?

A

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

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2
Q

How do cryoglobulins cause disease?

A

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

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3
Q

Specimens required for cryoglobulins and specific handling requirements

A

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

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4
Q

Difference between cryoglobulin and cryofibrinogen

A

Cryofibrinogen precipitates out of plasma but not serum

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5
Q

Significance of cryofibrinogen

A

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

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6
Q

Cause of brown serum

A

myoglobin, methemoglobin, methemalbumin

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7
Q

Where is AFP produced in the fetus?

A

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

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8
Q

Methods for AFP determination

A

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

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9
Q

Difference in sample processing of amniotic fluid and serum for AFP

A

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

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10
Q

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

A

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.

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11
Q

Indications for urate/uric acid measurement

A
  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
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12
Q

what is urate/uric acid?

A

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)

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13
Q

Methods for urate measurement

A
  1. Colorimetric
  2. Enzymatic
  3. HPLC (either ion exchange or reverse phase columns)
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14
Q

Colorimetric method for uric acid?

A

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

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15
Q

Enzymatic method for uric acid?

A

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

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16
Q

Reference and definitive methods for uric acid

A

Proposed definitive method - isotope dilution MS
Reference method - HPLC

17
Q

Causes of hyperuricaemia

A
  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
18
Q

Investigation of hyperuricaemia

A
  1. Look for secondary causes
  2. Renal function
  3. Assess for other features of metabolic syndrome
19
Q

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

A

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

20
Q

What is alpha1 antitrypsin?

A

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

21
Q

How is liver damage caused in AAT deficiency?

A

Accumulation of misfolded ptn

22
Q

What is the cause of lung disease in AAT deficiency?

A

Unchecked elastase activity

23
Q

Causes of increased AAT

A

Inflammation
Malignancy
Trauma
Pregnancy/ oestrogens
Neonates

24
Q

AAT method in your lab

A

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

25
Q

Limitations of AAT method

A

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

26
Q

Threshold concentration for determining normal PiMM allele of AAT?

A

20umol/L

27
Q

Protective threhold concentration of AAT (prevention of emphysema)?

A

11 umol/L

28
Q

Diagnosis of AAT deficiency?

A

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.

29
Q

Utility of faecal alpha-1-antitrypsin

A

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

30
Q

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

A

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.

31
Q

Gold standard for protein losing enteropathy?

A

Methods utilising administration of radioactive albumin or dextran

32
Q

How is alpha-1-antitrypsin genotyping performed?

A

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

33
Q

Gene encoding AAT?

A

SERPINA1

34
Q

Wild-type alpha-1-antitrypysin allele?

A

M

35
Q

AAT allele associated with severe deficiency?

A

Z

36
Q

Effect of acute inflammation on interpretation of AAT?

A

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.

37
Q

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

A

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.

38
Q

Challenges faced when developing vitamin D assays

A
  1. Highly lipophillic
  2. Multiple cross-reacting vit D metabolites
  3. High affinity of vit D to its binding protein
39
Q

Vitamin D methods

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

Analytes (15 decks)

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