FOCUS 2017 Training Day

Question 1 1.Name THREE signs/symptoms associated with

Haemochromatosis:

Liver cirrhosis, cardiomyopathy, diabetes mellitus, arthritis and skin pigmentation, low libido

2. What are the two common Haemochromatosis mutations and what amino acid substitutions do they arise from?

C282Y – Cysteine to Tyrosine

H63D – Histidine to Aspartate

Question 1 3. H63D - Using the information given above, what genotype

would you report for patient in lane:

a) 3 - Homozygous negative

b) 6 - Heterozygous positive

4. C282Y - Using the information given above, what genotype would you report for patient in lane:

a) 3 - Homozygous negative

b) 6 - Heterozygous positive

Question 1 5. State the calculation used for transferrin saturation?

Iron/Total Iron Binding capacity (TIBC) * 100 or 3.982 x Iron (umol/L) / Transferrin (g/L)

6. State two secondary conditions of Iron overload?

Anaemia with increased but ineffective erythropoiesis

Chronic liver disease: Alcoholism, hepatitis C, non-alcoholic steatohepatitis

Porphyria cutanea tarda

7. What is the mode of inheritance for primary haemochromatosis due to the mutations above?

Autosomal recessive

8. What is the Hardy-Weinberg equilibrium equation?

p2 + 2pq + q2 = 1

Question 2 a) Define U/L

Activity of an enzyme which transforms one micromole of substrate or forms one micromole of product per minute under defined conditions

b) State the Beer Lambert Equation

A = ε x c x l

A = Absorbance

ε = extinction coefficient

c = concentration

l = light path of cuvette (cm)

Reaction equations

ALT + P5P Activated ALT

L-alanine + α-ketoglutarate Pyruvate + L-glutamate

Pyruvate + NADH lactate + NAD

ALT

LDH

Question 2 c)Use the equation to calculate serum ALT activity from

the raw data measured at 340nm

A = ε x c x l – Rearrange!

c = A / ε x l

Path length = 10mm = 1cm so l = 1

Absorbance = A

Select part of the graph which gives a straight line (e.g. reading 26 to 38) and calculate ΔAbs

Remember absorbance readings are every 14 seconds

Δabs = 1.16754 – 1.08553 = 0.08201

Time = 12 readings * 14 = 168 seconds

Δabs/time = 0.08201/168 = 0.0004881 per second

Convert to mins by x 60 = 0.029286

Question 2 c = A / ε

Convert to concentration

need to use micromolar ε = 6.3 x 103/1000000 = 0.0063

A/ε

0.029286/0.0063 = 4.649

Correct for the 2 dilution steps

4.649 x ((80+16+25)/25) x ((120+30)/30) = 112.5U/L

d) What is the purpose of the second series of measurements at a wavelength of 410nm

Interference check

Question 3/1

Describe the Acid-Base disorder in each case:

a) Partially compensated metabolic acidosis. On 100% O2

b) Uncompensated respiratory acidosis. Hypoxaemic

c) Partially compensated metabolic alkalosis. Normal oxygenation.

Question 3/2 a) Calculate the bicarbonate

pH = 6.1 + log(HCO3-/0.225*PCO2) if PCO2 in kPa

pH = 6.1 + log(HCO3-/0.03*PCO2) if PCO2 in mmHg

HCO3- = 0.225 * PCO2 * 10 (pH-6.1)

7.4 mmol/L

b) Calculate the anion gap

(Sodium + Potassium) – (Chloride + Bicarbonate)

43.9 mmol/L

Question 3/2

c) Calculate the osmolal gap

2 x Na+ + urea + glucose

91-114mmol/L (depends on formula used)

d) What might the patient have ingested to give these results?

Ethylene glycol, methanol

Question 3/3 Define the following parameters:

a) Standard bicarbonate

Standard bicarbonate is the bicarbonate concentration that the patient would have if there was no respiratory component to the disorder, i.e. if pCO2 were 5.3kPa (the pCO2 of atmospheric air), body temperature = 37°C, and oxygen saturation is normal

b) Base excess

Base excess is the amount (mmol/L) of strong acid (if positive), or base (if negative) that would be required to bring the pH back to normal (7.4) under standard condition. Base excess reflects the metabolic component of the disorder.

c) Oxygen saturation

Oxygen saturation is the percentage of oxygen binding sites on haemoglobin which are carrying an oxygen molecule

Question 4/1 Write an interpretative report:

Paraprotein band present, typed as IgG lambda and concentration is 10.2g/L. The presence of clinical signs and symptoms (back pain, renal impairment and anaemia) warrant a referral to haematology.

Useful additional tests include urine Bence Jones protein and serum adjusted calcium (also accept ß2 microglobulin, ESR/ blood film, serum free light chains, bone marrow biopsy).

The sodium may be due to pseudohyponatraemia due to the presence of a paraprotein. Serum osmolality may be useful in differentiating true and pseudohyponatraemia.

Question 4/2 What is the most likely cause of this pattern of results?

Nephrotic syndrome – low albumin and IgG

What additional tests would be useful and what other biochemical abnormalities would you expect to see?

The most important additional test is urine protein: creatinine ratio alongside U&E and lipids.

You would expect to see high protein: creatinine ratio (or positive urine dipstick for protein) and high cholesterol and triglycerides.

What action would you suggest to the GP?

Urgent measurement of protein: creatinine ratio or urine dipstick test for protein would be advised and seek advice from renal team.

Question 4/3 Which condition is the following electrophoretogram

consistent with?

This chromatogram is consistent with alpha 1 anti-trypsin deficiency.

What further tests would you recommend to the requesting clinician?

Alpha 1 antitrypsin concentration should be measured, and phenotyping performed.

Question 5 1 - How well is this laboratory performing compared with all

other laboratories ?

The laboratory has a overall SDI of 0.88 which is higher than the best performing laboratories , (median 0.75) but is lower than the worst performing laboratories (97.5th centile 1.6). The overall performance is therefore satisfactory.

2 - Are there any performance issues identified in this distribution ?

Yes, the Sodium results are flagged as Poor with an SDI > 2 and need investigating. Although, the chloride SDI is < 2, the SDI of 1.49 would also require following up.

Question 5 3 - What does the individual and overall SDI for this analyte

suggest?

All 4 of the samples have an SDI score > 2, which suggests that the performance issue is across the analytical range.

4 - Identify the error. Is it imprecision or inaccuracy? The IS score of 4 and the Syx of 0.7 mmol/l suggests that the

imprecision is acceptable (0.5% CV at 130 mmol/l). The slope of 0.96 and intercept of +0.79 suggests a systematic

proportional negative bias of -4%. The Method mean and Analyser mean are in close agreement,

although have a small acceptable bias to the reference method at low and high concentration . The laboratory’s result is outside ± 2 SD of the method and analyser mean for 3 out of the 4 results.

Question 5 5 - Is this a new problem or is it longstanding?

The previous SDI scores are all < 2 suggesting that this is a new performance issue, although there is an increase in the SDI and high IS score for distribution OM. Results for previous distributions follows a general trend to the instrument mean.

6 - What is the cause of the performance issue?

Systematic proportional biases are usually due to calibration issues.

Question 5 7 - If this problem persists , how would you rectify the issue?

Check all calibrators are in date. Check that the calibrator’s integrity has not been compromised (check storage, reconstitution instructions if applicable). Check that the calibrator lot number has not changed and the correct value(s) have been entered on the analyser.

Contact WEQAS for a new set of samples to assist you in troubleshooting the issue.

Recalibrate. Run the new set of WEQAS samples to validate that the

performance issue has been resolved. Contact the manufacturer if you suspect that the calibrator

assigned value is incorrect. Obtain a different calibrator to validate the assigned value

is correct.

Question 6/1 1 - Using the data supplied, draw calibration curves for 25 OH

Vitamin D2 and 25 OH Vitamin D3.

Make sure you use the correct transitions

25 OH Vitamin D2 395→269

25 OH Vitamin D3 383→211

d6-25 OH Vitamin D3 (Internal Standard) 389→211

- (Deuterated 25 OH Vitamin D3)

Question 6/1 – D2 Calibration Conc:

nmol/L Counts IS

Ratio: Counts/IS

C1 0 66 12673 0.005

C2 32 2164 11263 0.192

C3 64 5470 12496 0.438

C4 122 10929 12614 0.866

y = 0.0071x - 0.014

-0.1

0

0.1

0.2

0.3

0.4

0.5

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0.7

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0.9

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0 20 40 60 80 100 120 140

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ign

l

Question 6/1 – D3 Calibration Conc:

nmol/L Counts IS

Ratio: Counts/IS

C1 10 623 12673 0.049

C2 49 2717 11263 0.241

C3 86 4218 12496 0.338

C4 169 8681 12614 0.688

y = 0.0039x + 0.0202

0

0.1

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0 20 40 60 80 100 120 140 160 180

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Question 6/2 – QC’s on calibration

D2 Counts IS Ratio:

Counts/IS

QCI 3992 13273 0.301

QCII 9052 12722 0.712

y = 0.0071x - 0.014

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120 140

Concentration

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tive

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l

85 - 10984 -1082

30-3938 - 471

25 OH

Vitamin D3

25 OH

Vitamin D2

Compound

Level

85 - 10984 -1082

30-3938 - 471

25 OH

Vitamin D3

25 OH

Vitamin D2

Compound

Level

Question 6/2 – QC’s on calibration

85 - 10984 -1082

30-3938 - 471

25 OH

Vitamin D3

25 OH

Vitamin D2

Compound

Level

85 - 10984 -1082

30-3938 - 471

25 OH

Vitamin D3

25 OH

Vitamin D2

Compound

Level

D3 Counts IS Ratio:

Counts/IS

QCI 2010 13273 0.151

QCII 4836 12722 0.380

y = 0.0039x + 0.0202

0

0.1

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0 20 40 60 80 100 120 140 160 180

Concentration

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gnal

Question 7

1 - At what absorbance should the net bilirubin absorbance (NBA) be measured?

476nm

2 - At what absorbance (s) should the net oxyhaemoglobin absorbance (NOA) be measured at?

The absorbance maximum for oxyhaemoglobin is between 410 – 418nm

Question 7

3 - Interpret the above scan above using the following measurements - CSF protein 0.82g/L, Serum bilirubin 38 µmol/L, Serum total protein 62 g/L

The NBA >0.007 (0.025) and the NOA is <0.02 (0.012)

In this case the serum bilirubin is >20µmol/L, so the following calculation needs to be applied:

Predicted Abs = CSF protein x serum bilirubin x 0.042 Serum protein

Then adjusted NBA = measured NBA – PA

Question 7

PA = 0.82g/L x 38µmol/L x 0.042 = 0.021 62 g/L

Adjusted NBA = 0.025 - 0.021 = 0.004AU The NBA is now <0.007 and the CSF protein 1.0 g/L, so a comment to accompany these results is: Increased CSF bilirubin, but probably totally accounted for by

the increase in serum bilirubin. Not supportive of SAH

Question 7

4 - Interpret this scan using the following measurements:

Serum bilirubin 10 µmol/L, CSF total protein 0.40g/L, Serum total protein 68g/L

The NBA >0.007 (0.012) and the NOA is >0.02 (0.024)

Bilirubin and oxyhaemoglobin increased. Consistent with SAH