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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=εxcxl
 A = Absorbance
 ε = extinction coefficient
 c = concentration
 l = light path of cuvette (cm)
Reaction equations

ALT + P5P Activated ALT

ALT

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

LDH

Pyruvate + NADH lactate + NAD


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/εxl
 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
 Write an interpretative report:
Question 4/1

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: Ratio:
nmol/L Counts IS 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

0.9
0.8

0.7
0.6
Relative Signl

y = 0.0071x - 0.014
0.5

0.4

0.3
0.2

0.1
0
0 20 40 60 80 100 120 140
-0.1

Conce ntration
Question 6/1 – D3 Calibration
Conc: Ratio:
nmol/L Counts IS 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
0.8

0.7

0.6
Relative Signal

0.5
y = 0.0039x + 0.0202
0.4

0.3

0.2

0.1

0
0 20 40 60 80 100 120 140 160 180

Concentration
Question 6/2 – QC’s on calibration
Ratio:
D2 Counts IS Counts/IS
QCI 3992 13273 0.301
QCII 9052 12722 0.712
1

0.9

0.8

0.7
0.6
Relative Signl

y = 0.0071x - 0.014
0.5

0.4

0.3
0.2

0.1

0
0 20 40 60 80 100 120 140
-0.1

Conce ntr ation

Compound 25 OH 25 OH
Vitamin D2 Vitamin D3
Level
1 38 - 47 30-39

2 84 -108 85 - 109
Question 6/2 – QC’s on calibration
Ratio:
D3 Counts IS Counts/IS
QCI 2010 13273 0.151
QCII 4836 12722 0.380
0.8

0.7

0.6
Relative Signal

0.5
y = 0.0039x + 0.0202
0.4

0.3

0.2

0.1

0
0 20 40 60 80 100 120 140 160 180

Conce ntration

Compound 25 OH 25 OH
Vitamin D2 Vitamin D3
Level
1 38 - 47 30-39

2 84 -108 85 - 109
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