p reliminary studies
DESCRIPTION
UK N ewborn S creening L aboratories N etwork. P reliminary studies. Imperfect bloodspots – what matters for the screening laboratory?. Alex Lawson* Kate Hall. * Birmingham H eartlands Hospital. Supersaturated. Quantity Insufficient for Testing. Specimen Not Dried Before Mailing. - PowerPoint PPT PresentationTRANSCRIPT
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Preliminary studies
Imperfect bloodspots – what matters for the screening laboratory?
UK Newborn Screening Laboratories Network
Alex Lawson*Kate Hall
* Birmingham Heartlands Hospital
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Poor Sampling – examples from S&S poster
Clotted or Layered
Serum Rings
Specimen Not Dried Before Mailing
Supersaturated
No Blood
Diluted, Discolored, or Contaminated
Scratched or Abraded
Quantity Insufficient for TestingSupersaturated
Specimen Not Dried Before Mailing
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UK Newborn Screening Laboratories Network
•Preliminary study prompted by discussions about widely differing avoidable repeat request rates for dbs between UK screening laboratories
•BCH has some of the highest rates and yet pride in high quality of bloodspots received
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UK Newborn Screening Laboratories Network
•Study prompted by emergence of Luminex Cardscan equipment and need for consensus criteria for spot rejection•Needed to be a scientific basis rather than using say mean Cardscan data from 40 visually good spots
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UK Newborn Screening Laboratories Network
What is known to affect measured results
•Variability of filter paper•Peripheral or central punching•Haematocrit of blood applied
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UK Newborn Screening Laboratories Network
Variability of filter paperNoted in 1970s by CDC blood absorption specifications created
Slazyk WE, Phillips DL, Therrell BL Jr,Hannon WH. Effect of lot-to-lot variabilityin filter paper on the quantification of thyroxin, thyrotropin, and phenylalanine in dried-blood specimens. Clin Chem 1988;34:53-8.
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UK Newborn Screening Laboratories Network
Punch site and haematocrit
Holub M, Tuschl K, Ratschmann R, Strnadová KA, Mühl A, Heinze G, Sperl W, Bodamer OA
Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measuredby tandem mass spectrometry
Clinica Chimica Acta 373 (2006) 27–31
15 mm spots
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UK Newborn Screening Laboratories Network
Haematocrit
Hill JB and Palmer P
Filter Paper Blood Collection and Punching AsA Means of Quantification
Clin Chem 1969, 15/5; 381-389
The hematocrit value was found to have an influence on the volume of blood containedin a punched spot.
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UK Newborn Screening Laboratories Network
Where do we punch?
•In the centre of a full spot?•Using a Multipuncher™ or Panthera ™?
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UK Newborn Screening Laboratories Network
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Typical first punch positions
UK Newborn Screening Laboratories Network
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Typical second punch positions
UK Newborn Screening Laboratories Network
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How do we evaluate quality?
•Visually•New – Luminex Cardscan
UK Newborn Screening Laboratories Network
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Need to study relevant factors for the UK screening programme:
• 10mm circle rather than 12.5 or 15• Ahlstrom 226• UK panel of screening tests
UK Newborn Screening Laboratories Network
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Experimental Design
UK Newborn Screening Laboratories Network
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• Blood from a single donor spiked with 6 analytes measured as part of the NBS programme at 3 concentrations
• Blood was then either– Spotted onto Perkin Elmer 226 cards in 4 different
volumes (10 µL, 20 µL, 50 µL & 75 µL)– n = 20 for each volume at each analyte
concentrationOR
– Manipulated using removal or addition of matched donor plasma to create haematocrits of 30%, 40%, 45%, 50% & 60%.
– 50 µL blood was then spotted onto Perkin Elmer 226 cards
– n = 6 for each haematocrit at each analyte concentration
UK Newborn Screening Laboratories Network
Level TSH Octanoyl Carnitine Leucine L-Methionine L-Phenylalanine L-Tyrosine
Low (endogenous) NA 0.09 199.84 18.38 74.86 72.33Medium (laboratory based alert) 4.15 0.34 291.89 55.68 216.27 225.77High (clinical alert level) 11.6 0.68 576.59 97.25 407.50 417.65
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UK Newborn Screening Laboratories Network
10 µL 20 µL 50 µL 75 µL
Spot Volume & Punch Location(Haematocrit 50%)
30% 40% 45% 50%
Haematocrit (Always 50 µL spot punched in middle)
60%
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• TSH measured by autoDELFIA™ fluoroimmunoassay (Perkin Elmer
• C8, leucine, methionine, phenylalanine and tyrosine measured by measured by underivatised tandem mass spectrometry
• Results compared using one-way ANOVA with post-hoc Tukey test (both experiments) and linear regression (haematocrit) (SPSS)
• Data displayed as percentage change when compared to a 50 µL centre punch
UK Newborn Screening Laboratories Network
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UK Newborn Screening Laboratories Network
10 µL 20 µL 50 µL 75 µL
Spot Volume & Punch Location(Haematocrit 50%)
30% 40% 45% 50%
Haematocrit (Always 50 µL spot punched in middle)
60%
50 µL spot punched in middle with haematocrit of 50% used as ‘standard’
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Results – Effect of spot size
and punch location
UK Newborn Screening Laboratories Network
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UK Newborn Screening Laboratories Network
C8 LEU MET PHE TYR
-25
-15
-5
5
15
25
35
45
10 ul Centre20 ul Centre75 ul Centre50 ul Edge75 ul Edge
Analyte
Perc
ent C
hang
e
Effect of spot size and punch location on new born screening results: Analytes at low (endogenous) concentrations
* = p <0.05 ** p = <0.001. n = 20 for each bar. Error bars represent +/- SEM.
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UK Newborn Screening Laboratories Network
TSH C8 LEU MET PHE TYR
-25
-15
-5
5
15
25
35
45
10 ul Centre20 ul Centre75 ul Centre50 ul Edge75 ul Edge
Analye
Perc
ent C
hang
e
****
**
**
Effect of spot size and punch location on new born screening results: Analytes at medium (lab alert) concentrations
*
** **
**** **
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* = p <0.05 ** p = <0.001. n = 20 for each bar.* = p <0.05 ** p = <0.001. n = 20 for each bar. Error bars represent +/- SEM.
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UK Newborn Screening Laboratories Network
TSH C8 LEU MET PHE TYR
-25
-15
-5
5
15
25
35
45
10 ul Centre20 ul Centre75 ul Centre50 ul Edge75 ul Edge
Analye
Perc
enta
ge C
hang
e
Effect of spot size and punch location on new born screening results: Analytes at high (clinical alert) concentrations
* = p <0.05 ** p = <0.001. n = 20 for each bar.* = p <0.05 ** p = <0.001. n = 20 for each bar. Error bars represent +/- SEM.
*
****
****
**
*
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**
****
**
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Results – Effect of
haematocrit
UK Newborn Screening Laboratories Network
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UK Newborn Screening Laboratories Network
-50
-40
-30
-20
-10
0
10
20
30
Octonyl Carnit-ineLeucineMethioninePhenylalanineTyrosine
**
*
Effect of haematocrit on new born screening results: Analytes at low (endogenous) concentrations
* = p <0.05 ** p = <0.001. n = 6 for each data point. Error bars represent +/- SEM.
LowAnalyte R2 p
TSH NA NAC8 0.08 0.181Leu 0.398 <0.001Met 0.297 0.006Phe 0.759 <0.001Tyr 0.337 0.003
30% 40% 45%60%
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UK Newborn Screening Laboratories Network
-20
-10
0
10
20
30
TSHOctonyl Carnit-ineLeucineMethioninePhenylalanineTyrosine
Effect of spot size and punch location on new born screening results: Analytes at medium (lab alert) concentrations
* = p <0.05 ** p = <0.001. n = 6 for each data point. Error bars represent +/- SEM.
** **
*
* Medium
Analyte R2 pTSH 0.815 <0.001C8 0.661 <0.001Leu 0.015 0.572Met 0.27 0.009Phe 0.31 0.005Tyr 0.283 0.008
30% 40% 45% 60%
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UK Newborn Screening Laboratories Network
-15
-10
-5
0
5
10
15
20
TSH Octonyl CarnitineLeucine MethioninePhenylalanine Tyrosine
Effect of spot size and punch location on new born screening results: Analytes at high (clinical alert) concentrations
* = p <0.05 ** p = <0.001. n = 6 for each data point. Error bars represent +/- SEM.
**
**
*
*
**
**
**
HighAnalyte R2 p
TSH 0.863 <0.001C8 0.734 <0.001Leu 0.36 0.002Met 0.581 <0.001Phe 0.805 <0.001Tyr 0.651 <0.00130% 40% 45% 60%
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UK Newborn Screening Laboratories Network
Analyte‘True’ result
Centre punch Hct
30%
Centre punch Hct
60%
Centre Punch 10 µL spot
TSH 4.6 6 4.1 4.15
C8 0.34 0.36 0.31 0.29
Leu 282.5 278.5 280.5 238.7
Met 34.5 32.7 34.9 27.6
Phe 216.8 204.5 220.4 179.5
Tyr 221.7 211.1 225.7 189.1
Laboratory alert level
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UK Newborn Screening Laboratories Network
Analyte‘True’ result
Centre punch Hct
30%
Centre punch Hct
60%
Centre Punch 10 µL spot
TSH 12 13.8 10.3 10.9
C8 0.69 0.75 0.64 0.64
Leu 531.8 494.0 551.0 469.3
Met 99.9 90.6 104.1 83.7
Phe 410.7 368.8 442.5 351.4
Tyr 418.4 383.2 448.9 352.4
Clinical alert level
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UK Newborn Screening Laboratories Network
0 5 10 15 20 250
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Hcts of babies in hospital
hct
Age in days
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UK Newborn Screening Laboratories Network
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.000.00
0.10
0.20
0.30
0.40
0.50
0.60
Hcts in patients monitored for PKU and Tyrosinaemia
hct
Age in years
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• Work with Finlay Mackenzie from UKNEQAS to confirm
our preliminary findings and extend haematocrit work• Prove that washed red cells with added plasma behaves
similarly• Extend analytes to include those for expanded nbs UK• Collect all Cardscan data on cards in study• Determine which Cardscan measurements will signal
significant departure from true results
UK Newborn Screening Laboratories Network
Future plans
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• Deliberately falsify manufactured bloodspots• Evaluate in Cardscan then measure analytes
UK Newborn Screening Laboratories Network
Later plans