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  1. UK Newborn Screening Laboratories Network Preliminary studies Imperfect bloodspots – what matters for the screening laboratory? Alex Lawson* Kate Hall * Birmingham Heartlands Hospital

  2. Supersaturated Quantity Insufficient for Testing Specimen Not DriedBefore Mailing Scratched or Abraded Serum Rings Diluted, Discolored, or Contaminated Clotted or Layered No Blood Poor Sampling – examples from S&S poster Supersaturated Specimen Not Dried Before Mailing

  3. 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

  4. UK Newborn Screening Laboratories Network Study prompted by emergence of LuminexCardscan 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

  5. UK Newborn Screening Laboratories Network What is known to affect measured results Variability of filter paper Peripheral or central punching Haematocrit of blood applied

  6. UK Newborn Screening Laboratories Network Variability of filter paper Noted in 1970s by CDC  blood absorption specifications created Slazyk WE, Phillips DL, Therrell BL Jr, Hannon WH. Effect of lot-to-lot variability in filter paper on the quantification of thyroxin, thyrotropin, and phenylalanine in dried-blood specimens. ClinChem 1988;34:53-8.

  7. 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 measured by tandem mass spectrometry ClinicaChimicaActa 373 (2006) 27–31 15 mm spots

  8. UK Newborn Screening Laboratories Network Haematocrit Hill JB and Palmer P Filter Paper Blood Collection and Punching As A Means of Quantification ClinChem 1969, 15/5; 381-389 The hematocrit value was found to have an influence on the volume of blood contained in a punched spot.

  9. UK Newborn Screening Laboratories Network Where do we punch? In the centre of a full spot? Using a Multipuncher™ or Panthera ™?

  10. UK Newborn Screening Laboratories Network

  11. UK Newborn Screening Laboratories Network Typical first punch positions

  12. UK Newborn Screening Laboratories Network Typical second punch positions

  13. UK Newborn Screening Laboratories Network How do we evaluate quality? Visually New – LuminexCardscan

  14. UK Newborn Screening Laboratories Network 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

  15. UK Newborn Screening Laboratories Network Experimental Design

  16. UK Newborn Screening Laboratories Network • 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 concentration OR • 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

  17. UK Newborn Screening Laboratories Network Spot Volume & Punch Location (Haematocrit 50%) 10 µL 20 µL 50 µL 75 µL Haematocrit (Always 50 µL spot punched in middle) 30% 60% 40% 45% 50%

  18. UK Newborn Screening Laboratories Network • 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

  19. UK Newborn Screening Laboratories Network Spot Volume & Punch Location (Haematocrit 50%) 50 µL spot punched in middle with haematocrit of 50% used as ‘standard’ 10 µL 20 µL 50 µL 75 µL Haematocrit (Always 50 µL spot punched in middle) 30% 60% 40% 45% 50%

  20. UK Newborn Screening Laboratories Network Results – Effect of spot size and punch location

  21. UK Newborn Screening Laboratories Network 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.

  22. UK Newborn Screening Laboratories Network 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 = 20 for each bar. * = p <0.05 ** p = <0.001. n = 20 for each bar. Error bars represent +/- SEM.

  23. UK Newborn Screening Laboratories Network 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. Error bars represent +/- SEM. * = p <0.05 ** p = <0.001. n = 20 for each bar.

  24. UK Newborn Screening Laboratories Network Results – Effect of haematocrit

  25. UK Newborn Screening Laboratories Network Effect of haematocrit on new born screening results: Analytes at low (endogenous) concentrations 40% 30% 45% 60% * = p <0.05 ** p = <0.001. n = 6 for each data point. Error bars represent +/- SEM.

  26. UK Newborn Screening Laboratories Network Effect of spot size and punch location on new born screening results: Analytes at medium (lab alert) concentrations ** ** * * 40% 45% 60% 30% * = p <0.05 ** p = <0.001. n = 6 for each data point. Error bars represent +/- SEM.

  27. UK Newborn Screening Laboratories Network Effect of spot size and punch location on new born screening results: Analytes at high (clinical alert) concentrations ** * ** ** * ** ** 40% 45% 60% 30% * = p <0.05 ** p = <0.001. n = 6 for each data point. Error bars represent +/- SEM.

  28. UK Newborn Screening Laboratories Network Laboratory alert level

  29. UK Newborn Screening Laboratories Network Clinical alert level

  30. UK Newborn Screening Laboratories Network Age in days

  31. UK Newborn Screening Laboratories Network Age in years

  32. UK Newborn Screening Laboratories Network Future plans • 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

  33. UK Newborn Screening Laboratories Network Later plans • Deliberately falsify manufactured bloodspots • Evaluate in Cardscan then measure analytes