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

2. 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 Testing 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 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

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. Clin Chem 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 Clinica Chimica Acta 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 Clin Chem 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. Typical first punch positions UK Newborn Screening Laboratories Network

12. Typical second punch positions UK Newborn Screening Laboratories Network

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

14. 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

15. Experimental Design UK Newborn Screening Laboratories Network

16. 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 UK Newborn Screening Laboratories Network LevelTSHOctanoyl CarnitineLeucineL-MethionineL-PhenylalanineL-Tyrosine Low (endogenous)NA0.09199.8418.3874.8672.33 Medium (laboratory based alert)4.150.34291.8955.68216.27225.77 High (clinical alert level)11.60.68576.5997.25407.50417.65

17. UK Newborn Screening Laboratories Network 10 µL20 µL50 µL75 µL Spot Volume & Punch Location (Haematocrit 50%) 30% 40%45%50% Haematocrit (Always 50 µL spot punched in middle) 60%

18. 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

19. 10 µL20 µL50 µL75 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’

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

21. 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.* = p <0.05 ** p = <0.001. n = 20 for each bar. Error bars represent +/- SEM. * ** *

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

25. 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. Low AnalyteR2R2 p TSHNA C80.080.181 Leu0.398<0.001 Met0.2970.006 Phe0.759<0.001 Tyr0.3370.003 30% 40% 45% 60%

26. 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 = 6 for each data point. Error bars represent +/- SEM. ** * * Medium AnalyteR2R2 p TSH0.815<0.001 C80.661<0.001 Leu0.0150.572 Met0.270.009 Phe0.310.005 Tyr0.2830.008 30% 40% 45% 60%

27. 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 = 6 for each data point. Error bars represent +/- SEM. ** * * High AnalyteR2R2 p TSH0.863<0.001 C80.734<0.001 Leu0.360.002 Met0.581<0.001 Phe0.805<0.001 Tyr0.651<0.001 30% 40% 45% 60%

28. UK Newborn Screening Laboratories Network Analyte ‘True’ resultCentre punch Hct 30% Centre punch Hct 60% Centre Punch 10 µL spot TSH4.664.14.15 C80.340.360.310.29 Leu282.5278.5280.5238.7 Met34.532.734.927.6 Phe216.8204.5220.4179.5 Tyr221.7211.1225.7189.1 Laboratory alert level

29. UK Newborn Screening Laboratories Network Analyte ‘True’ resultCentre punch Hct 30% Centre punch Hct 60% Centre Punch 10 µL spot TSH1213.810.310.9 C80.690.750.64 Leu531.8494.0551.0469.3 Met99.990.6104.183.7 Phe410.7368.8442.5351.4 Tyr418.4383.2448.9352.4 Clinical alert level

30. UK Newborn Screening Laboratories Network Age in days

31. UK Newborn Screening Laboratories Network Age in years

32. 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

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