P reliminary studies

Preliminary studies

Imperfect bloodspots – what matters for the screening laboratory?

UK Newborn Screening Laboratories Network

Alex Lawson*Kate Hall

* Birmingham Heartlands Hospital

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


•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


•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


What is known to affect measured results

•Variability of filter paper•Peripheral or central punching•Haematocrit of blood applied


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.


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


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.


Where do we punch?

•In the centre of a full spot?•Using a Multipuncher™ or Panthera ™?


Typical first punch positions


Typical second punch positions


How do we evaluate quality?

•Visually•New – Luminex Cardscan


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


Experimental Design


• 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


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


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%

• 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



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’

Results – Effect of spot size

and punch location



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.

***

*

* **

*

*


TSH C8 LEU MET PHE TYR

-25

-15

-5

5

15

25

35

45


Analye

Perc

ent C

hang

e

****

**

**

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.


TSH C8 LEU MET PHE TYR

-25

-15

-5

5

15

25

35

45


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.

*

****

****

**

*

**

****

**

****

**

**

Results – Effect of

haematocrit



-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%


-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


** **

*

* 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%


-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


**

**

*

*

**

**

**

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%


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


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


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


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

• 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


Future plans

• Deliberately falsify manufactured bloodspots• Evaluate in Cardscan then measure analytes


Later plans

Documents

P reliminary studies