Specimen by Immunoassay

Transcription

1 SCID Detection in the Guthrie Specimen by Immunoassay By David Janik Wadsworth Center Division of Translational Medicine Albany, NY

2 SCID Screening Challenge Detection of SCID in a single 3mm punch (1.5 µl of serum) Exceptional Outcome Condition detectable by NBS for which an effective treatment is available, when identified and treated in early infancy The only test currently available with the potential of detecting SCID in the Guthrie specimen is the TREC assay DNA as First Tier testing Uncommon, usually more costly, most labs are not equipped Immunoassays are used routinely in NBS as a first- tier screening protocol

3 SCID Screening Challenge Detection of SCID in a single 3mm punch (1.5 µl of serum) Exceptional Outcome Condition detectable by NBS for which an effective treatment is available, when identified and treated in early infancy The only test currently available with the potential of detecting SCID in the Guthrie specimen is the TREC assay DNA as First Tier testing Uncommon, usually more costly, most labs are not equipped Immunoassays are used routinely in NBS as a first- tier screening protocol

4 SCID Screening Challenge Detection of SCID in a single 3mm punch (1.5 µl of serum) Exceptional Outcome Condition detectable by NBS for which an effective treatment is available, when identified and treated in early infancy The only test currently available with the potential of detecting SCID in the Guthrie specimen is the TREC assay DNA as First Tier testing Uncommon, usually more costly, most labs are not equipped Immunoassays are used routinely in NBS as a first- tier screening protocol

5 SCID Screening Challenge Detection of SCID in a single 3mm punch (1.5 µl of serum) Exceptional Outcome Condition detectable by NBS for which an effective treatment is available, when identified and treated in early infancy The only test currently available with the potential of detecting SCID in the Guthrie specimen is the TREC assay DNA as First Tier testing Uncommon, usually more costly, most labs are not equipped Immunoassays are used routinely in NBS as a first- tier screening protocol

6 SCID Screening Challenge Detection of SCID in a single 3mm punch (1.5 µl of serum) Exceptional Outcome Condition detectable by NBS for which an effective treatment is available, when identified and treated in early infancy The only test currently available with the potential of detecting SCID in the Guthrie specimen is the TREC assay DNA as First Tier testing Uncommon, usually more costly, most labs are not equipped Immunoassays are used routinely in NBS as a first- tier screening protocol

7 Intent D l d bl i lti l Develop an upgradeable primary multiplex immunoassay for SCID screening

8 Biomarkers CD3 Specific marker for T Cells CD45 Common leukocyte antigen. Used as internal control

9 Goals for our SCID Immunoassay Immunoassay for the detection of T cell deficiency in DBS Multiplex formatting (Luminex), allowing for the addition of other biomarkers Use only one 3mm punch (1.5 μl of serum) for sample and internal control Ghost spots can be used in the TREC assay Fast, Easy, Cost effective Kit and Automation

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11 TREC-positive specimens TREC tested specimens and Controls* Number of cells x 10 6 CD3 Classification Diagnosis Low Positive ADA 4.1 Negative Ctrl 10.8 Negative Ctrl Low Positive PNP 5.5 Negative Ctrl 0.4 Positive X-linked Low Positive X-linked Low Positive X-linked 8 coded specimens from MA-NBS; 5 positive; one, maternal engrafted; all correctly identified

12 Confirmation of TREC results Ghost specimens were returned to MA-NBS for TREC analysis and all designations remained the same Our immunoassay does not interfere with the TREC screening protocol as used in the MA-NBS program

13 CD3 antibody detection comparison Commercial Custom The Custom antibody has less deviation among replicates, increased lower limit of detection, and decreased background

14 Liquichip Generated Std Curves Standard curves from a multiplex of designed CD3 antibody pair and the CD45 antibody pair

15 Cross-reaction reaction test CD3 x CD45 det MFI Number of Leukocytes x 10^6/mL Blood MFI CD45 x CD3 det Number of Leukocytes x 10^6/mL Blood Lack of cross-reaction reaction between CD3 and CD45 antibodies

16 Statens Serum Institut Specimens 127 coded specimens from Statens Serum Institut, Denmark Positives were diagnosed clinically

17 127 Coded Guthrie Specimens

18 Staten Serum Institut Samples Number sex (m/f) Diagnosis CD3 MFI 1 x 10^6 CD3 + Cells/mL 1 m Wiskott Aldrich Syndrome m Omenn Syndrome 394 LOW 17 m SCID 180 LOW 29 m SCID 282 LOW 41 f SCID 125 LOW 81 f SCID f SCID m SCID m SCID 420 LOW 113 m SCID m SCID 289 LOW 127 specimens from Denmark, 11 positive, all correctly identified.

19 Distribution of Normal and SCID CD3 Follows a Normal distribution amples mber of Sa Num MFI

20 Newborn weight greater than 1750g, N = 448 Newborn weight less than 1750g, N = 128

21 Detectable T cell deficiencies Absence of T and B cells (SCID) Absence of T cells (normal B cells) (SCID) SCID other unspecified form Adenosine deaminase deficiency (ADA) Purine Nucleoside Phosphorylase deficiency (PNP) X-Linked and X-Linked with maternal engraftment MA-NBS Wiskott Aldrich Syndrome Omenn Syndrome

22 Conclusions Our immunoassay can distinguish between low and normal T cell counts CD45 works well as an internal control Our immunoassay has the potential to indicate SCID or other T cell deficient variants Elution does not interfere with the MA-NBS TREC assay Future Expand assay to include biomarkers for B cells Expand assay to include biomarkers for B cells and NK cells

23 Acknowledgements The Lab of Kenneth Pass, PhD Barbara Shepard, PhD Daniela Hila Keith Friedman New York State Newborn Screening Program Michele Caggana, PhD Statens Serum Institut, Denmark Bent Nørgaard-Pedersen, MD, PhD Massachusetts Newborn Screening Program Anne Comeau, PhD and Laboratory

24 Funding This work was supported by NIH Contract ADB-NO1-DK (HHSN , K. Pass PI), Novel Technologies in Newborn Screening, and Luminex, Corp Additional support was provided by CDC Cooperative Agreement (1U01EH000362, A. Comeau PI), Implementing SCID NBS with Multiplexed Assays in an Integrated Program Approach.

25 Fin