Improved Immunoassay for the Detection of Severe Combined Immunodeficiency

To the Editor:

Testing for severe combined immunodeficiency (SCID) has recently become of interest to the newborn screening (NBS) community, because the detection of this condition in early infancy is crucial for effective treatment. Several newborn screening programs have implemented screening for this condition. The current method of screening for SCID is by T cell receptor excision circles (TREC) assay (1), which detects the presence or absence of an excised segment of DNA. However, molecular techniques are not routinely adapted in many NBS laboratories, where an immunoassay would be preferred. As recently reported (2), we developed an immunoassay that quantifies CD3, a component of the T cell receptor complex (3), to provide an alternative or complementary method to TREC testing. Low or absent T cells in peripheral blood is a common characteristic of T cell immunodeficiencies and in all but one form of SCID.

We have previously described an immunoassay using the Luminex microsphere technology that displays excellent potential for the detection of CD3 from dry blood spots specimens (2). Here we report the validation of an improved immunoassay on 124 dry blood specimens from the Danish Newborn Screening Biobank (4).

In our previous study we used a pair of commercial antibodies (2). However, because of lot to lot variation and availability issues we felt a more reliable supply of antibody was necessary to make a dependable assay. We contracted (Invitrogen) for the production of antigen specific avian antibodies to human CD3 because IgY antibodies have been shown to be stable for long term storage and do not bind to human Fc receptors, rheumatoid factor or complement. Therefore, nonspecific binding of interfering substances is greatly reduced. IgY antibodies are purified from chicken egg yolks with an egg laying hen producing enough eggs in a week to purify antibodies equivalent to the amount of antibodies in 75-90 mL of mammalian serum (5). Our study demonstrates that the custom IgY antibodies to CD3 have low background and are highly specific.

When we compared the commercial anti-CD3 detection antibody to the avian anti-CD3 detection antibody, the avian IgY antibody proved to have a more robust performance than the IgG antibody; less antibody is required per assay, and the IgY has an expended linear range from 0.2 × 10⁶ to 15 × 10⁶ T cells/mL, while the commercial antibody had a linear range of 1.0 × 10⁶ to 7.5 × 10⁶ T cells/mL. Other improvements to the protocol from the previously published assay are: reduction of incubation time of blood eluate with microspheres from 3 hours to 2 hours and removal of the amplification step and its associated wash steps. This enhanced assay protocol consisted of seven steps instead of 11 steps in the previous assay, these include washes between each incubation, incubation of eluate with microspheres, incubation with detection antibody, incubation with streptavidin phycoerythrin, resuspention of microspheres, and data collection on the Luminex 100 (Luminex Corp.).

In order to validate this improved assay we measured the CD3 concentration of 124 coded neonatal dry blood spots obtained from The Danish Newborn Screening Biobank. These results were sent to Drs. Bent Nørgaard-Pedersen and Carsten Heilmann for decoding. The decoded results revealed that normal infant samples had T cell counts which ranged from 2.14 × 10⁶/mL to >16 × 10⁶/mL. Eleven samples were from infants with T cell related immunodeficiencies; nine of these eleven samples were from infants diagnosed with SCID, of which one contained 25% maternal engraftment of T cells. The remaining two specimens were from infants diagnosed with Omenn syndrome and Wiskott Aldrich syndrome. The affectd infants had lower estimated T cell counts than controls, they ranged from below our limit of detection of 0.2 × 10⁶/mL to 1.07 × 10⁶/mL.

To date, the CD3 immunoassay we developed using these antibodies has been able to correctly identify specimens with low or undetectable T cells from various forms of SCID and other immunodeficiencies. Because of its enhanced performance, ease of use and ability to correctly identify T cell deficiency, this assay should be considered a complimentary or alternative to other dry blood spot T cell detection techniques.