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Introduction to Severe Combined Immunodeficiencies (SCID) and Newborn Screening for SCID

Part 2

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Published: March 2013

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Once diagnosed accurately, newborns with SCID/T cell lymphopenia can appropriate management including life-sustaining treatment. Delays in identification of affected infants rapidly reduce the success of such treatments and significantly increase mortality. In Part 2 of this 2-part presentation, Dr. Abraham discusses the use of appropriate laboratory tests and test interpretation tools for follow-up of abnormal newborn screen for SCID/T cell lymphopenia cases.

Presenter: Roshini Abraham, PhD

  • Associate Professor of Laboratory Medicine/Pathology and Medicine at Mayo Clinic
  • Consultant in the Division of Clinical Biochemistry and Immunology at Mayo Clinic in Rochester, Minnesota
  • Director of the Cellular and Molecular Immunology Laboratory within the Department of Laboratory Medicine and Pathology
  • Director of the Jeffrey Modell Foundation for Primary Immunodeficiencies Diagnostic and Research Center at the Mayo Clinic

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Part 2

To follow-up on abnormal TREC results obtained on the newborn screen for SCID/T-cell lymphopenia, we have identified a set of tests that we call Tier 1, that are used for the immediate postabnormal newborn screen result. The tests used for this level of follow-up testing can vary from state to state. In our Tier 1 testing, we include 3 tests: T-cell, B-cell, and NK-cell subset quantitation by flow cytometry (TBBS/9336), CD4 Recent Thymic Emigrant quantitation (RTE) by flow cytometry (CD4RT/89504), and (TREC/87959 TREC) quantitation in whole blood (as opposed to dried blood spots which is what is typically used in the newborn screening program) where the TREC result is reported as TREC copies per million CD3 T cells. Additionally, from the CD4 recent thymic emigrants (RTE) flow assay, the relative proportions of CD4+45RA+ naïve T cells and CD4+45RO+ memory T cells can also be provided.

Using an example of a patient with ADA-SCID, the value of T-cell, B-cell, and NK-cell quantitation by flow cytometry is demonstrated. In this assay, the absolute lymphocyte count (which is the CD45+ lymphocytes) is obtained in addition to the percent and absolute counts for CD3 T, CD4, and CD8 T cells, CD19 B cells, and CD16+56+ NK cells. As indicated in part 1 of this presentation, there are several genetic defects associated with SCID. The use of TBNK quantitation by flow cytometry can narrow down the list of possible genetic defects for a specific patient. In the example shown in this slide, T, B, and NK cells are almost close to absent, therefore, the phenotype is T-B-NK- SCID, of which the most common genetic defect is ADA deficiency, which was confirmed in this patient by genetic testing and measurement of ADA enzyme levels.

This slide shows another example of T-cell, B-cell, and NK-cell quantitation, however, the emphasis here is on the modest T-cell lymphocytosis as well as the presence of an unusual population of CD3+CD4-CD8- T cells circled in red on the slide. This patient had undetectable TREC in whole blood analysis. Further investigations revealed a diagnosis of SCID with Omenn syndrome, no evidence of maternal engraftment, and the molecular defect was not identified.

This slide shows an example of CD4RTE (recent thymic emigrant) analysis by flow cytometry. The upper set of figures shows the stepwise identification of CD4 T cells, segregation of CD4 T cells into CD45RA+ and CD45RO+, which in the upper left quadrant of the panel and the third panel demonstrates the presence of naïve T cells by evaluating CD31 expression on CD4+CD45RA+T cells demonstrated in the box within the third panel. The lower half represents the patient’s data (same patient as described on the previous slide). As the first panel shows, CD4 T cells are present in this patient; however, unusually for a young infant, there are almost no naïve CD45RA+ T cells and the majority of the CD4 T cells express the memory marker CD45RO, shown in the upper left quadrant of the middle panel, and there are no cells within the CD45RA box, which is the lower right quadrant. This is further substantiated by the absence of CD31 expression on the CD45RA+ T cells. This phenotype is consistent with the patient’s previously described Omenn syndrome where there is oligoclonal expansion of T cells with few to no naïve T cells, and the majority expressing the CD45RO memory T-cell marker.

This slide is a continuation of the analysis of the previous patient; however, this is data from studies performed posthematopoietic cell transplant. This data is Day + 70 posttransplant. Again, in the lower panel, the second and third graphs show a small but noticeable increase in naïve T cells, but certainly not yet evidence of complete naïve T-cell recovery or thymic reconstitution.

This patient was reassessed periodically posthematopoietic cell transplantation and at almost 2 years posttransplant, the patient shows evidence thymic-derived T-cell reconstitution with naïve CD45RA+ T cells that also express the recent thymic emigrant marker, CD31, panels 2 and 3. This is a success story for this patient!

The TREC assay at Mayo was initiated in 2007 and uses whole blood as the sample of choice. The assay is performed by quantitative real-time PCR. The result is expressed as copies per million CD3 T cells so that TREC copies can be assessed relative to the patient’s T-cell count. In the TREC report, the flow cytometric quantitation is also provided for CD3, CD4, and CD8 T cells. Further, a new reference range, which specifically encompasses greater granularity in the pediatric population, has been included in the assay.

This slide shows the pediatric TREC reference values and, as expected, there is an age-related decrease in TREC from infancy to postpuberty, whether looking at the graph showing the 95% confidence interval on the mid-95% range, on the left-hand side, or the 95% confidence interval on the fifth percentile on the right-hand side.

Though adult TREC data is not really relevant to a discussion of newborn screening, the normal range reference data is shown to draw a comparison with TREC data in the pediatric population. It is useful to note that thymic activity in adults does not cease after puberty, but continues to contribute to T-cell homeostasis, albeit at a much reduced level, till the sixth to seventh decade of life.

For patients who have abnormal results on the Tier 1 follow-up tests, it is reasonable to proceed to Tier 2 testing. Again, the definition of what comprises Tier 2 testing can vary from state to state and clinician to clinician. However, it would seem logical to follow-up the Tier 1 quantitative flow testing with functional assessment of lymphocytes by measuring proliferative response to mitogens, specifically phytohemagglutinin (PHA). Also, if the TBNK quantitation, in addition to the clinical and/or family history, is suggestive of a specific genetic defect or group of defects, genetic testing may be simultaneously initiated. If there is evidence of T cells in circulation or T-cell lymphocytosis as in the case described previously, maternal engraftment should be excluded by performing FISH analysis for sex chromosomes in male infants, or short-tandem repeat (STR) analysis in female infants. If there is suspicion of enzyme deficiencies, such as ADA-SCID, ADA levels can be quantitated and this testing is available through the laboratory of Dr. Michael Hershfield at Duke University).

An example is provided in this slide of measurement of lymphocyte proliferation to mitogens, specifically PHA, by flow cytometry. The bar graph shows the quantitative data of percent proliferating CD3+ T cells in a freshly prepared normal control, control samples that have been aged for various time points, and the patient sample, indicated in the black bar. The patient sample clearly shows less than 10% proliferation to PHA, which would be consistent with a SCID phenotype, if other relevant features and test results are also present.

The flow cytometric plots below merely demonstrate the advantage of visual confirmation of cellular proliferation by the presence of blast cells as seen in the top panel. Also, the use of specific antibodies allows discrimination of specific cell populations for example, total CD45+ lymphocytes demonstrated in the green histogram, and CD3+ T cells depicted in the blue histogram. The bottom panel shows the quantitative component where the percent of proliferating cells, whether they are total lymphocytes or CD3+ T cells, can be estimated. And the little box to the left of the lower panel shows the percent values that can be used for quantitation.

The flow cytometric assay for measuring lymphocyte proliferation using Edu has distinct advantages over the standard and still widely used tritiated thymidine (3H-t) assay. In the case of SCID and T-cell lymphopenia patients, it is particularly valuable, because cellular dilution caused by lymphopenia can create a “false-negative” result with abnormally low proliferation. The thymidine assay cannot discriminate such false-negative results because it is an assay performed on a “bulk cell” population with no specific marker or modality to identify cellular subsets. Since specific cellular markers can be used in the flow assay, it provides an enhanced level of sensitivity, in addition to visual assessment of proliferating cells. The flow assay measures proliferation for both total CD45+ lymphocytes and CD3+ T cells. The CD45+ lymphocyte data in the flow assay is broadly comparable to the data obtained from the thymidine assay. It is the CD3+ T-cell proliferation that provides the greater sensitivity in the flow assay for measuring proliferation. If the percent CD3+ T-cell proliferation is <10% to PHA, it should trigger a possible diagnosis of SCID or a severe functional T-cell defect in children ≤2 years of age.

An example of the increased sensitivity of the flow proliferation assay is shown in this slide. This sample was obtained from a female patient with profound T-cell lymphopenia. As expected, the CD45+ lymphocyte proliferation is significantly decreased; however, the CD3+ T-cell proliferation is within normal limits. If the thymidine assay had been performed on this patient, she would have been considered to have an abnormal proliferative response to PHA (due to the cellular dilution from the lymphopenia); however, the ability to further analyze the T-cell compartment by flow, provided additional value to the analysis by revealing the presence of normal, functional T cells. Therefore, it is important to have the means to discriminate between true nonfunctional T-cell responses and diminished numbers of T cells with preserved T-cell function.

Newborn screening for SCID can result in identification of infants with T-cell anomalies that do not fit neatly into any of the standard categories and, therefore, additional analyses may be required to identify or classify the immunological defect. Therefore, Tier 3 testing may be necessary for some infants. Tier 3 tests could include additional measurement of lymphocyte function, such as anti-CD3 stimulation of T cells and assessment of proliferation poststimulation; measurement of cytokine production in activated mitogen-stimulated T cells called polyfunctional T-cell analysis; multicolor flow cytometry to look at the T-cell subsets in greater detail by immunophenotyping; measurement of T-cell receptor repertoire diversity; chromosomal studies; lymphocyte proliferation to antigens, which is relevant to older and not newborn infants; radiation sensitivity (identification of radiosensitive SCID); quantitative immunoglobulins; NK-cell function and phenotyping; and other immunological analyses as deemed relevant by clinical history and previous workup. The assays colored in pink are not yet orderable clinically from Mayo, but the anti-CD3 proliferation panel, NK cell-subset immunophenotyping panel, and T-cell receptor analysis by Vb spectratyping will become clinically orderable from Mayo by the end of the year. The radiation sensitivity analysis is currently available only from the laboratory of Dr. Richard Gatti at UCLA.

An example is shown of the T-cell receptor Vb spectratyping assay used to measure TCR-repertoire diversity. In this assay, 24 different TCR Vb families are assessed by PCR and fragment-length analysis. The spectratypes (also called T-cell immunoscope) for the patient is depicted in red, while the data for an adult normal control is shown in green. A limited number of families are shown here for purpose of illustration. Typically, in normal individuals there is a polyclonal, which means multiple peaks, with Gaussian distribution of CDR3 length in the spectratype analysis. However, in this patient with DiGeorge syndrome and an Omenn-like phenotype, there is CD8 T-cell lymphocytosis with CD4 T-cell lymphopenia, and the T-cell receptor spectratype is dramatically skewed with either no representation, or oligoclonal or monoclonal peaks for several families. This analysis is very helpful for patients with leaky SCID or Omenn syndrome, DiGeorge syndrome, and other partial T-cell lymphopenias, as well as for assessment of T-cell reconstitution and diversity posthematopoietic cell transplantation.

This slide shows CD4RTE flow analysis for the patient described in the previous slide. As mentioned earlier, there is evidence of little to no CD45RA+ CD4+ naïve T cells with the majority expressing CD45RO (memory marker, as shown in middle panel). The few CD45RA+ T cells that are present are negative for CD31, which is a marker associated with naïve T cells that typically have not undergone cell division as seen in the third panel. This phenotype is supportive of a leaky SCID, which is substantiated also by the skewed TCR-diversity analysis as shown on the previous slide.

This slide shows an example of additional Tier 3 testing for newborn screening SCID follow-up. This patient presented with a T-B-NK- phenotype, an absolute lymphocyte count of 100, severe pneumonia, and a diffuse, erythematous skin rash. Subsequent analysis revealed this patient had ADA-SCID deficiency. The patient was initiated on PEG-ADA, while evaluation for a suitable donor for hematopoietic cell transplantation was underway. On treatment with steroids for the skin rash, there was a dramatic and sudden increase in the NK-cell counts in blood (lower bar graph with each color representing a date of testing). To identify which type of NK cells were associated with this influx of NK cells in blood, NK-cell subset immunophenotyping, mentioned in a previous slide, was performed. Typically, the majority (90%) of circulating NK cells are of the cytotoxic that is CD16+56+ phenotype, while a minority (10%) are cytokine-producing (CD56+++CD16+/-) NK cells. In this patient, NK-subset immunophenotyping revealed a significant increase in the cytokine-producing NK cells. There was also fairly dramatic clearing of the skin lesion after steroid treatment (lower-right photograph), suggesting that the increase in NK cells in blood was a direct consequence of release of NK cells from the skin rash into circulation that is due to steroid-associated NK cell retrafficking.

A report by Shibata et al in the literature indicated that a similar phenomenon was previously seen in a patient with X-linked SCID, which is caused by mutations in the common gamma chain, who had a diffuse skin rash with Omenn-like features, very similar to the above-mentioned ADA-SCID patient. In the Shibata report, there were cytokine-producing (CD56+++CD16+/-) NK cells infiltrating the skin, and flow cytometric analysis revealed a pattern very similar to that seen in our ADA-SCID patient, the flow data shown on right of the slide. Therefore, there may be a spectrum of manifestations of SCID or T-cell lymphopenia, which necessitates multistep testing, as described in this presentation.


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