TREC screening in preterm infants: navigating borderline results

In preterm infants, however, the same result carries a different diagnostic weight than it does in a full-term newborn. Immature thymic output, low circulating lymphocyte numbers, placental disease, respiratory illness, and the practical conditions of intensive care can all reduce the signal on a dried blood spot.

Reported median TREC concentrations are lower in infants born at 30–36 weeks of gestation than in those born at 37–42 weeks. One clinical comparison placed the median at approximately 237 TRECs/µL in the preterm group versus approximately 300 TRECs/µL in full-term peers. The difference is not merely laboratory noise. It reflects developmental biology: the preterm thymus is still expanding its capacity to generate recent thymic emigrants, and the peripheral T-cell pool is being established under conditions of substantial physiologic stress.

That is why t-cell receptor excision circles interpretation in preterm infants cannot rest on a single number. A borderline result is a screening signal, not a diagnosis. The result has to be read alongside gestational age, birth weight, the infant’s clinical course, the timing and quality of the blood spot, the laboratory cutoff, and—when indicated—the absolute lymphocyte profile measured by flow cytometry.

The Physiology of Immature Thymic Output: Why Preterm TREC Levels Differ

What TREC quantification actually measures

T-cell receptor excision circles are small, circular DNA fragments formed during T-cell receptor rearrangement in the thymus. They are produced when developing T cells assemble their receptor genes and are released into the cell as byproducts of that process. Because TRECs do not replicate when a T cell divides, they are most informative as markers of recent thymic emigrants rather than of the entire T-cell population.

Newborn screening laboratories generally quantify TRECs by real-time quantitative PCR using DNA extracted from a dried blood spot collected on a Guthrie card. The assay is usually normalized to a single-copy reference gene, such as albumin or β-actin, to indicate whether the specimen contains an adequate amount of amplifiable DNA. The result is reported as a TREC concentration or copy number, depending on the platform.

In a healthy newborn, a substantial proportion of circulating T cells are naive cells that have recently left the thymus. The TREC signal therefore provides an indirect view of thymic production. It does not count functional T cells directly, does not identify a specific genetic cause, and does not establish whether the immune system can respond normally to stimulation.

That distinction matters in the NICU. A low signal can arise because the thymus is producing fewer new T cells, because fewer lymphocytes are circulating in the sample, because the infant is acutely ill, or because the specimen does not contain enough usable DNA. The assay is valuable precisely because it is sensitive to major defects in T-cell production—but that sensitivity also makes it vulnerable to developmental and clinical confounders.

Gestational age as a direct modifier of TREC values

Gestational age is one of the most consistent biological factors affecting TREC levels. The thymus is active before birth, but its structure and output continue to mature during late gestation. Infants born very early therefore enter extrauterine life with a smaller and less mature thymic system than full-term infants.

The effect is not an abrupt switch at one precise week of gestation. TREC values tend to fall as gestational age decreases, with the lowest values seen among the most premature infants. Studies often identify particularly low results in infants born before 28 weeks, but the relevant interpretation depends on the screening program and the population used to establish its reference range.

A preterm infant can consequently have a TREC concentration below the routine cutoff without having a pathogenic disorder of T-cell development. The result is biologically plausible even when the infant has no infection, no family history of immunodeficiency, and no other clear sign of immune dysfunction.

This is the central reason that low TREC levels in premature newborns are often managed differently from an absent or nearly absent result in a full-term infant. The question is not simply whether the value is below the cutoff. The question is how far below it is, whether it is detectable, whether the infant is clinically stable, and whether the result persists as the infant matures.

Platform variability and cutoff heterogeneity

TREC values are not interchangeable across screening platforms. Laboratories use different extraction methods, amplification systems, reference genes, reporting units, and decision thresholds. A number that is considered borderline on one platform may fall into a different interpretive category on another.

For example, commonly used assays may operate with thresholds in different ranges:

These values should be treated as examples of platform-specific practice, not as universal clinical limits. The laboratory’s own cutoff and reporting language take precedence. A result labeled “borderline,” “inconclusive,” or “repeat required” is not equivalent to a result reported as “no TREC detected.”

A low TREC result in a preterm infant is a signal of reduced or uncertain thymic output—not a diagnosis by itself.

The quality of the dried blood spot also matters. An underfilled card, an uneven blood application, a specimen collected very soon after birth, or DNA degradation can affect the reliability of the measurement. When the result and the clinical picture do not fit, the laboratory may need to assess specimen adequacy before the clinical team interprets the number as a genuine immunologic abnormality.

Perinatal Stressors and Their Impact on Thymic Involution

Prematurity is only one part of the explanation for borderline screening results. The fetal and neonatal environment can suppress thymic output temporarily, particularly when the infant has experienced prolonged inflammation, hypoxia, nutritional compromise, or severe respiratory illness.

Intrauterine growth restriction and maternal disease

Intrauterine growth restriction (IUGR) and maternal hypertensive disorders have been associated with lower TREC values. These conditions can expose the fetus to chronic placental insufficiency and altered endocrine and inflammatory signaling. The effect on the thymus is not necessarily permanent. It may reflect a period in which immune-cell production is reduced while the infant adapts to a stressful perinatal environment.

A similar caution applies when several factors occur together. A very preterm infant with IUGR and a history of maternal hypertension may have a lower baseline TREC value than an otherwise comparable preterm infant. The combined effect can make the screen look more concerning without proving a primary T-cell disorder.

Leukopenia at the time of specimen collection is another important modifier. Since the assay is performed on DNA from whole blood, a low white-cell or lymphocyte count can contribute to a reduced TREC concentration. The result should therefore be interpreted with the complete blood count, including the absolute lymphocyte count, rather than in isolation.

Respiratory distress and acute neonatal illness

Respiratory distress syndrome (RDS), sepsis evaluations, surgery, and other intensive-care events can complicate interpretation. Acute illness activates stress pathways and inflammatory mediators that may contribute to transient thymic involution. The infant may also receive treatments or undergo procedures that change circulating cell counts over a short period.

Placental pathology can add another layer. Severe placental lesions, including massive perivillous fibrin deposition, have been associated with fetal stress and reduced thymic output. In such cases, the low TREC result may reflect the combined effects of gestational immaturity and a difficult intrauterine course.

None of this means that a sick preterm infant with a low TREC result can be assumed to have a benign explanation. SCID and other primary T-cell deficiencies can occur in infants who are also premature or critically ill. The point is narrower and more practical: perinatal stressors change the pretest probability and often justify a staged approach for detectable, non-urgent abnormalities.

Timing is part of the result

The day of life on which the blood spot was collected affects interpretation. A specimen taken very early in the neonatal period may capture the infant before thymic output and circulating lymphocyte numbers have stabilized. A later specimen may be influenced by transfusion, severe illness, medication exposure, or recovery from the initial stress.

The screening laboratory and the NICU team should therefore document:

• Gestational age and birth weight
• Postnatal age at collection
• Whether the sample was adequate and collected before or after transfusion
• The infant’s white-cell and absolute lymphocyte counts
• Respiratory, infectious, or inflammatory complications
• Maternal hypertension, placental disease, or IUGR
• Whether the infant has a known family history of immunodeficiency

This information does not replace confirmatory testing. It helps determine how quickly testing should proceed and which result is most informative next.

Navigating Borderline Results: Clinical Decision-Making in the NICU

Repeat DBS sampling at corrected gestational age

For a detectable but borderline result in a clinically stable preterm infant, repeat dried blood spot sampling is commonly used to determine whether the abnormality persists. The timing is set by the screening program and the infant’s clinical circumstances, often around a later corrected gestational age or term-equivalent period.

The biological rationale is straightforward. As the thymus matures and the peripheral T-cell pool expands, TREC values may rise into the expected range. A repeat specimen can therefore separate developmental immaturity from a persistent failure of T-cell production without immediately subjecting every preterm infant to venipuncture and extensive testing.

Repeat DBS is not an excuse to delay evaluation indefinitely. It is appropriate only when the result is non-urgent and the infant does not have clinical or laboratory features that increase concern. A repeat sample should be brought forward, or bypassed in favor of flow cytometry, when the first result is undetectable or nearly undetectable, the absolute lymphocyte count is very low, the infant has concerning infections, or there is a relevant family history.

Immediate referral for urgent or near-zero results

A TREC result at or near zero is handled differently. Profoundly low TREC production is less easily explained by ordinary prematurity alone and may indicate SCID or another severe T-cell lymphopenia. These infants generally require prompt contact with a pediatric immunologist and confirmatory testing rather than waiting for maturation.

The urgent pathway typically includes:

1. Flow cytometry with absolute T-, B-, and NK-cell counts.

2. Separate assessment of naive and memory T-cell populations.

3. A complete blood count with an absolute lymphocyte count.

4. Review of transfusion history, medications, and acute illness.

5. Infection-prevention guidance while the evaluation is underway.

6. Additional functional and genetic testing when the phenotype supports it.

The urgency is clinical, not merely administrative. If SCID is confirmed, exposure to live vaccines, transfusion-associated lymphocytes, and preventable infections can have serious consequences. Early recognition also allows the care team to plan definitive treatment before the infant develops a severe infection.

A practical decision matrix

The exact categories vary by program, but the logic is consistent:

The table is a framework, not a substitute for the laboratory’s protocol. Some programs repeat testing more than once in very premature infants; others proceed directly to flow cytometry when the initial value is sufficiently low. The result category, not the label “preterm,” determines the pace.

The cutoff is not the diagnosis. Repeat sampling, absolute cell counts, flow cytometry, and clinical context determine what the first TREC result means.

Distinguishing Transient Lymphopenia from Primary Immunodeficiency

What flow cytometry adds

When a repeat screen remains abnormal or the first result is urgent, flow cytometry provides information that DBS screening cannot. It measures the distribution of lymphocyte populations directly and reports absolute numbers as well as percentages.

An initial immunophenotyping panel commonly includes:

• CD3-positive total T cells
• CD4-positive and CD8-positive T-cell subsets
• Naive T-cell markers, often including CD45RA
• CD19-positive B cells
• CD16/CD56-positive natural killer (NK) cells
• Absolute lymphocyte counts calculated from the complete blood count

The pattern is more informative than any single percentage. A premature infant may have a low total lymphocyte count with detectable naive T cells and gradual recovery over time. That pattern can be compatible with transient lymphopenia, particularly when the infant’s clinical course and repeat testing are reassuring.

By contrast, severe T-cell lymphopenia with very few or absent naive T cells requires urgent evaluation for a primary immunodeficiency. It is essential not to infer the underlying SCID subtype from the TREC result alone. Different genetic forms produce different combinations of T-, B-, and NK-cell abnormalities.

The X-linked SCID pattern must be stated precisely

Classic X-linked SCID caused by pathogenic variants in IL2RG has a T−B+NK− phenotype. T cells are absent or profoundly reduced, B cells are present in number, and NK cells are absent or markedly reduced. The presence of B cells does not mean that antibody production is normal: B-cell function is impaired because the cells lack adequate T-cell help.

This pattern should not be generalized to all forms of SCID. Other molecular subtypes can produce different immunophenotypes:

• T−B−NK+ patterns may occur with defects affecting antigen-receptor rearrangement, including some RAG1, RAG2, or related disorders.
• T−B−NK− patterns may occur with defects affecting shared signaling pathways or lymphocyte development.
• Some conditions produce residual or atypical T-cell populations rather than a complete absence of T cells.
• Omenn syndrome and other leaky forms of SCID can show abnormal, sometimes oligoclonal T cells rather than the classic complete T-cell absence seen in more profound disease.

Therefore, preserved B cells with absent NK cells points toward the classic X-linked pattern only in the appropriate clinical and laboratory context. B- and NK-cell counts cannot be used as a universal shorthand for SCID.

Longitudinal recovery is informative, but not self-sufficient

A rise in TREC values on repeat screening supports transient developmental immaturity or stress-related lymphopenia. It does not, by itself, exclude every primary immunodeficiency. Some disorders are partial, leaky, or associated with fluctuating counts. Similarly, a persistently low result increases concern but still requires confirmation with cell enumeration, functional testing, and—when indicated—molecular analysis.

The trajectory should be read together with:

• Absolute CD3, CD4, and CD8 counts
• The proportion and absolute number of naive T cells
• Evidence of maternal lymphocyte engraftment when relevant
• Infection history and physical findings
• Growth, feeding, and recovery from neonatal illness
• Family history of early deaths, recurrent infections, or known immunodeficiency

The practical distinction is between a developing immune system that is moving in the expected direction and one that remains quantitatively or functionally abnormal.

Diagnostic Escalation: When to Move from DBS to Flow Cytometry

Start with absolute counts, not percentages

Flow cytometry reports can be misleading if percentages are read without absolute counts. A high percentage of T cells can coexist with a severely reduced total lymphocyte count. Conversely, a low percentage may reflect expansion of another lymphocyte population rather than a major deficit in absolute T-cell numbers.

For this reason, the clinical team should review absolute CD3, CD4, and CD8 counts alongside B- and NK-cell counts. There is no single universal neonatal threshold that identifies every infant who needs the same intervention. Reference ranges vary with gestational and postnatal age, and the interpretation of a count around a decision boundary depends on the entire phenotype.

A markedly reduced absolute T-cell count, absent or nearly absent naive T cells, or a near-zero TREC result should prompt urgent specialist management. Less severe reductions can sometimes be followed with repeat testing, but only when the infant is clinically stable and the screening laboratory or immunologist considers the result non-urgent.

Proliferation testing and functional assessment

When flow cytometry confirms severe or persistent T-cell lymphopenia, functional testing may be needed. Lymphocyte proliferation assays, including responses to mitogens such as phytohemagglutinin (PHA), assess whether the available T cells can activate and divide. Anti-CD3 stimulation may provide additional information in selected cases.

Functional results require careful interpretation. A low response can be affected by the number and maturity of circulating lymphocytes, recent illness, medications, and technical conditions. It should not be used as a stand-alone substitute for immunophenotyping or genetic testing.

Genetic confirmation

Genetic testing is selected according to the phenotype and the available clinical information. A targeted primary-immunodeficiency panel may include genes such as IL2RG, JAK3, RAG1, RAG2, DCLRE1C, and LIG4, among others. If a targeted panel is negative or the phenotype is atypical, broader sequencing may be considered.

The purpose of genetic testing is not only to assign a label. The molecular diagnosis can clarify prognosis, guide infection-prevention measures, inform the choice and timing of hematopoietic stem-cell transplantation or gene therapy, and support testing of relatives. While that work is in progress, precautions should be based on the level of clinical suspicion rather than postponed until a variant is found.

In an infant who may have SCID, the team may avoid live vaccines, review the need for irradiated and appropriately selected blood products, and limit exposure to infectious contacts. These measures are individualized by the treating clinicians. They are not triggered by every borderline TREC result, but they become increasingly important when the result is near zero or flow cytometry shows profound T-cell deficiency.

Why preterm screening needs a layered protocol

Preterm infant newborn screening accuracy is not a fixed property of the assay. It depends on the interaction between biology, specimen quality, platform calibration, and the clinical pathway used after an abnormal result. A program that applies a full-term cutoff without accounting for gestational age will generate more borderline screens. A program that ignores very low or undetectable results because prematurity is considered an adequate explanation risks delaying the diagnosis of genuine disease.

The most useful model is layered:

• TREC screening identifies infants who may have reduced thymic output.
• Clinical and laboratory context distinguishes a detectable borderline signal from a high-risk result.
• Repeat DBS allows maturation to resolve some low results in stable premature infants.
• Flow cytometry measures the actual lymphocyte phenotype when the screen remains abnormal or is urgently low.
• Functional testing and genetic analysis determine whether the abnormal phenotype represents a defined primary immunodeficiency.

This architecture also explains why a false-positive screen is not necessarily a failure of the program. In a population with low baseline TREC values, a sensitive first-line test will identify infants who need clarification. The quality of screening is judged by whether the follow-up pathway separates transient physiology from clinically important disease without losing time in infants who need urgent treatment.

Clinical significance of borderline TREC results

A borderline TREC result in a preterm infant usually raises one of three possibilities: immature thymic output, transient lymphopenia related to illness or perinatal stress, or an underlying primary T-cell disorder that has not yet declared its full phenotype. The first two are more common explanations in the NICU, but the third is the reason the result cannot simply be dismissed.

The most reassuring pattern is a clinically stable infant with detectable TRECs, no major lymphopenia, no concerning infection history, and increasing TREC values or lymphocyte numbers on follow-up. The more concerning pattern is an infant with an undetectable or nearly undetectable result, severe absolute lymphopenia, absent naive T cells, recurrent or opportunistic infection, persistent thrush, poor growth, or a family history suggestive of SCID.

Between those extremes, interpretation remains deliberately cautious. The clinical significance of borderline TREC results lies in their trajectory and in the phenotype that follows—not in the initial copy number alone.

TREC screening does not replace clinical judgment. It gives the NICU a sensitive entry point into the evaluation of severe T-cell deficiency, while gestational age and perinatal stress explain why the first signal may be difficult to interpret. The safest approach is neither to overdiagnose every low result nor to normalize every abnormality as prematurity. It is to match the response to the strength of the signal: repeat a non-urgent borderline screen at the appropriate stage of maturation, escalate promptly when TRECs are near zero or lymphopenia is severe, and use flow cytometry and molecular testing to define the immune phenotype.

The assay is not specific. The protocol is what makes it clinically useful.