2 Testing for TBD

Published

August 19, 2026

2.1 Flow-FISH (flow cytometry with fluorescence in situ hybridization)

2.1.1 Overview

Flow-FISH (fluorescence in situ hybridization combined with flow cytometry) is the gold standard method for clinical telomere length (TL) measurement.
This technique measures absolute telomere length in peripheral blood leukocyte subsets and is essential for diagnosing telomere biology disorders (TBDs).

2.1.1.1 Why Flow-FISH?

Method	Accuracy	Reproducibility	Cell Subset Analysis	Clinical Validation
Flow-FISH	High	Excellent (CV ~2-5%)	Yes	Gold standard
qPCR	Moderate	Poor (CV ~8-25%)	No	Not recommended for diagnosis
Southern blot (TRF)	High	Good	No	Research standard

Key Point

Flow-FISH is the only recommended method for clinical diagnosis of telomere biology disorders per consensus guidelines.

2.1.2 Clinical Indications

2.1.2.1 Primary Indications

Suspected dyskeratosis congenita or telomere biology disorder
Idiopathic bone marrow failure / aplastic anemia
Familial or early-onset idiopathic pulmonary fibrosis
Cryptogenic liver cirrhosis / hepatopulmonary syndrome
Unexplained cytopenias with suggestive features
MDS/AML with clinical features suggestive of TBD

2.1.2.2 Secondary Indications

Evaluation of potential HCT donors in families with known TBD
Variant interpretation (VUS in telomere-related genes)
Risk stratification in pulmonary fibrosis

2.1.2.3 Clinical Clues Suggesting TBD

Red Flags for Short Telomeres

Premature graying (age <30 years)
Family history of IPF, BMF, liver disease, or MDS/AML
Mucocutaneous triad (nail dystrophy, skin pigmentation, oral leukoplakia)
Multi-organ involvement (lung + liver, lung + marrow, etc.)
Poor response to immunosuppression in aplastic anemia

2.1.3 Laboratory Options

2.1.3.1 RepeatDx (Vancouver, BC)

Website: repeatdx.com

Accreditation: CLIA-certified, CAP-accredited, licensed in all 50 US states + Canada

Test	Cell Types Analyzed	Use Case
2-Panel	Lymphocytes, Granulocytes	Initial screening, most cases
6-Panel	Lymphocytes, Granulocytes, B-cells, Naïve T-cells, Memory T-cells, NK cells	Complex/atypical cases, VUS interpretation

Optional Add-on: Medical consultation with hematopathologist (+$250)

2.1.3.1.1 Specimen Requirements (RepeatDx)

Adults:     5-10 mL whole blood in EDTA (lavender top)
Pediatric:  5 mL whole blood in EDTA (lavender top)

2.1.3.1.2 Shipping Instructions

Collect and ship same day (morning collection preferred)
Ship Monday–Wednesday only (to avoid weekend delays)
Room temperature — no ice packs
Use FedEx International Priority
Contact lab with tracking number

2.1.3.1.3 Ordering

Download requisition form from repeatdx.com/order-telomere-test
Complete and sign by ordering physician
Ship specimen with requisition

2.1.3.2 Johns Hopkins Pathology

Contact: Johns Hopkins Genomics-MDL
Phone: 410-955-1438
Fax: 410-955-0484

Address:

Johns Hopkins Genomics-MDL
1812 Ashland Ave, Room 245
Baltimore, MD 21205

2.1.3.2.1 Specimen Requirements (Johns Hopkins)

Patient	Tube	Volume
Adults	ACD (yellow top)	12-18 mL
Infants/Children	EDTA (lavender top)	≥3 mL

Important

Fill tubes to maximum volume to prevent hemolysis. Receipt preferred within 24 hours (acceptable up to 72 hours in ACD tubes).

2.1.3.2.2 Test Information

CPT Code: 88182
Turnaround: ~2 weeks
Cost: ~$400 (contact lab for current pricing)

2.1.3.2.3 Ordering

JHH/Affiliates: Order through EPIC
External: Use requisition form from pathology.jhu.edu

2.1.4 Interpretation Guidelines

2.1.4.1 Percentile Thresholds

Percentile	Interpretation	Clinical Action
<1st	Very short — highly specific for TBD	Genetic testing strongly recommended
1st–10th	Short — may be significant	Clinical correlation required; consider genetic testing
10th–50th	Low-normal	Unlikely TBD unless strong clinical suspicion
>50th	Normal	TBD effectively ruled out (100% NPV)

2.1.4.2 Age-Dependent Interpretation

Age Considerations

Children/adolescents: <1st percentile highly specific
Adults 40-60: 1st-10th percentile may still indicate TBD
Adults >60: Telomere length overlaps with normal aging; clinical context critical
Genetic anticipation: Successive generations present earlier with shorter telomeres

2.1.4.3 Cell Subset Patterns

Pattern	Suggests
Short in all subsets	Classic TBD
Short lymphocytes, normal granulocytes	May indicate acquired process
Discordant results across subsets	Consider 6-panel for clarification

2.1.5 Special Considerations

2.1.6 Post-Transplant Patients

Critical Limitation

Flow-FISH measures donor telomere length after allogeneic HCT. Testing must be performed before transplant to assess recipient’s native telomere biology.

2.1.7 Active Hematologic Malignancy

Consider whether circulating tumor cells may affect results
For germline genetic testing: use skin biopsy (cultured fibroblasts) instead of blood

2.1.8 Transfusion History

Recent transfusions may affect results
Ideally test before transfusion or wait adequate washout period

2.1.9 Integration with Genetic Testing

2.1.10 Recommended Workflow

flowchart TD
    A[Clinical Suspicion for TBD] --> B[Flow-FISH Telomere Length]
    B --> C{Result}
    C -->|<1st %ile| D[Genetic Testing Panel]
    C -->|1st-10th %ile| E[Clinical Correlation]
    C -->|>10th %ile| F[TBD Unlikely]
    E --> G{Strong clinical features?}
    G -->|Yes| D
    G -->|No| H[Monitor/Reassess]
    D --> I{Pathogenic variant?}
    I -->|Yes| J[Confirmed TBD]
    I -->|VUS| K[Functional studies / Segregation]
    I -->|Negative| L[Clinical TBD diagnosis if <1st %ile + features]

Diagnostic algorithm for suspected TBD

2.1.10.1 Genetic Testing Panels

Laboratory	Panel	Genes
Mayo Clinic (TELDP)	Telomere Biology Disorders	18 genes
Invitae	Telomere Biology Disorders	13 genes (+2 add-on)
UChicago	TBD/DC Panel	19 genes
GeneDx	Dyskeratosis Congenita	12 genes

2.1.11 Quick Reference Card

2.1.11.1 Ordering Checklist

Confirm clinical indication
Select appropriate laboratory
Obtain correct tube type and volume
Complete requisition form with clinical history
Coordinate same-day collection and shipping (Mon–Wed)
Ship at room temperature
Notify lab of shipment with tracking number

2.1.11.2 Key Contact Information

Lab	Phone	Fax
RepeatDx	Contact via website	—
Johns Hopkins	410-955-1438	410-955-0484
Cleveland Clinic	1-800-628-6816	—

2.1.11.3 References

Knuth, Donald E. 1984. “Literate Programming.” Comput. J. (USA) 27 (2): 97–111. https://doi.org/10.1093/comjnl/27.2.97.

2.1.11.4 Key Literature

Alder JK, et al. Diagnostic utility of telomere length testing in a hospital-based setting. PNAS. 2018;115(10):E2358-E2365. PMID: 29463756
Alter BP, et al. Telomere length is associated with disease severity and declines with age in dyskeratosis congenita. Haematologica. 2012;97(3):353-359. PMID: 22058220
Savage SA, et al. Dyskeratosis Congenita and Related Telomere Biology Disorders. GeneReviews. Updated 2023.
Mangaonkar AA, Patnaik MM. Short Telomere Syndromes in Clinical Practice: Bridging Bench and Bedside. Mayo Clin Proc. 2018;93(7):904-916. PMID: 29804727

Last updated: 2026-08-19

2.2 Telomere Biology Disorders Panel

2.3 Overview

Germline genetic testing is a critical component of the diagnostic workup for telomere biology disorders (TBDs). Pathogenic variants in telomere maintenance genes can be identified in approximately 60-80% of patients with TBD, confirming diagnosis and enabling family screening, genetic counseling, and informed treatment decisions.

2.3.1 When to Order Genetic Testing

Indications for TBD Genetic Panel

Telomere length <1st percentile by Flow-FISH
Telomere length 1st-10th percentile with clinical features suggestive of TBD
Classic dyskeratosis congenita phenotype
Familial pulmonary fibrosis (especially early-onset)
Aplastic anemia / bone marrow failure of unknown etiology
MDS/AML with TBD-suggestive features
Variant of uncertain significance (VUS) interpretation support
Cascade testing of at-risk family members

2.4 Gene Panel Comparison

2.4.1 Summary Table

Laboratory	Panel Name	# Genes	TAT	Del/Dup	NY Approved
Mayo Clinic	TELDP	18	~2 weeks	Yes	Yes
Invitae	TBD Panel	13 (+2 add-on)	10-21 days	Yes	Yes
UChicago	TBD/DC Panel	19	6 weeks	Yes	—
GeneDx	DC Panel	12	4 weeks	Yes	—
PreventionGenetics	DC & TBD Panel	17	Varies	Yes	—

2.4.2 Detailed Gene Coverage

Table 2.1: Gene coverage across major TBD panels

Gene	Function	Mayo	Invitae	UChicago	GeneDx
TERT	Telomerase catalytic subunit	✓	✓	✓	✓
TERC	Telomerase RNA component	✓	✓	✓	✓
DKC1	Dyskerin (telomerase stabilization)	✓	✓	✓	✓
TINF2	Shelterin complex	✓	✓	✓	✓
RTEL1	Telomere helicase	✓	✓	✓	✓
PARN	Poly(A) ribonuclease	✓	✓	✓	✓
CTC1	CST complex	✓	✓	✓	✓
STN1	CST complex	✓	✓	✓	—
WRAP53	Cajal body localization	✓	✓	✓	✓
ACD	Shelterin complex	✓	✓	✓	✓
NHP2	H/ACA RNP complex	✓	✓	✓	✓
NOP10	H/ACA RNP complex	✓	✓	✓	✓
POT1	Shelterin complex	✓	✓	✓	—
NAF1	H/ACA RNP biogenesis	✓	—	✓	—
ZCCHC8	NEXT complex	✓	—	—	—
USB1	RNA processing	✓	—	✓	✓
RPA1	DNA replication	✓	—	✓	—
LIG4	DNA ligase IV	✓	—	✓	—
DCLRE1B	Apollo nuclease	—	—	✓	—
NPM1	Nucleophosmin	—	—	✓	—

Gene	Function	Mayo	Invitae	UChicago	GeneDx
TERT	Telomerase catalytic subunit	✓	✓	✓	✓
TERC	Telomerase RNA component	✓	✓	✓	✓
DKC1	Dyskerin (X-linked)	✓	✓	✓	✓
TINF2	Shelterin complex	✓	✓	✓	✓
RTEL1	Telomere helicase	✓	✓	✓	✓
PARN	Poly(A) ribonuclease	✓	✓	✓	✓
CTC1	CST complex	✓	✓	✓	✓
STN1	CST complex	✓	✓	✓	—
WRAP53	Cajal body localization	✓	✓	✓	✓
ACD	Shelterin (TPP1)	✓	✓	✓	✓
NHP2	H/ACA RNP complex	✓	✓	✓	✓
NOP10	H/ACA RNP complex	✓	✓	✓	✓
POT1	Shelterin complex	✓	✓	✓	—
NAF1	H/ACA RNP biogenesis	✓	—	✓	—
ZCCHC8	NEXT complex	✓	—	—	—
USB1	RNA processing	✓	—	✓	✓
RPA1	DNA replication	✓	—	✓	—
LIG4	DNA ligase IV	✓	—	✓	—
DCLRE1B	Apollo nuclease	—	—	✓	—
NPM1	Nucleophosmin	—	—	✓	—

2.4.3 Key Genes by Clinical Phenotype

Phenotype	Most Commonly Implicated Genes
Adult-onset IPF	TERT, TERC, PARN, RTEL1, NAF1, ZCCHC8
Classic pediatric DC	DKC1, TINF2, TERT, TERC
Severe DC (HH, Revesz)	TINF2, DKC1, RTEL1
Coats Plus syndrome	CTC1, STN1
Aplastic anemia / BMF	TERT, TERC, TINF2, RTEL1
MDS/AML	TERT, TERC, RTEL1
Immunodeficiency	DKC1, RTEL1

2.5 Laboratory Details

2.5.1 Mayo Clinic Laboratories (TELDP)

Test Code: TELDP
CPT Codes: 81479
Genes: 18

Phone: 800-533-1710
Website: mayocliniclabs.com

Type	Volume	Container
Whole blood	1 mL minimum	EDTA (lavender top)
Skin biopsy	—	Sterile container with saline
Cultured fibroblasts	—	T-25 flask

Whole blood: ~2 weeks
Extracted DNA: ~3 months
Cultured fibroblasts: ~1 month

Mayo Advantages

Most comprehensive coverage (18 genes)
Includes NAF1 and ZCCHC8 (important for pulmonary fibrosis)
Fast turnaround for blood specimens

2.5.2 Invitae (Labcorp Genetics)

Test Code: 05314
Genes: 13 primary + 2 preliminary-evidence add-ons

Phone: 800-436-3037
Website: invitae.com/test-catalog/test-05314

Type	Volume	Container
Whole blood	3 mL	EDTA (lavender top)
Saliva	2 kits	Invitae collection kit
Buccal swab	—	Invitae collection kit
Extracted DNA	1 µg minimum	—

10-21 calendar days (average 14 days)

Invitae Advantages

Fastest average turnaround (~14 days)
Widely accessible, easy ordering
Multiple specimen options
NY state approved

Invitae Limitations

Missing NAF1, ZCCHC8, USB1 (relevant for pulmonary fibrosis)
Fewer genes than Mayo or UChicago

2.5.3 University of Chicago Genetic Services

Test Code: 5114
CPT Codes: 81345, 81479
Genes: 19

Phone: 773-834-0555
Fax: 773-702-9130
Website: dnatesting.uchicago.edu

Type	Notes
Blood	Standard
Saliva	2 kits required
Buccal	—
Cultured cells	—
Extracted DNA	—

~6 weeks

UChicago Advantages

Most genes (19) including DCLRE1B, NPM1
Academic laboratory with research expertise
Includes deletion/duplication analysis

2.5.4 GeneDx

Test Code: TB47
CPT Codes: 81479
Genes: 12

Website: genedx.com

Type	Volume	Container
Whole blood	2-5 mL	EDTA (lavender top)
Buccal swabs	—	—
Extracted DNA	—	—

~4 weeks

GeneDx Limitations

Fewer genes (12) — missing STN1, POT1, NAF1, ZCCHC8, RPA1, LIG4
May miss some adult-onset pulmonary fibrosis variants

2.6 Specimen Selection

2.6.1 When to Use Non-Blood Specimens

Use Skin Biopsy (Cultured Fibroblasts) When:

Patient has active hematologic malignancy (MDS, AML, other)
Patient has undergone allogeneic HCT (blood would test donor DNA)
Concern for somatic mosaicism confounding results
Circulating tumor cells may be present

2.6.2 Specimen Comparison

Specimen	Pros	Cons
Blood (EDTA)	Easy collection, fast processing	Not suitable post-HCT or with active malignancy
Saliva	Non-invasive, can ship at room temp	May have low DNA yield; some labs require 2 kits
Buccal swab	Non-invasive	Lower DNA quality/quantity
Skin biopsy	True germline DNA	Requires procedure, longer TAT for culture
Extracted DNA	Flexible	Quality dependent on extraction method

2.7 Interpretation Framework

2.7.1 Variant Classification (ACMG Guidelines)

Classification	Definition	Clinical Action
Pathogenic	Disease-causing	Confirms diagnosis; cascade testing indicated
Likely Pathogenic	>90% probability disease-causing	Treat as pathogenic clinically
VUS	Uncertain significance	Does not confirm or rule out; may need functional studies
Likely Benign	>90% probability benign	Unlikely to be causative
Benign	Not disease-causing	Not causative

2.7.2 Integrating Telomere Length with Genetics

flowchart TD
    A[TL <1st %ile + Pathogenic variant] --> B[Confirmed TBD]
    C[TL <1st %ile + VUS] --> D[Likely TBD - functional studies may help]
    E[TL <1st %ile + Negative genetic testing] --> F[Clinical TBD diagnosis<br/>~40% have no identifiable mutation]
    G[TL 1st-10th %ile + Pathogenic variant] --> H[Confirmed TBD]
    I[TL 1st-10th %ile + VUS] --> J[Uncertain - clinical correlation critical]
    K[TL 1st-10th %ile + Negative] --> L[TBD less likely but not excluded]
    M[TL >10th %ile + Pathogenic variant] --> N[Possible incomplete penetrance<br/>or variant may cause long telomeres]

Integrating Flow-FISH and genetic testing results

2.7.3 VUS Interpretation Support

When a VUS is identified, consider:

Telomere length — Very short TL supports pathogenicity
Segregation analysis — Does variant track with phenotype in family?
Functional studies — Telomerase activity assays (research)
Clinical phenotype — Classic features support pathogenicity
In silico predictions — CADD, REVEL, SpliceAI scores
Population frequency — Rare variants more likely pathogenic

2.8 Inheritance Patterns

2.8.1 By Gene

Pattern	Genes
X-linked recessive	DKC1
Autosomal dominant	TERT, TERC, TINF2, RTEL1, PARN, NAF1, ACD, POT1, ZCCHC8
Autosomal recessive	TERT, RTEL1, PARN, CTC1, STN1, NHP2, NOP10, WRAP53, USB1, ACD
AD or AR	TERT, RTEL1, PARN, ACD

Genetic Anticipation

TBDs demonstrate genetic anticipation — successive generations present at younger ages with more severe phenotypes due to progressive telomere shortening across generations. A parent with IPF may have a child with aplastic anemia.

2.9 Panel Selection Algorithm

flowchart TD
    A[Clinical Scenario] --> B{Primary phenotype?}
    B -->|Pulmonary fibrosis| C[Mayo or UChicago<br/>Need NAF1, ZCCHC8 coverage]
    B -->|Classic pediatric DC| D[Any panel adequate]
    B -->|Adult BMF/MDS| E[Mayo or UChicago preferred]
    B -->|Coats Plus suspected| F[Ensure CTC1, STN1 covered]
    B -->|Fastest result needed| G[Invitae ~14 days]
    B -->|Maximum gene coverage| H[UChicago 19 genes]
    B -->|Post-HCT patient| I[Use skin biopsy specimen]
    B -->|Active malignancy| I

Recommended panel selection by clinical scenario

2.9.1 Quick Recommendations

Scenario	Recommended Panel
Adult-onset pulmonary fibrosis	Mayo (TELDP) or UChicago
Classic pediatric DC	Any panel (all cover core genes)
Fastest turnaround needed	Invitae (~14 days)
Maximum gene coverage	UChicago (19 genes)
Post-HCT or active malignancy	Any panel with skin biopsy specimen
Cost-sensitive	Invitae (often lowest cost)

2.10 Family Testing & Genetic Counseling

2.10.1 Cascade Testing Indications

Once a pathogenic variant is identified in a proband:

First-degree relatives should be offered testing
Potential HCT donors must be tested to avoid using affected donor
Reproductive counseling for family planning

2.10.2 Pre-Test Counseling Points

TBD may be identified in asymptomatic family members
Results have implications for medical management and surveillance
Approximately 40% of clinical TBD cases have no identifiable genetic cause
VUS results may create uncertainty

2.10.3 Post-Test Counseling Points

Positive result: Surveillance recommendations, family testing, reproductive options
Negative result: Does not exclude TBD if telomeres are short
VUS result: Explain uncertainty, offer segregation analysis, periodic reclassification review

2.11 Quick Reference

2.11.1 Ordering Checklist

Confirm clinical indication and phenotype
Select appropriate panel based on phenotype
Determine specimen type (blood vs. skin biopsy)
If post-HCT or active malignancy → use skin biopsy
Complete requisition with clinical history
Arrange genetic counseling (pre- and post-test)
Document informed consent

2.11.2 Laboratory Contact Information

Lab	Phone	Website
Mayo Clinic	800-533-1710	mayocliniclabs.com
Invitae	800-436-3037	invitae.com
UChicago	773-834-0555	dnatesting.uchicago.edu
GeneDx	See website	genedx.com

2.12 References

2.12.1 Key Literature

Savage SA, et al. Dyskeratosis Congenita and Related Telomere Biology Disorders. GeneReviews®. Updated January 2023. PMID: 20301779
Niewisch MR, Beier F, Savage SA. Clinical manifestations of telomere biology disorders in adults. Hematology Am Soc Hematol Educ Program. 2023;2023(1):563-572. PMID: 38066924
Schratz KE, et al. Cancer spectrum and outcomes in the Mendelian short telomere syndromes. Blood. 2020;135(22):1946-1956. PMID: 32076701
Mangaonkar AA, Patnaik MM. Short Telomere Syndromes in Clinical Practice: Bridging Bench and Bedside. Mayo Clin Proc. 2018;93(7):904-916. PMID: 29804727
Alder JK, et al. Diagnostic utility of telomere length testing in a hospital-based setting. PNAS. 2018;115(10):E2358-E2365. PMID: 29463756

2.12.2 Guidelines

Telomere Biology Disorders: Diagnosis and Management Guidelines, 2nd Edition — Available at teamtelomere.org

Document created for clinical reference. Verify current laboratory requirements, gene content, and pricing before ordering.

Last updated: 2026-08-19