Molecular Diagnostic Testing Department

In 1997, our Molecular Diagnostics Department was established at DNA Reference Lab. To date, the department has focused on two critical areas in molecular diagnostics: infectious diseases and molecular pathology. Under molecular pathology DRL has established procedures for genetic disease testing, leukemia detection, HLA typing and cell engraftment determinations. Our scientists have developed and adopted several PCR based diagnostic tests for infectious agents such as Human Papilloma virus, Varicella Zoster virus, Cytomegalovirus, HIV and HCV genotyping, HIV phenotyping; and HIV-1 and HCV viral loads. Most of these procedures were in vitro diagnostic assays approved by FDA. Our commitment and strategy is to keep up with all new technologies. The department has experienced exponential growth for each year in its existence and is now poised to expand into other areas of Molecular Pathology and diagnostics such as viral testing for transplant and immune-compromised patients. DNA Reference Lab has been CAP and CLIA accredited. We offer long-standing experience in Molecular Biology Research and Clinical Laboratory Science. Laboratory Molecular Diagnostic testing is becoming pivotal in Clinical Laboratory Science and personalized medicine. DRL is committed to utilizing the most advanced technology and applications available to provide and pioneer testing in this new field. Our instrumentation includes several genetic analyzers, real time PCR machines and numerous support equipments to develop and establish molecular diagnostic tests. With the installed capacity, we have the ability to analyze a large number of samples and the expertise to develop and validate all aspects of new molecular diagnostic assays.

Testing Service Currently Offered by DRL

  • BKV qualitative (blood, plasma, urine)
  • BKV quantitative (blood, plasma, urine)
  • CMV qualitative (blood, plasma, urine)
  • CMV quantitative (blood, plasma, urine)
  • EBV qualitative (blood, plasma)
  • EBV quantitative (blood, plasma)
  • Herpes Simplex 1 & 2 in cerebrospinal fluid (CSF)
  • Herpes Simplex 1 & 2 in vaginal swabs.

Stat & Same-Day Results for Quantitative Viral DNA for CMV, EBV and BKV

DNA REFERENCE LAB is pleased to announce that it is offering Same-Day Results for all our viral transplant panels to serve the following purpose:

  • Qualitative and quantitative DNA testing for CMV, EBV and BKV
  • Same day results. Samples by 9:00 AM results by 5:00 PM
  • Generate a baseline for the viral load in your patient.
  • Assess the need to administer anti-viral agents and therapy
  • Assess the efficacy of your anti-viral agent and therapy
  • Determine viral loads for BKV in blood and urine from adults or children
  • Monitor and quantify the three viruses in post transplant patients
  • Apply them as part of your pre-transplant work out for candidate patients

BK Virus (BKV) DNA by PCR – Quantitative

DRL Test Code # 1

Method:   Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Specimens Note:    It is recommended that blood and urine be serially tested.
Whole Blood (ACD or EDTA): 5.0 (min 3.0) mL, ambient (4 days),
refrigerated (7 days).

Urine:    10.0 (min 5.0) mL, refrigerated (7 days).

Plasma (ACD, EDTA, or PPT)    3.0 (min 1.0) mL, separated/centrifuged within 6 hours, refrigerated or frozen (do not freeze in PPT). If storing longer than 24 hours, store frozen.

Serum:   2.0 (min 1.0) mL, refrigerated (7 days) or frozen.

CSF:    1.0 (min 0.2) mL, refrigerated (7 days) or frozen.

Bone Marrow:     3.0 (min 2.0) mL, refrigerated (7 days).

Causes for Rejection:     Quantity not sufficient (QNS) for analysis; time and/or temperature instructions not followed; blood in heparin; plasma frozen in PPT.

Reference Range:     Not Detected (< 500 copies/mL)

Quantitative Range:    500 to 1.0 x 1010 BKV DNA copies/mL

Turnaround Time:     Same or Next Day

CPT Code:     87799

Description:

BK virus (BKV) DNA quantification is based upon the real-time PCR amplification and detection of BKV
genomic DNA. A value of less than 500 BKV DNA copies/mL indicates that the patient’s viral load is
below the quantitative limit of this assay, and does not indicate that the patient is not infected with BKV.

Clinical Utility:

Real-time PCR detection for BKV is a sensitive and specific method to diagnose viral nephropathy and
primary infection associated with respiratory illness in children. The detection and monitoring of BKV,
common in children undergoing stem cell transplantation (SCT) and renal transplant patients, allows the
physician to appropriately treat the primary and reactivated infection.
Clinical studies support the parallel testing of urine and blood plasma by PCR to confirm the presence of
BKV. Mild immune impairment can lead to increased virus replication and the presence of the virus in
urine. Testing of urine and blood from immunosuppressed patients alerts the physician to asymptomatic
reactivation of BKV. Blood and urine viral loads tend to decrease after treatment by antiviral therapies.
The viral load of urine is typically 4-6 log orders higher than the viral load of blood. The viral load from
urine may be detected earlier than the blood viral load and tends to take longer to decrease compared to the blood viral load. Thus, it is recommended that both blood and urine be serially tested.

References:

Watzinger, et al. Real-Time Quantitative PCR Assays for Detection and Monitoring of Pathogenic Human Viruses in Immunosuppressed Pediatric Patients. Journal of Clinical Microbiology Nov. 2004; 42/11: 5189-5198.
Reploeg, et al. BK Virus: A Clinical Review. Clinical Infectious Diseases 2001; 33: 191-202.
Hirsch et al. Testing for polyomavirus type BK DNA in plasma to identify renal-allograft recipients with viral nephropathy. N Engl J Med 2000; 342:1309-15.

Cytomegalovirus (CMV) Quantitative Real-time PCR

DRL TEST CODE:     3

CPT CODE:     87497 (x1)

CLINICAL UTILITY:

CMV is an important pathogen in the transplant setting causing pneumonitis, colitis, hepatitis, CNS disease, neutropenia, and disseminated disease. Prior to the availability of rapid and sensitive DNA PCR, CMV was a leading cause of morbidity and mortality in the transplant population. Quantitative CMV DNA PCR can be used for early detection of CMV reactivation, primary infections, and monitoring response to treatment.

Methodology:     Quantitative Polymerase Chain Reaction

PROCEDURE

Extraction of CMV DNA from specimen followed by amplification and detection using real-time, quantitative PCR. An internal control is added to ensure the extraction was performed correctly and the PCR reaction was not inhibited. The assay design includes multiple targets to account for viral mutations, which significantly reduces the chance of false negative results. This test has not been cleared or approved for diagnostic use by the U.S. Food and Drug Administration.

SPECIFICITY

The primers and probes used in this assay are specific for known CMV strains based on similarity search algorithms. Additionally, no cross reactivity was detected when tested against adenoviruses, BKV, EBV, HSV-1, HSV-2, HHV-6 variant A, HHV-6 variant B, HHV-7, HHV-8, JCV, parvovirus B19, SV-40, and VZV.

Ordering Recommendation:    Preferred test for detecting cytomegalovirus infection and guiding posttransplant therapy.

CAUSES FOR REJECTION

Whole blood frozen, specimens beyond their acceptable length of time from collection as listed in the specimen handling, specimens received in trap containers, or specimen types other than those listed.

TURNAROUND TIME

Same day (specimens must be received in the laboratory by 9:00 AM), Monday through Friday.

SHIPPING

Ship Monday through Thursday. Friday shipments must be labeled for Saturday delivery. All specimens must be labeled with patient’s name and collection date. A DRL test requisition form must accompany each specimen. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight® to: DNA REFERENCE LAB, 5819 NW Loop 410, Ste #166, San Antonio, Texas 78238

Specimen Required:     Collect: Lavender (EDTA) or pink (K2EDTA).

Specimen Preparation: Separate plasma from cells. Transfer 1 mL plasma to a sterile container. (Min: 0.5 mL).

Storage/Transport Temperature: Frozen.

Unacceptable Conditions: Serum: Heparinized specimens.

Stability (collection to initiation of testing): Ambient: 24 hours; Refrigerated: 5 days; Frozen: 1 year

Reference Interval:

The quantitative range of this test is 2.6- 6.6 log copies/mL (390-3,900,000 copies/mL) or 2.4- 6.4 log IU/mL (227- 2,270,000 IU/mL). One IU/mL of CMV DNA is approximately 1.72 copies/mL.

Interpretive Data:

A negative result (less than 2.6 log copies/mL [less than 390 copies/mL] OR less than 2.4 log IU/mL [less than 227 IU/mL]) does not rule out the presence of PCR inhibitors in the patient specimen or CMV DNA concentrations below the level of detection of the test. Inhibition may also lead to underestimation of viral quantitation.

Note:

The limit of quantification for this DNA test is 2.6 log copies/mL (390 copies/mL) or 2.4 log IU/mL (227 IU/mL). If the test DID NOT DETECT the virus, the test result will be reported as ”

CPT Code(s): 87497

Epstein-Barr Virus (EBV) Viral Load Testing by Real-Time PCR Now Available

DRL TEST CODE:     4

CPT CODE:     87798

Summary

Effective Epstein Barr virus (EBV) viral load testing by real-time PCR is available now from DRL at 5819 NW Loop 410, Ste #166, San Antonio, Texas 78238. This comes as the result of extensive analytical verification and validation studies for the laboratory developed test (LDT) using Focus Diagnostic ASR reagents.

Benefits:

  • Assists in early diagnosis of infectious mononucleosis when serology testing is inconclusive.
  • Evaluation of pre-transplant patients
  • Assists in diagnosing and monitoring of patients with post-transplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma or AIDS-related brain lymphoma
  • Monitoring of viral loads in immune-compromized patients
  • Monitoring the efficacy of anti-viral therapy.
  • Exceptional turnaround time (same day results).

Background

By the time adults reach the age of 35-40, 95 % of them will test positive for EBV. It persists in the host after primary infection and may reactivate at any time. Since EBV may replicate without causing any harm, it is important to distinguish asymptomatic infection from EBV disease. EBV has been associated with malignant proliferative disorders of both epithelial and lymphoid origins to include:

  • Hodgkin’s and Burkett’s lymphoma
  • B-and T-cell non-Hodgkin’s lymphoma
  • Nasopharyngeal and gastric carcinoma

EBV is also involved in causing substantial disease from lymphoproliferative disorders among immunocompromised individuals such as transplant recipients and AID’s patients.

Monitoring EBV infections and therapeutic treatment

Quantitative methods for measuring EBV are becoming widely used to diagnose, monitor and treat EBV-related diseases. To illustrate this situation, PTLD is a particular case. In addition, studies have indicated that preemptive treatment of EBV and reducing immunosuppressive therapy can lead to reduced incidence of PTLD in immunocompromised patients. EBV –related PTLD is usually accompanied by increased EBV DNA in the peripheral blood. EBV viral load monitoring is used to guide initiation of preemptive or anti-EBV-related tumor therapy.

Successive monitoring

An increased EBV DNA level compared to stable elevated levels is a reliable marker for PTLD. Therefore, successive monitoring of the same specimen type is important in the identification of patients at risk of disease because a high EBV DNA load alone cannot always predict impeding PTLD.

Whole blood versus plasma

The choice of the optimal clinical specimen may differ based on the type of EBV infection due to difference in the disease status of EBV DNA.

Specimen Requirements

Collection:      One 4 ml lavender top tube (EDTA) or CSF in sterile leak-proof container

Handling:      Whole blood: Do not freeze. Ship refrigerated at 2-8oC
Plasma: Centrifuge and immediately separate plasma into a plastic vial. Refrigerate, freeze at -20oC if specimen cannot be assayed within 3 days.
CSF: Refrigerate or freeze CSF within 30 minutes. For delayed transport freeze at -20oC.

Stability:      Whole blood: Ambient 24 hours, frozen unacceptable and refrigerated for 72 hours.
Plasma: Ambient 24 hours, refrigerated 24 hours, frozen 14 days
CSF: Ambient 30 minutes, refrigerated 72 hours, frozen 14 days

Standard Volume:      One ml for plasma, whole blood or CSF

Transport:     Whole blood: Ship refrigerated at 2-8 oC.
Plasma: ship frozen at -20oC.CSF: Ship frozen at -20oC.

Rejection Criteria:     Non-sterile or leaking container amniotic fluid, bone marrow, biopsy tissue, heparinized specimen, frozen whole blood, hemolyzed or clotted whole blood.

Reference Range:     Not detected

REFERENCES

  1. Pitetti RD, et al. Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary EBV infection in children. Pediatr Infect Dis J 2003;22:736-9.
  2. Cohen JI. Clinical aspects of EBV infection. In E. Robertson (ed.), Epstein-Barr virus. Caister Academic Press, Norfolk, England, 2005:35-54.
  3. Gulley ML, et al. Using Epstein-Barr viral load assays to diagnose, monitor, and prevent PTLD. Clin Micro Reviews 2010;23:350-66.
  4. Bakker NA, et al. EBV-DNA load monitoring late after lung transplantation: a surrogate marker of the degree of immunosupression and a safe guide to reduce immunosuppression. Transplantation 2007; 83:433-8.
  5. Van Esser JW, et al. Prevention of EBV-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood 2002;99:4364– 69.
  6. Funk GA, et al. Viral dynamics in transplant patients: implications for disease. Lancet Infect Dis 2007;7:460–72.
  7. Gärtner B, et al. EBV viral load detection in clinical virology. J Clin Virol 2010;48:82-90.
  8. Abbate I, et al. Multicenter comparative study of EBV DNA quantification for virological monitoring in transplanted patients. J Clin Virol 2011;50: 224–9.
  9. Hakim H, et al. Comparison of various blood compartments and reporting units for the detection and quantification of EBV in peripheral blood. J Clin Microbiol 2007;45:2151-5.
  10. Ruf S, et al. Comparison of six different specimen types for Epstein-Barr viral load quantification in peripheral blood of pediatric patients after heart transplantation or after allogeneic hematopoietic stem cell transplantation. J Clin Virol 2012;53: 186–194.800

DISCLAIMER

Specimens are approved for testing according to the Specimen Information field above. The CPT codes provided are based on DRL interpretation of the American Medical Association’s Current Procedural Terminology (CPT) codes and are provided for informational purposes only. CPT coding is the sole responsibility of the billing party. Questions regarding coding should be addressed to your local Medicare carrier. DRL assumes no responsibility for billing errors due to reliance on the CPT codes illustrated in this material.

The advantage of molecular diagnostic testing at DRL

We understand that you have many laboratories to choose from when deciding where to go with your test. That is why we have made it our goal to give our laboratory significant advantage over larger laboratory corporations and other laboratories.

  • #1 Our Promise – At the DNA Reference Lab, you are a human being, and we know it. We do not staff our phones with robots. The administrative staff is knowledgeable and friendly. We believe that automated services are impersonal, and waste your time and money. Therefore, when calling the DNA Reference lab, you will never speak to a robot.
  • #2 Our Size – Our small size in comparison to large corporate labs allows for easier communication between staff, allowing us to implement new standards, technology, and processes much easier. This means that we will work harder to give your test the extra attention it deserves.
  • #3 Our Accreditation – Our elaborate accreditation activity is unsurpassed by laboratories our size. DNA Reference lab is AABB, CAP, CLIA accredited.
  • #4 Our Technology – DNA REFERENCE LAB utilizes cutting-edge technologies for all its validated testing procedures. Our technologies include the latest in RNA/DNA extraction, STR, SNP, Real-time PCR, CE and NGS systems.
  • #5 Our Location – Located in San Antonio, our laboratory sits in the heart of a city of 1.5 million, meaning that San Antonio and most Texas residents have a convenient place to test directly. We collect samples in our main lab, and skipping the time required for delivery of samples means that you obtain your results faster.
  • #6 Our Education – Testing is performed by MD, PhDs, PhDs and MS professionals who have extensive experience in high complexity laboratory testing.
  • #7 Our Prices – We strive to provide the most competitive pricing within the industry.