BONE MARROW ENGRAFTMENT - WHOLE BLOOD/MARROW FIRST POST
Label Name: BME 1ST POST
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6120 
Last Review:  3/17/2017 10:02:10 AM
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 6  ML
Label Reminders
  Be sure to include patient's name, history #, date and time of collection, and collector's initials.
Collection Notes
  Adult:
  • Bone Marrow: One lavender-top EDTA tube (minimum of 1 ml) is required for testing. Forward unprocessed bone marrow promptly at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.

    Peripheral Blood: One lavender-top EDTA tube (minimum of 3 mls) is required for testing. Forward unprocessed peripheral blood promptly to the laboratory at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.

    Buccal swab (using the MDL specimen collection kit): Scrape the inside of the mouth using 10 strokes with a sterile nylon bristle cytology brush. Dip the brush up and down 10 times in the provided solution (Cell Lysis Solution) contained in the 1.5mL microfuge tube. Detailed instructions and collection kits are available on request from the Clinical Molecular Diagnostics Laboratory.


 
Transport
  Deliver peripheral blood and bone marrow specimens to lab at ambient temperature. If there is a delay of more than 24 hours in delivery, refrigerate the sample. DO NOT FREEZE.
Turn Around Time -  Routine: 7 days   Stat: N/A
Reference Values
BME 1ST POST
Methodology
  This assay uses PCR-mediated amplification and subsequent size analysis of short tandem repeats (STRs) to determine the recipient and/or donor cell composition of a transplant patient’s peripheral blood or bone marrow. For fractionated samples, lymphocytes or granulocytes are first isolated from post-transplant samples using magnetically labeled anti-human CD3 or CD15 antibodies (isotype: mouse IgG1 and IgMkappa respectively) and the RoboSep automated cell separator (StemCell Technologies, Vancouver, BC, Canada). Genomic DNA is then extracted from the purified fractionated cells, and/or the whole unfractionated sample. This DNA is used in a multiplexed PCR-mediated amplification reaction targeting a total of fifteen autosomal STRs and one STR marker on the pseudo autosomal region of the X and Y chromosomes. Following PCR amplification, fluorescently labeled PCR products are resolved by capillary electrophoresis on the ABI 3130xl Genetic Analyzer. GeneMapper software (ABI) is used to calculate the number of repeats and relative abundance of each repeat for each STR locus. This data is then used to calculate the percentage of donor and recipient cells in the original sample using the donor and pre-transplant recipient STR profiles (Donor: MD-_-_, Recipient pre-transplant: MD-_-_) utilizing the ChimerMarker Analysis Software (Softgenetics LLC, State College, PA). As little as a 2% population of donor or recipient cells can be detected with this assay.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    BACKGROUND:

The success or failure of a bone marrow transplant can be assessed by analysis of short tandem repeats (STRs). STRs are short, repetitive well defined DNA sequences that consist of multiple tandem copies of a 2-7 base pair core element. Distinct STRs can be found scattered throughout the human genome. Between individuals, the number of repeats for any given STR (and thus, its length) can vary greatly. Determining the length of STR loci, therefore, can serve as a convenient way of determining whether a DNA sample belongs to a specific individual. This method of ‘DNA Fingerprinting’ is most well known for its use in paternity and forensic molecular testing. However, in our laboratory we use the same approach to determine if the white blood cells from an individual who has received a bone marrow transplant are derived from the donor’s transplanted marrow or from the recipient’s residual bone marrow. The methods we use for determining the length of an STR are straightforward. The lengths of multiple STRs are measured by amplifying the STRs in a multiplexed PCR reaction with fluorescently tagged primers. Amplicons are then resolved by capillary gel electrophoresis. The relative integrated intensities of the resulting peaks can be used to determine the percentage of cells that are donor derived.

CLINICAL SIGNIFICANCE AND UTILITY:

The efficacy of a patient’s bone marrow transplant can be monitored using this test. Early evidence of failure or rejection of a patient’s transplant can be used to guide further therapy. This is particularly true with the increased use of allogeniec bone marrow transplantation following incomplete marrow ablation coupled with donor lymphocyte infusions (the ‘mini-allo’ transplant). For patients who have received transplants for hematological malignancies, this engraftment test occasionally has the secondary benefit of providing evidence of recurrence. This test can also be used to determine whether transplacental maternal engraftment has occurred in infants with immunodeficiencies. We rarely perform this test to exclude the possibility of switched surgical pathology specimens when this need arises. Finally, this assay can be used to determine whether or not cytogenetic analysis of chorionic villus samples are compromised by maternal cell contamination. We do not perform paternity or forensic testing.

The percent bone marrow engraftment is determined by analyzing 15 different STR loci. For each STR, donor and recipient alleles are identified, the relative proportions of each are determined and these proportions are averaged to give a final value for percent engraftment (percent donor cells in the sample).

REFERENCES

Bader et al (2005) How and when to monitor chimerism. Bone Marrow Transplantation. 35; 107-119.

Fernandez-Aviles et al (2003) Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of STR markers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood. Leukemia. 17; 613-620.

Miura et al (2006) Analysis of donor-type chimerism in lineage-specific cell populations after allogeneic myeloablative and nonmyeloablative stem cell transplantation. Bone Marrow Transplantation. 37; 837-843.

Nuckols et al (2000) Evaluation of an automated technique for assessment of marrow engraftment after allogeneic bone marrow transplantation using a commercially available kit. Coagulation and Transfusion Medicine. 113; 135-140.

5. Keil et al (2003) Rapid establishment of long-term culture-initiating cells of donor origin after nonmyeloablative allogeneic hematopoietic stem-cell transplantation, and significant prognostic impact of donor T-cell chimerism on stable engraftment and progression-free survival. Transplanation. 76; 230-236.
   
Indications
    This test can be used to:

• Monitor the status of an allogeneic bone marrow transplant for early detection of transplant failure in a patient with a non-neoplastic indication for transplantation.

• Monitor the status of an allogeneic bone marrow transplant for early detection of recurrence in a patient with a history of a hematologic malignancy.

• Determine whether the cytogenetic findings of a chorionic villus sample are maternal or fetal (exclude the possibility of maternal cell contamination)

• Exclude the possibility of switched surgical pathology specimens when there is a question of sample identity.
   
Limitations
    Determining a percentage of donor cells is dependent upon differences between the transplant recipient and donor in at least one of the STR loci that we analyze. Rarely, the donor and recipient will have identical numbers of STRs for both alleles for all eight loci that we routinely test. In this case, donor and recipient will be indistinguishable and the assay will not be informative. This would always be the case for transplants between identical twins, and rarely occurs by chance with related or even unrelated donors. An extended STR panel is available which allows for the analysis of four different/additional microsatellite markers. If our panel of eight markers is not informative, the extended panel is performed as a reflex test.


   
Test Synonyms
  Synonym(s): BME
Synonym(s): BONE MARROW ENGRAFTMENT ANALYSIS
Synonym(s): RFLP-IDENTITY
Synonym(s): SHORT TANDEM REPEAT ANALYSIS
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times:
  Monday through Friday