Bone Marrow Engraftment Studies

Cindy L. Vnencak‐Jones1

1 Vanderbilt University Medical Center, Nashville, Tennessee
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.17
DOI:  10.1002/0471142905.hg0917s62
Online Posting Date:  July, 2009
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Abstract

Bone marrow engraftment studies are used to evaluate the level of donor versus recipient cells in post‐transplant peripheral blood or bone marrow specimens. Unique DNA fingerprints identified from the recipient and the donor are used to determine the proportion of each contained within the total DNA extracted from the post‐transplant specimen. These percentages correspond to relative amounts of donor and recipient cells in the specimen. Engraftment studies are sequentially performed on transplant patients to monitor closely the levels of donor and recipient cells so that appropriate therapeutic intervention can proceed if and when needed. This unit describes the use of fluorescent PCR for amplification of genomic short tandem repeats (STR). STR analysis is now considered the gold standard for engraftment studies and provides a quick and accurate assessment of the contribution of both donor and/or recipient hematopoietic cells in post‐transplantation specimens. Curr. Protoc. Hum. Genet. 62:9.17.1‐9.17.34. © 2009 by John Wiley & Sons, Inc.

Keywords: allogeneic bone marrow transplantation; DNA typing; fluorescent PCR; chimerism; short tandem repeats

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: STR Amplification by PCR and Detection by Capillary Electrophoresis on an ABI 3130xl Genetic Analyzer
  • Basic Protocol 2: Interpretation of STR PCR Products for Bone Marrow Engraftment Studies
  • Support Protocol 1: Extraction of DNA from Peripheral Blood or Bone Marrow
  • Support Protocol 2: DNA Extraction from Buccal Scrapings
  • Support Protocol 3: DNA Extraction from Lineage‐Specific Sorted Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: STR Amplification by PCR and Detection by Capillary Electrophoresis on an ABI 3130xl Genetic Analyzer

  Materials
  • Patient DNA samples (stored at 4°C): pre‐ and post‐transplant recipient DNA, and donor DNA (Support Protocols protocol 31, protocol 42, or protocol 53)
  • AmpFLSTR Profiler Plus ID PCR Amplification kit (Applied Biosystems, cat. no. 4330284), including:
    • AmpFLSTR PCR Reaction Mix (store at 4°C)
    • AmpFLSTR Profiler Plus ID Primer Set (store protected from light at 4°C)
    • AmpliTaq Gold DNA Polymerase (store at –20°C)
    • AmpFLSTR Control DNA 9947A (store at 4°C)
    • AmpFLSTR Allelic Ladder Control DNA (store at 20°C in PCR area as these represent amplified products)
  • Sterile H 2O
  • Running buffer (Applied Biosystems, cat. no. 402824)
  • 3130 POP‐7 (Applied Biosystems, cat. no. 4363785)
  • HiDi formamide (Applied Biosystems, cat. no. 4311320, stored in 1‐ml working aliquots at –20°C)
  • GeneScan ROX‐500 size standards (Applied Biosystems, cat. no. 401734, stored at 4°C)
  • 50°C water bath or heat block
  • 200‐µl PCR amplification tubes
  • 20‐ and 200‐µl plugged pipet tips
  • DNA thermal cycler
  • ABI 3130xl Genetic Analyzer connected to computer
  • 96 microcentrifuge tube holder (USA Scientific, cat. no. 2324‐2046)
  • 96‐well reaction plate (Applied Biosystems, cat. no. P15080A51)
  • 96‐well plate septa (Applied Biosystems, cat. no. 4315933)
  • Tabletop centrifuge with 96‐well plate adaptor
NOTE: Open each patient DNA tube using a clean, disposable, biohazard pad to prevent carryover of liquid onto your glove, which may then be inadvertently deposited in the adjacent patient PCR reaction tube.

Basic Protocol 2: Interpretation of STR PCR Products for Bone Marrow Engraftment Studies

  Materials
  • Data from capillary electrophoresis ( protocol 1)
  • GeneMapper v3.7 software (Applied Biosystems)

Support Protocol 1: Extraction of DNA from Peripheral Blood or Bone Marrow

  Materials
  • RBC Lysis Solution (Qiagen, cat. no. 158904; store at room temperature)
  • Patient sample: peripheral blood or bone marrow collected in EDTA, sodium heparin, acid citrate dextrose, or sodium citrate
  • Cell Lysis Solution (Qiagen, cat. no. 158908; store at room temperature)
  • Protein Precipitation Solution (Qiagen, cat. no. 158912; store at room temperature)
  • DNA Hydration Solution (Qiagen, cat. no. 158196; store at room temperature)
  • 100% 2‐propanol (store at room temperature in fire cabinet)
  • 70% ethanol (store at room temperature)
  • 1% agarose minigel containing ethidium bromide (unit 2.7)
  • Lambda DNA/HinfIII size standards (Invitrogen, cat. no. 15612013)
  • 50‐ml disposable polypropylene tubes
  • Gauze pads
  • Transfer pipets, sterile
  • Centrifuge
  • Wooden, cotton applicator stick, sterile
  • 50oC incubator
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7) and quantitation of DNA ( appendix 3D)

Support Protocol 2: DNA Extraction from Buccal Scrapings

  Materials
  • Patient
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2D), sterile
  • Cell Lysis Solution (Qiagen, cat. no. 158908; store at room temperature)
  • Protein Precipitation Solution (Qiagen, cat. no. 158912; store at room temperature)
  • 100% 2‐propanol (store in a fire cabinet at room temperature)
  • 20 mg/ml glycogen (Qiagen)
  • 70% ethanol
  • DNA Hydration Solution (Qiagen, cat. no. 158916; store at room temperature)
  • 1% (w/v) agarose minigel containing ethidium bromide (unit 2.7)
  • Lambda DNA/HinfIII size standards (Invitrogen, cat. no. 15612013)
  • Cotton‐tipped wooden applicators
  • 1.5‐ml microcentrifuge tubes, sterile
  • 50°C water bath
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7) and quantitation of DNA ( appendix 3D)\

Support Protocol 3: DNA Extraction from Lineage‐Specific Sorted Cells

  Materials
  • Lineage‐specific sorted cells from bone marrow or peripheral blood
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2D)
  • Qiagen QIAamp DNA Mini Kit, including:
    • Proteinase K (mix thoroughly before use)
    • Cell Lysis Buffer (AL): mix thoroughly prior to use (store individual buffers up to 1 year at room temperature)
    • QIAamp spin columns (store at room temperature for 1 year) 2‐ml collection tubes
    • Wash buffer (AW1; store up to 1 year at room temperature; dilute with 100% ethanol prior to use as directed by manufacturer)
    • Wash buffer (AW2; store up to 1 year at room temperature; dilute with 100% ethanol prior to use as directed by manufacturer)
    • Elution buffer (AE): mix thoroughly prior to use (store up to 1 year at room temperature)
  • 100% ethanol
  • 56°C water bath
CAUTION: Cell lysis buffer (AL) and wash buffer 1 (AW1) contain guanidine hydrochloride, which is an irritant to the eyes and skin. Wear gloves and eye protection when handling these reagents. These buffers can form highly reactive compounds when mixed with disinfecting agents containing bleach.CAUTION: Wash buffer 2 (AW2) contains 0.04% sodium azide as a preservative, which is highly toxic. Wear gloves when handling this reagent.
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Figures

  •   FigureFigure 9.17.1 Example of a Profiler worksheet.
  •   FigureFigure 9.17.2 Example of capillary electrophoresis plate loading form. IP, instrument protocol.
  •   FigureFigure 9.17.3 Electropherograms from pre‐transplant, donor, and post‐transplant DNA specimens following PCR amplification by AmpFLSTR Profiler Plus ID PCR Amplification kit. Numbered lines correspond to STR markers (1) Amelogenin, (2) D3S1358, (3) D8S1179, (4) D5S818, (5) vWA, (6) D21S11, (7) D13S317, (8) FGA, (9) D7S820, (10) D18S51 (also see Table ).

Videos

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Internet Resources
  http://www.appliedbiosystems.com
  The Applied Biosystems User Guides pertaining to materials used in the protocols listed above serve as excellent resources for these studies. These manuals can be downloaded by going to the Applied Biosystems Web site (also see SUPPLIERS APPENDIX).
  http://www.qiagen.com/goto/products/DNAclinical
  Qiagen QIAamp DNA Blood Mini Kit procedure with troubleshooting assistance available (also see SUPPLIERS APPENDIX).
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