Which blood collection tube should be used for flow‑sorted chimerism testing after allogeneic stem cell transplantation (allo‑SCT) in an adult patient?

Blood Collection Tube for Flow-Sorted Chimerism Testing Post-Allograft

For flow-sorted chimerism analysis after allogeneic stem cell transplantation, collect blood in EDTA (lavender/purple top) tubes, specifically K₂EDTA or K₃EDTA anticoagulant tubes. 1

Rationale for EDTA Tube Selection

• EDTA tubes preserve nucleic acids and cellular integrity by chelating divalent cations (calcium, magnesium) that serve as cofactors for DNase enzymes, thereby protecting DNA from enzymatic degradation during collection, transport, and processing—critical for subsequent PCR-based chimerism analysis. 1

• Flow cytometry cell sorting requires viable, intact cells with preserved surface antigens, and EDTA anticoagulation maintains cell viability without the PCR inhibition concerns associated with heparin tubes. 1

• International consensus guidelines for hematologic malignancies explicitly state that EDTA anticoagulant is appropriate for PCR-based assays, which are the standard method for chimerism quantification after cell sorting. 1

Collection Specifications

Volume and Tube Type

• Collect a minimum of 5 mL of peripheral blood in K₂EDTA or K₃EDTA tubes to provide sufficient nucleated cells for flow sorting followed by chimerism analysis. 1

• Bone marrow aspirate (1–2 mL from the first or second draw) in sodium heparin or EDTA is the preferred specimen type when plasma cell enrichment protocols are adapted for chimerism testing, though peripheral blood is standard for routine post-transplant monitoring. 2

Immediate Handling

• Invert the EDTA tube 8–10 times (180-degree turns) immediately after collection to ensure thorough mixing of blood with anticoagulant and prevent clot formation. 1

• Fill tubes completely until the vacuum is exhausted to maintain the correct blood-to-anticoagulant ratio; underfilling below approximately 67% of nominal volume causes DNA degradation and spurious results. 1

Processing Timeline

• Process specimens within 24 hours of collection to avoid significant cell degradation; for maximal sensitivity in detecting low-level mixed chimerism, processing within 2 hours is preferred. 1

• If immediate processing is not feasible, refrigerate samples at 4°C for no longer than 24 hours before cell sorting and DNA extraction. 1

• Samples should be processed within 24–120 hours after collection depending on laboratory validation of sample stability, with CD138 and other cell surface markers potentially internalizing over extended time periods. 2

Order of Draw

• Follow the standardized order of draw when collecting multiple tube types: blood culture tubes first, sodium citrate tubes second, serum tubes third, heparin tubes fourth, EDTA tubes fifth, and sodium fluoride tubes last—this prevents cross-contamination of additives that could interfere with flow cytometry or PCR. 1

Common Pitfalls to Avoid

• Do not use heparin tubes as the primary collection method because some investigators consider heparin to inhibit PCR reactions, although this concern is mitigated by adequate cell washing during processing. 1

• Avoid underfilling EDTA tubes, as the incorrect blood-to-anticoagulant ratio introduces significant bias and can cause hemolysis that compromises both flow cytometry gating and DNA quality. 1

• Use a 21-gauge needle to minimize hemolysis and cellular damage that could compromise both cell viability for sorting and DNA quality for chimerism PCR. 1

• Do not delay processing beyond validated stability windows, as prolonged storage at room temperature (>36 hours) leads to marked cell degradation and reduced sensitivity for detecting low-level mixed chimerism. 1

Clinical Context

• Chimerism monitoring practices vary widely among transplant centers, with the most consistent indication being engraftment monitoring within the first 30 days post-transplant, followed by early detection of impending relapse and identification of HLA-loss relapse cases. 3

• Lineage-specific chimerism analysis (requiring flow-sorted cell populations) is more sensitive than whole blood chimerism in predicting impending relapse, particularly in diagnoses with high relapse risk. 4

• High-sensitivity methods such as next-generation sequencing are increasingly used for chimerism quantification after cell sorting, making preservation of high-quality DNA in EDTA tubes even more critical for detecting low-level mixed chimerism (<1% sensitivity). 4