Chimerism Testing

Chimerism testing measures the proportion of donor derived and recipient derived cells in the blood or bone marrow after allogeneic haematopoietic stem cell transplantation. The usual method is analysis of short tandem repeat (STR) polymorphisms by PCR and capillary electrophoresis, which distinguishes donor from recipient alleles and reports donor percentage.

Quantitative real time PCR for insertion deletion polymorphisms, digital PCR and next generation sequencing detect lower levels (0.01 to 0.1%). Testing is performed on whole blood or marrow and on sorted lineages (CD3-positive T cells, CD33 or CD15-positive myeloid cells, CD19 B cells, CD34-positive progenitors).

Full donor chimerism (over 95% donor cells) is the usual goal after myeloablative transplant. Mixed chimerism (5 to 95% recipient cells) is common after reduced intensity conditioning and carries risk of graft rejection or relapse.

Falling donor chimerism in the T-cell or CD34 compartment predicts relapse in leukaemia, particularly in myeloid malignancy, where each 1% fall in donor myeloid chimerism is associated with higher relapse risk. In non-malignant diseases (thalassaemia, sickle cell disease, immunodeficiency) stable mixed chimerism of 20 to 30% donor myeloid cells may be enough to correct disease.

Indications. Routine monitoring after allogeneic stem cell transplantation (engraftment around day 14 to 30, then day 60, 100, 180 and 365, and then yearly). Suspected graft failure, falling blood counts or poor graft function. Early detection of relapse in leukaemia, with lineage specific testing (CD34 or myeloid fraction).

Decision on donor lymphocyte infusion or immunosuppression withdrawal. Monitoring after non-malignant disease transplants. Assessment of recurrence after cord blood transplantation with double units (dominance of one unit). Confirming identity of cell products and excluding sample mix up. Transfusion associated graft-versus-host disease, microchimerism in solid organ transplants (research).

Specimen. EDTA peripheral blood 3 to 5 mL or bone marrow aspirate 1 to 2 mL for DNA extraction (and for cell sorting, fresh sample within 24 hours). Pre-transplant DNA samples of donor and recipient are required to identify informative markers. Results depend on a pre-transplant baseline.

Reference values and categories.

• Full donor chimerism: over 95% donor cells.
• Mixed chimerism: 5 to 95% recipient cells (some use 1 to 5% as low level).
• Graft failure: under 5% donor cells or return of recipient haemopoiesis.
• Sensitivity of STR analysis is about 1 to 5%. Quantitative PCR methods detect 0.1 to 0.01%, and digital PCR and sequencing under 0.01%.

Interpretation.

• Full donor chimerism with count recovery: successful engraftment, with continued monitoring.
• Stable mixed chimerism: acceptable after reduced intensity conditioning, and consistent with disease control in non-malignant disease.
• Falling donor chimerism in CD3 T cells: risk of graft rejection, and immunosuppression is reduced and donor lymphocyte infusion considered (balanced against graft-versus-host disease).
• Falling donor chimerism in the myeloid or CD34-positive fraction in a patient with leukaemia: impending relapse, with measurable residual disease testing by flow cytometry or molecular markers, bone marrow examination, and pre-emptive treatment (immunosuppression withdrawal, donor lymphocyte infusion, hypomethylating agents, targeted drugs).
• Rising recipient cells with dropping counts: graft failure, and a second transplant or stem cell boost may be required.
• Cord blood double unit transplants: usually one unit dominates by day 100.
• A trend over at least two to three consecutive samples is more reliable than a single value.

Critical values and findings needing immediate action.

• Rapid fall in donor chimerism (for example a drop of 10% or more, or under 90% in a myeloid cell compartment) in leukaemia: bone marrow assessment, measurable residual disease testing and early intervention before haematological relapse.
• Loss of donor chimerism with pancytopenia: graft failure, stop myelosuppressive drugs, check viral reactivation and drugs, and consider a donor stem cell boost.
• Increasing mixed chimerism with new graft-versus-host disease signs (rash, jaundice, diarrhoea): evaluate and treat for acute GvHD.
• New cytopenia on immunosuppression withdrawal: repeat chimerism and review.

Interferences and pitfalls.

• Informative markers must be chosen from pre-transplant samples of both donor and recipient. A donor and recipient who are siblings share many alleles, so more markers are required. Pre-transplant samples are stored.
• Recent blood transfusion in the first weeks can add third party DNA, and red cell chimerism differs from nucleated cell chimerism (ABO mismatch).
• Whole blood chimerism misses lineage specific changes, so T-cell and myeloid fractions are analysed separately, especially in leukaemia.
• STR sensitivity is limited, and a stable value near the detection limit should not be over interpreted.
• Preferential amplification of smaller alleles, stutter peaks and low DNA can cause bias, and replicate analysis is required.
• Bone marrow samples are more informative than blood for CD34-positive cells, although a marrow aspirate is invasive.
• Chimerism is not a replacement for measurable residual disease testing of leukaemia specific markers (NPM1, BCR-ABL1, flow cytometry).
• Haploidentical and cord blood transplant interpretation requires attention to the dominant unit.
• Results are only valid for the transplant under study, and a second transplant needs a new baseline.

Special groups.

• Children: monitoring in non-malignant diseases (thalassaemia, sickle cell disease, severe combined immunodeficiency, inborn errors) where mixed chimerism may be adequate, and growth of mixed chimerism needs close follow up.
• Pregnancy: not applicable to routine care, though microchimerism from pregnancy exists in healthy women.
• Older adults: reduced intensity regimens produce mixed chimerism more often, and tailored monitoring is used.
• Regions: access to STR analysis is limited, and chimerism is used where transplant centres exist, with frequent monitoring as a way to detect relapse early.

Follow up tests. Full blood count with differential and reticulocytes, bone marrow aspirate and trephine, measurable residual disease testing (flow cytometry, NPM1, BCR-ABL1, WT1), cytogenetics or FISH for known abnormalities, lineage specific chimerism (CD3, CD33, CD19, CD34), cytomegalovirus, Epstein-Barr virus and adenovirus PCR, immunosuppressant levels, immunoglobulin levels,

lymphocyte subsets, HLA antibody screening for graft failure, and review for graft-versus-host disease.