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Flow cytometry consignment services

Services for medical professionals

Mixed chimerism test

Introduction

Since the wide implementation of bone marrow transplants and umbilical-cord blood transplants as treatments for leukemia, the outcome of leukemia treatments has been significantly improved. On the other hand, poor engraftment and disease recurrence after transplantation are still serious issues to date. The incidence of poor engraftment is 1% in the case of bone marrow transplants and 5-20% in the case of umbilical-cord blood transplants. The incidence of disease recurrence is as high as 10-50% in both types of transplants. While replantation is one of the most promising measures to address these issues, it is still the first priority to detect poor engraftment and recurrence effectively at an early stage to obtain better outcomes for the bone marrow or umbilical-cord blood transplants.

Recognizing this need, ReproCELL developed a new mixed chimerism test method within the framework of a joint project with the Institute of Medical Science at the University of Tokyo. This method allows early detection of poor engraftment and disease recurrence with a sensitivity (>10^-4), quantitative accuracy and speed that are unfeasible with the conventional test method.

Measurement principle

*Antibody selection:
Focusing on the inconsistency of HLA between donor and recipient, an anti-HLA antibody is selected that responds to each HLA in a specific manner. In the above example, an anti-HLA-A11 antibody (donor-specific marker) and anti-HLA-A24 antibody (recipient-specific marker) are shown.

Comparison with conventional test method

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Early stage mixed chimerism test of peripheral blood

A factor of poor engraftment is attributable to the propagation of remaining recipient-originated bone marrow immunocytes after the transplant, resulting in inhibition of the donor-originated bone marrow cells. To detect poor engraftment, it is essential to monitor the change in the ratio between the recipient-originated leukocytes and donor-originated leukocytes (chimerism) in the peripheral blood. However, there are some challenges in the conventional test method in terms of sensitivity, quantitative accuracy and speed.

Our new mixed chimerism test is a method that has successfully resolved all these difficulties and is best suited for a mixed chimerism test at an early post-transplant stage. With the simultaneous analysis of the other antigen markers, mixed chimerism tests for the leukocyte fraction (CD45⁺) and T-cell fraction (CD3⁺) that play an important role in the post-transplant engraftment are possible. Moreover, growing interest is seen recently in the post-transplant dynamic changes of mixed chimerism in leukocyte subpopulation as a new field of study. This mixed chimerism test may be a promising technique in this aspect.

Analysis result

Example of analysis result

Chronological change of mixed chimerism in T-cell faction in the engraftment process

Test details

Options

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Bone marrow mixed chimerism test

The recurrence refers to the status where leukemia cells increase again after remission through a treatment. Accordingly, it is critical in the recurrence screening to detect an increase of MRD (minimal residual disease) at an early stage.

Conventional tests for recurrence check are grouped into the 3 following types:"Identification of tumor cell based on morphological observation", which is the classic method, "Indirect recurrence test based on chimerism analysis (XY-FISH, STR-PCR, etc)"utilizing the fact that the recurrence is attributable to the recipient-originated cell, and, "MRD identification based on tumor-specific gene analysis (PCR, Southern Blot, FISH, etc.) utilizing the specific marker.
Each of these methods has its own strengths and weaknesses in the aspects of sensitivity, labor, patient coverage and cost. Medical professionals select a test method that seems most suitable to each individual patient.

The mixed chimerism test can be used as a potent option of the recurrence screening because of its unprecedented level of advantages.

Analysis result

Recipient cell: HLA-A11 / Donor cell: HLA-A9

Example of analysis result

Result from bone marrow mixed chimerism test on Day 159 and Day 357 after umbilical-cord blood transplant

Test details

Options

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Available options for mixed chimerism test

2 types of options are available for our mixed chimerism test.

Absolute number count

The percentage of each subpopulation is shown in the flow cytometry analysis result.
However, an option to obtain the absolute number of each subpopulation is also available.
In our absolute number measurement, the absolute number of each leukocyte subpopulation is measured by using the ProCOUNT reagent kit (manufacturer: BD).

Sorting of specific cells

Donor-originated cells or recipient-originated cells specified in the mixed chimerism test are sorted.
The cell specimen after the sorting is returned using the method specified by the client.

Application of Primary Culture Cells
By sorting the recipient-originated cells identified in the mixed chimerism test and by combining the following types of tests, the sensitivity of detecting recurrence and poor engraftment can be significantly improved.

Case study: Sorting + FISH analysis

Specimen : Bone marrow specimen on Day 357 after umbilical-cord blood transplant
Tumor marker [AML1/ETO]

Because the tumor marker of this specimen was evident, fractional sorting of the recipient-oriented cells was carried out by using a mixed chimerism test. The sorted fraction underwent FISH analysis.
The analysis result revealed that the fraction considered as originated from the recipient cells had tumor markers with 99% incidence, while no tumor marker was identified in the fraction from donor-originated cells.

*Note: Prior consultation is necessary for consignment of our sorting service.

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