Assessing quantitative chimerism longitudinally: technical considerations, clinical applications and routine feasibility

Abstract

In this review, we describe the current laboratory approach to quantitative chimerism testing based on short tandem repeats (STRs), focusing on a longitudinal analysis. The latter is based on relative changes appearing in the course of sequential samples, and as such exploits the ultimate potential of this intrinsically semiquantitative platform. Such an analysis is more informative than single static values, less likely to be confused with platform artifacts, and is individualized to the particular patient. It is particularly useful with non-myeloablative conditioning, where mixed chimerism is common. Importantly, longitudinal monitoring is a routinely feasible laboratory option because multiplex STR-polymerase chain reaction kits are available commercially, and modern software can be used to perform computation, reliability testing and longitudinal tracking in a rapid, easy to use format. The ChimerTrack application, a shareware, user friendly program developed for this purpose, produces a report that automatically summarizes and illustrates the quantitative temporal course of the patient's chimeric status. Such a longitudinal perspective enhances the value of quantitative chimerism monitoring for decisions regarding immunomodulatory post transplant therapy. This information also provides unique insights into the biological dynamics of engraftment underlying the fluctuations in the temporal course of a patient's chimeric status.

Quantitative monitoring of multi-donor chimerism: a systematic, validated framework for routine analysis

“…23,32 STR-loci for validation analysis Six DD chimeric STR loci, which are technically indistinguishable from mixed DNA clinical cases, were each fabricated from sets of three unrelated, normal DNA samples; these DNAs would correspond to D1, D2 and R in clinical material. Thus, each DD-CHM locus will have six alleles, but because of the high frequency of some of these alleles in our population, the alleles were often shared between DNA sources, that is, have the same STR repeat structure and size (bp).…”

“…At such loci, hundreds of possible configurations for allelic sharing are possible, many of which will result in the masking of critical recipient and donor measurements. Such extensive allelic sharing also interferes with assessing measurement error; 23,46 therefore, even if %CHM could be estimated, its accuracy would be in doubt. Indeed, one could question whether it is ever possible to accurately assess CHM in such DD cases.…”

“…47 The second obstacle to quantitation with STRs is the phenomenon of allelic measurement error-typically 5-20%-which will naturally influence the accuracy of %CHM. 23,32,46 The process of calculating %CHM in DD cases is substantially more complex than it is for single-donor HSCTs. The presence of two donor DNA sources in the patient can produce chimeras with up to six alleles, instead of four.…”

“…[20][21][22][23][24] STRs are short sequences of nucleotides that are randomly and variably repeated throughout the genome. 25 Using this quantitative approach, it is possible to assess the full range of CHM values, from 0 to 100%.…”

Assessment of CD4 T-lymphocyte reactivity by the Cylex ImmuKnow assay in patients following allogeneic hematopoietic SCT

“…Currently, post-alloHSCT laboratory tests that assess engraftment consist primarily of complete blood count (CBC) and chimerism studies, 4 but there are no routine functional tests that evaluate the immune function of the new graft. The Cylex ImmuKnow assay, a laboratory test approved by the FDA in 2002, determines the cellular immune function in immunosuppressed patients by quantitatively measuring the intracellular ATP levels in PHAstimulated CD4 þ T-helper (Th) cells in whole blood.…”