Predicting Outcomes of Rat Vascularized Composite Allotransplants through Quantitative Measurement of Chimerism with PCR-Amplified Short Tandem Repeat

Abstract

Chimerism has been associated with the induction and maintenance of tolerance to vascularized composite allotransplants (VCA). Although most VCA studies have examined chimerism using flow cytometry, we proposed that precision in the measurement of chimerism may be better approximated when complimentary polymerase chain reaction (PCR) is applied to a specific short tandem repeat (STR). We identified a STR, D10Rat25, which exhibited a ~20 bp difference in length between two rat strains (BN and LEW) often utilized as the donor and recipient in many allotransplantation studies. D10Rat25 was PCR-amplified and quantified with capillary electrophoresis. With pure LEW and BN DNA, a standard curve was constructed to measure chimerism with good linearity. When applied to rat VCA, the relationship between systematic (in peripheral blood) or local (at specific organ/tissues) chimerism to allograft outcomes was noted. We found that peripheral chimerism was elevated by up to ~9% postoperative month 1 (POM 1) but then reduced regardless of the final VCA outcome. However, differences in VCA skin chimerism between early rejection and POM 1 (shown as ΔChimerismPOM1-ER) were notable with respect to VCA outcomes. ROC analysis identified the optimum cutoff value as 17.7%. In summary, we have developed a reliable method to quantify the percentage of BN cells/DNA in BN-LEW chimeras. The detection limit was characterized, and the acquired data were comparable with flow cytometry. This method can be applied to solid organ and composite tissue allotransplantation studies.

Vascularized Composite Allotransplantation of the Hand

“…The majority of centers (19/40) placed specific anatomic requirements for eligibility: 5 centers mentioned bilateral amputation or dysfunction as a prerequisite for surgery 17,34,38,54,110 ; others (5) allowed for unilateral transplantation as long as the extremity to be transplanted corresponded to the patient's dominant 20,63,77,79,99 . Nine centers allowed for unilateral nondominant amputation on a case-by-case basis 12,19,28,42,50,66,69,70,75,88,103,105,115 …”

Reciprocal Donor-recipient Strain Combinations Present Different Vascularized Composite Allotransplantation Outcomes in Rodent Models

“…The immunosuppression combination of ALS, short-course CsA, and ADSCs induced donor-specific tolerance in LEW recipients. 9,18 However, when the same regimen was applied in BN recipients, all allografts were rejected within the first month. Furthermore, the ADSCs had no effects on VCA survival in BN recipients ( P = 0.069), but significantly prolonged the VCA survival in LEW recipients under the same regimen ( P = 0.003) (Fig.…”

“…In contrast, tolerant rats exhibited only transient (up to 2 months) peripheral chimerism. 18,39 Such divergence may reflect the species differences in the level of alloreactive cell suppression required to maintain allograft survival, and could be clarified by further systematic investigation.…”

“…Previously, we successfully performed VCA from a BN donor to a LEW recipient. 18 The osteomyocutaneous allografts isolated from the donor's hindlimb were transplanted to the groin area of the recipient. Serious damage, aside from minor scratches, rarely occurred during the 5-month observation period.…”

The intragraft vascularized bone marrow induces secondary donor-specific mystacial pad allograft tolerance

“…It differed from the previous protocols where the secondary VCA transplantation was performed >200 days ( 8 ). According to our previous studies with murine models, 1 month is a crucial period for the induction of tolerance when the chimerism level peaked ( 25 , 26 ). The secondary allograft remained viable even after removing the VBM component ( 9 ).…”