Noninvasive diagnosis for allograft rejection
AssigneeEmory University
InventorsAftab A. Ansari, Francois Villinger, Kenneth W. Sell
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Abstract
The present invention provides a noninvasive method for diagnosing and/or predicting a rejection episode in a transplant patient, for example, in a heart transplant patient, by determining a rise in the frequency of thioguanine-resistant mutant peripheral blood mononuclear cells. Those resistant mutant cells have a deficiency in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase. The relative frequency of the mutant cells in the population from a sample can be determined by direct plating in the presence of a selection medium and by determining of the mutation by polymerase chain reaction technology.
Claims14
A method for diagnosis or prediction of an allograft rejection episode in a patient having an allograft of a solid organ or organ tissue, said method comprising the steps of:
- (a) obtaining a sample of peripheral blood of said patient;
- (b) culturing an aliquot of peripheral blood mononuclear cells from the peripheral blood sample obtained in step (a) in a medium which is selective for those cells which lack a functional hypoxanthine-guanine phosphoribosyl transferase;
- (c) culturing a second aliquot of the peripheral blood mononuclear cells from the peripheral blood sample obtained in step (a) in a medium which allows enumeration of total clonable cells;
- (d) determining a frequency of cells lacking a functional hypoxanthine-guanine
- whereby a rejection episode is diagnosed or predicted when the frequency of cells lacking a functional hypoxanthine-guanine phosphoribosyl transferase is greater than 6 per 10.sup.6 peripheral blood mononuclear cells.
A method for diagnosis or prediction of an allograft rejection episode in a patient having an allograft of a solid organ or tissue, said method comprising the steps of:
- (a) obtaining a sample of peripheral blood of said patient;
- (b) extracting ribonucleic acid from an aliquot of peripheral blood mononuclear cells from the peripheral blood sample obtained in step (a);
- (c) performing multiplex polymerase chain reaction using oligonucleotide primers having nucleotide sequences as given in SEQ ID NOs:1-16 to amplify each of eight exons of a messenger ribonucleic acid (mRNA) encoding hypoxanthineguanine phosphoribosyl transferase on the ribonucleic acid of step (b);
- (d) analyzing the products of the multiplex polymerase chain reaction of step (c) to determine the relative abundances of the amplification products corresponding to each of the eight exons of the mRNA,
- (e) determining a frequency of cells lacking functional hypoxanthine-guanine phosphoribosyl transferase by a relative decrease in the abundance of the amplification product of one or more exons in comparison to the results obtained in carrying out steps (a) through (d) for peripheral blood mononuclear obtained from a normal individual who did not have an allograft, and
- (f) correlating the frequency of cells lacking functional hypoxanthine-guanine phosphoribosyl transferase in the sample to results obtained from a normal individual, whereby an allograft rejection episode is diagnosed or predicted when there is a greater frequency of cells lacking functional hypoxanthine-guanine phosphoribosyl transferase in the sample than in the normal individual.
Dependencies are inferred from the claim text.