Luminex Technology

We discussed a review article during our transplant clinic morning session about Luminex technology for HLA antibody detection in organ transplantation published in Nephrology 2009; and would like to share it with you.

Pre-formed donor-specific human leukocyte antigen (HLA) class 1 antibodies (DSA) in renal transplant recipients causes hyperacute rejection; there has been an imperative to test all potential recipients prospectively for HLA antibodies to avoid transplanting incompatible grafts.

Since was first developed until recently, the method universally used for antibody detection was the complement-dependent cytotoxicity (CDC) assay. In recent years, solid phase assays have been introduced as methods for HLA antibody screening which have again redefined the definition of pre-sensitization. Luminex is a solid phase assay which consists of a series of polystyrene microspheres (beads), containing fluorochromes. Test is run as shown in the Figure.

Another important concept is PRA (Percent Reactive Antibody); it is the percentage of cells in a random panel which contains all known HLA types; giving positive results with anti-serum. PRA is useful in estimating the probability of a patient receiving a crossmatch-negative kidney.

By referring to this review study, the comparison and advantages of Luminex over CDC:
1-CDC checks for cell death caused by complement fixing antibodies against any cell antigen, while DSA by Luminex checks for the presence of the antibody against HLA type ONLY.
2-Luminex DSA is much more sensitive and can detect Donor-Specific HLA antibodies which in some cases associated with rejection in the presence of negative donor CDC cross-match. Large studies by Gibney et al and Smith et al indicated that a further subset of patients with DSA detectable only by Luminex is at high risk of graft rejection
3-CDC method can detect any Ab directed at the cell surface of a B or T cells; HLA and non-HLA antibodies and these antibodies are complement dependent in causing cell death. Luminex on the other hand detects ONLY the HLA antibodies itself, it doesn’t discriminate between complement fixing or non-compliment fixing HLA-Ab.

The question is: whether those non-compliment fixing Ab are detrimental to the graft and, if not, are we excluding patients from receiving compatible graft by using Luminex tech.
As per Washerman et al C4d+ “complement fixing Ab”, Flow + “Luminex positive” HLA class 1 antibodies graft survival is inferior to C4d -, flow + and flow – patients.

Now, what about those non-compliment fixing antibodies that are detected by Luminex, do they have an effect on the graft?
Smith et al showed that 1 year survival of renal graft of patients with C4d+ DSA was 20%, C4d – DSA was 54%, C4d + non-DSA patients was 91%. The interesting observations are the profound negative effect of the compliment fixing antibody on graft survival and also the reduction in graft survival of the C4d- DSA+

Other significant benefit of Luminex is detecting HLA-class 2 Ab in pre- and post-transplant with high level of sensitivity.

Having the fact that Luminex is far more sensitive than CDC, and at least in many cases detects additional clinically relevant Ab, the question is Can Luminex HLA replace CDC?

Also, keep in mind that on the other hand some patients with CDC-neg and Luminex positive DSA had uneventful post transplant course without evidence of rejection! So, having too much sensitive test can potentially contribute to denying some patients a suitable Graft?!

Nephrectomy or No Nephrectomy?

A recent study in JASN Feb 2010 issue, a study reports that patients who have failed allografts and who return to dialysis and undergo allograft nephrectomy have improved survival as compared to patients that have retained the allografts. This is going to spring a lot of debate in Transplantation field as it was always thought that if you take out the allograft, you have more chances of making antibodies and you will have a higher risk of rejection.
The authors in this argue that being on low dose immunsuppresion along with dialysis might be more of an infectious and cardiovascular risk and hence worse outcomes.
Its a good paper and a good study to challenge the current notion. But do we take this to practice yet?
Probably not yet, this is retrospective, only medicare patients and a more methodological study needs to be done.
This is going to bring in a lot of debate. Looking forward to see what happens next

New drugs for Hepatitis C

The latest AJT report introduces us to a new line of drugs for treatment of Hepatitis C.  The two drugs that are closest to coming to the market are in Phase 3 trials are will be submitted to FDA soon.  They are telaprevir and boceprevir. Protease inhibitors have been discussed in treating hepatitis C for years now and this brings it to the forefront.
Allowing to treat patients prior to liver transplantation might be the most benefit as the interactions post transplants with immunosuppresive medications is always a challenge. We see that in HIV patients who get kidney transplants.
The article summarizes that the future of treating primary Hep C infection or recurrence in the liver graft is going to be a cocktail of medications that will include standard medications like interferon and ribavarin but also protease inhibitors.  Recurrence of Hep c in the transplanted liver is a commonly encountered problem and this might hopefully help in the near future.
Treatment of Hep C might tag behind how HIV is being treated.

CDC HIGH RISK DONORS

What is a CDC high risk donor? A recent article in AJT Feb 2010 issue talked about a study that wanted to see how centers of disease control high risk (CDCHR) status of organ donors affects the recipients and if the organs are being used appropriately.  An observational study showed that after 2 years, the median survival of those kidneys was no different then non CDCHR kidneys.  Labeling these kidneys as CDCHR or high risk resulted in wastage of 41 kidneys per year.

What constitutes CDC criteria for high risk:- a donor who falls under one of the categories:- IV drug user, hemophiliac, prostitution history, high risk sexual activity, exposure to HIV and jail sentencing.  If this information is known, that organ is considered CDC high risk and post transplant care also includes regular checking of hepatitis and HIV viral loads every few months.

Something to ponder on!

Adrenal Gland Transplantation

The recent issue of AJT Feb 2010 describes a case report of human intramuscular adrenal gland transplantation.
Intramuscular endocrine gland transplantations have been described and we do have cases of that in the renal world of parathyroid glands being retransplanted in the muscles forearms to help continue some function on ESRD patients.  Adrenal transplantation is less common. This case describes a story of a young girl who had adrenal insufficiency and had renal failure and received a renal and adrenal transplantation from her mother.
This is the first case of a successful adrenal transplant for adrenal insufficiency.
Chronic medical treatment for adrenal insufficiency includes steroids and treatment for the transplant also includes steroids, the risk and benefits have to be weighed eventually.
At this point, the authors recommend that adrenal gland allotransplantation is only indicated in patients who already are maintained on immunosuppression for either concomitant or prior solid organ transplants.

JOURNAL CLUB: DNA Methylation controls Foxp3 gene expression

A basic science article was discussed at immunology journal club. It discusses the role of T regs and its how they come from naive T cells and what other factors triggers them.
The vast experiments done elegantly in the study concluded the following:
1. TGF-B induces FoxP3 expression in T regs but its unstable in vitro
2. Azacytidine derivatives( demethylation agent) stabalizes foxp3 expression in induced Tregs.
3. The T reg specific demthylated region (TSDR) determines transcriptional activity
4. Azacytidine promotes stable Foxp3 expression in vitro
5. In vivo induced Tregs exhibit stable Foxp3 expression and complete TSDR demethylation
6. Foxp3 Treg generated invivo by targeting of agonist ligands to dendritic cells showed long term survival in the absence of the inducing antigen and exhibited efficient TSDR demythylation.

Besides the specifics of epigenomic imprinting in the mehylation region of the T cell region which might be critical for T cell lineage, this paper I thought highlighted something very alarming. One is that TGF-B is an important promoter of T reg cells. There are drugs out there currently under development that are inhibitory to TGF-B for anti fibrotic effects. Again, we have to think  of these molecules as possible bimodal and perhaps they have different functions in different disease entities.
Second is that the dendritic cells might be important players in the long term survival of Tregs. Since there are studies that show that the patients that has increase Foxp3+ Tregs, those patients post transplant do better.
Perhaps, sustaining that positive effect should be the goal in transplantation. We can perhaps then achieve tolerance. Perhaps!!
This is interesting to me as a clinician as there might be promise ahead for tolerance.
The article source is here.
A review of the Foxp3 T regs is nicely shown in recent Nature Immunology as well
Image source: http://www.scielo.br/img/revistas/abd/v81n3/e10f1.jpg

Sirolimus and tubular toxicity

The Feb 2010 issue of AJKD talks about a study that nicely depicts the toxicities of Sirolimus.  Since its arrival to the market for preventing rejection, it has met with resistance mostly due to its severe proteinuria.  This paper describes the toxicities of sirolimus - tubules as well as in glomerulus.  Most well know toxicity of this drug is FSGS or severe proteinuria ( sometimes collapsing variant, majority the podocytopenic type of FSGS).  Severe TMA has also been noted with sirolimus use. Most of the reports of sirolimus toxicity are in kidney transplant recipients although there have been 2 reports of proteinuria in a islet cell transplant and BMT patient who had normal kidney function prior to the drug initiation.
This was a single center study done in Switzerland.  But it was open label and randomized prospective trial comparing the tubular toxic affects and rejection outcomes in cyclosporine vs sirolimus group. There were 63 patients randomly assigned to cyclosporine-based regimens, and 64, to sirolimus-based regimens. Kidney function was similar in both groups, but the elevation in markers associated with glomerular damag and tubular damage were statistically significantly more in sirolimus group. Glucosuria incidence was higher in patients randomly assigned to sirolimus therapy.  On histologic examination, the overall severity of tubular lesions was significantly higher in patients randomly assigned to sirolimus therapy. Compared with a cyclosporine-based immunosuppression regimen, a sirolimus-based regimen is associated with de novo low-grade glomerular proteinuria, increased excretion of markers associated with tubular damage, and evidence of tubular damage on kidney biopsy.
Small but nicely done study. Just brings to mind again the utility of sirolimus. Clinically, the KDIGO guidelines have a grade 2C suggestion to use this drug for patients with Kaposi sarcoma, along with reduction in immunosuppresion.
Image source: http://upload.wikimedia.org/wikipedia/commons/d/d4/Sirolimus1.gif

Positive Cross Match, Desensitization

The recent AJT Report talks about fighting the positive Cross Match or desensitization.  Currently, many centers are using the combination of pheresis and low dose IVIG or high dose IVIG and rituximab is thrown in once in a while as well.  The value of rituximab is still questioned.  No one really knows how it is working in some and not in others.
There are few new agents:- one is bortezomib, a proteasome inhibitor that has shown some data in treating antibody mediated rejection.  There are some centers using one cycle pre and one cycle post transplant as densitization technique.  This has become a concern for many as people are starting to use it for indications that have not been systematically tested.  The side effect profile of this drug is not trivial. It includes peripheral neuropathy, pyrexia, anemia, leukopenia  and GI side effects.
Finally, the latest player in the market is eculizumab ( complement protein c5 inhibitor).  It has worked in some centers to treat antibody mediated rejection. Is it a good idea to use it for desensitization. Its early to say.

Lets wait and watch.