C1q assay to detect complement fixing antibodies

Complement

In the HLA lab we are consistently confronted with the issue of whether a detected donor-specific antibody is present or absent.  Even if a DSA is found the ultimate question still remains: is this antibody clinically relevant?   The advent of the C1q Assay now allows labs to screen sera for a sub-set of immunoglobulin G (IgG) antibodies which bind complement.  This assay is easy to run in parallel with and a single antigen bead assay and informs the lab which antibodies present in the sera are complement-fixing.  Studies have should this assay can be predictive of antibody mediated rejection.

IgM antibodies are the best at recuiting complement, however their role in transplant rejection is unclear.   IgM DSA do not lead to hyperacute rejection however, IgM antibodies against donor HLA have been shown to decrease both kidney and heart allograft survival.  The C1q asssay only detects complement fixing IgG so the role of IgM fixing complement and its clinical relevance will have to wait.

Of the IgG only certain subclasses are capable of activating the complement cascasde.  This includes IgG 1, 2, 3  of which IgG3 is best and IgG4 does not fix complement.   Using this assay will bypass IgG4 as not clinically relevant.   Although this has not be proven more studies need to be done to address the subclasses of IgG and clinical outcome.  A recent 2011 study demonstrated patients with weak or no complement-activating DSA had a lower incidence of antibody mediated rejection.

ATC Meeting in Philadelphia 2011

On Monday May 2, 2011 the TxDxCOP met to discuss the progress we have made over the year from its inception.  Some of the highlights from this year include.

• Increasing membership to our community of practice to over 80 professionals.  
• The executive committee drafted a letter for AST to address the FDAs new directive to regulate laboratory develop testing.  
• Developed a website and blog to post community information and foster an interactive environment to discuss transplant diagnostics.
• Establish transplant diagnostic curriculum for transplant conferences 

By the numbers:  

All abstracts for the ATC meeting are available online once you quickly register for free.

40 abstracts submitted to the ATC contain the keyword: diagnostic

19 abstracts submitted to the ATC contain the keyword: biomarker

HIV NAT testing

HIV Viral Components

The area of molecular diagnostics in transplantation is an emerging field.  Its impact on viral detection can be felt in a recent case where a patient contracted HIV after receiving a kidney transplant from a live donor.  In this case the donor was HIV tested using the Enzyme Immune Assay ~3 months (79 days) prior to transplant.  This assay was unable to detect a HIV exposure which occurred closer to the day of transplant.  After the incidence the CDC NAT (nucleic acid testing) tested the donor leukocytes collected 57 days pre-transplant and found they were negative for the presence of HIV genes (gp41, pol, p17), however these genes were amplified from donor leukocytes at day 11 pre-transplant.   In light of the first case in over two decades, the CDC has created new recommendations for HIV testing organ donors no more than seven days before transplantation.  These new guidelines should eliminate or dramatically reduce the risk for transplant-transmitted HIV infection

This situation emphasizes the strength of NAT testing as a diagnostic tool to shorten the gap between HIV infection and detection to 8-10 days compare to serological techniques (EIA/ELISA/Western Blot) which measure the development of HIV-specific antibodies after 3-8 weeks.  The ability of NAT testing to detect the presence of HIV genes in a relatively short period of time will increase support within the transplant community to immediately rescreen living donors with identified behavioral risk factors  prior to organ transplant.

Currently, the US Public Health Service guidelines only recommend serologic screening of HIV infection in potential living donors.  Revising these guidelines to include NAT testing and a standardized testing timeline before live donor transplant is essential. A 2009 study in AJT found that incorporation of NAT testing was able to significantly increase the utilization of certain (high risk donor) HRD organs by OPOs. (organ procurement organizations)  They also surveyed OPO clinical directors found 52% of centers always performed HIV NAT testing whereas 24% do not test donors for HIV.  It is possible these low numbers refect the cost-benefit analysis being performed for NAT testing where several disadvantages including loss of donor organs due to a false-positive NAT result need to be addressed. 

FDA Approves Test for Everolimus Immunoassay

Nature Reviews Drug Discovery 8, 535-536 (July 2009)

The FDA has approved the first test to monitor blood levels of Everolimus in  kidney transplant patients.  This is a competitive inhibition assay using a Quantitative Microsphere System (QMS®).  An agglutination reaction between antibody and microsphere particle bound Everolimus is inhibited in the presence of free Everolimus in the patient sample.  From this a concentration-dependent classic aggultination inhibition curve can be acquired where a maximum rate of agglutination corresponds with the lowest Everolimus concentration.  When evalutated by Dasgupta et. al  the assay produced a direct linear correlation between 1.5 and 20ng/mL and a lower limit of 1.3ng/mL.  The researchers also noted a 46% cross-reactivity with sirolimus which is structurally similar to everolimus.  The QMS Everolimus Immunoassay from Thermofisher  provides a new immunoassay for the routine monitoring of Everolimus in whole blood.

FDA Oversight of Laboratory Developed Tests (LDTs)

The FDA has held a 2 day workshop to hear from the pubic on how best to regulate LDTs.   In the past the FDA has had the right to oversee LDT development, however not until recently decided to enforce regulation.  Multiple assays involved in transplantation (ie. HLA typing, crossmatching, C4d staining) would all fall under this new regulation.  Without federal oversight the FDA is concerned that LDTs not properly validated will put patients at risk for a missed or wrong diagnosis.  In addition, there is pressure from industry sources who want a level playing field whose tests need to be approved or cleared by the FDA.  The current lack of FDA oversight in academic labs allows for quick turn around time at a rapid bench to bedside pace. Currently, the FDA wants a risk-based application of oversight to all LDTs.  It is unclear how the FDA will enforce this regulation or how it will effect transplantation in the US.

NGAL test to predict early acute kidney injury

NGAL

The ideal for diagnostic testing would be knowing early when a transplant patient is undergoing a rejection episode.  Searching for news on biomarkers I found recently the NGAL Test™ was released for diagnostic use in Europe (currently unavailable in United States) as a biomarker to diagnose acute kidney injury.  NGAL (Neutrophil Gelatinase-Associated Lipocalin), a small protein expressed in specific granules of neutrophils and at lower levels in other organ systems including the kidney.   NGAL is up-regulated in cells under a variety of stressors and is involved in the sequestering of bacterial products such as iron-containing siderophores during an inflammatory response.

Other great equalities in addition to being an early biomarker of acute renal failure,  NGAL is resistant to protease degradation and can be detected in either urine or blood.

Triage® NGAL Test