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Saving time on HLA testing’s final compatibility check

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Kevin B. O’Reilly

February 2014—What would you give for a little more time? Take a vital task that you do every day, every week, or every month, and do it about three times faster—with no effect on the quality of the outcome. No, this is not another comfort-food recipe from Rachael Ray’s best-selling series of 30-Minute Meals cookbooks. This is the story of how a growing number of histocompatibility laboratories are optimizing a test that can detect low concentrations of donor-specific human leukocyte antigen antibodies and help avoid post-transplant organ graft failure.

The flow cytometric crossmatch is one of several tests that histocompatibility labs can use, after HLA typing is done, to determine the risk of hyperacute rejection or other early graft failure. It is a final compatibility check before giving the transplant team the green light. The test, which reveals how serum from the recipient reacts against donor lymphocytes, is more sensitive than cytotoxicity-based methods. A positive flow cytometric crossmatch—even in the presence of a negative complement-dependent cytotoxic crossmatch or a negative antihuman globulin-augmented cytotoxic crossmatch—is a strong predictor of graft rejection.

Use of the flow cytometric crossmatch has grown, according to the CAP. Between 2002 and 2007, there was a 7.1 percent growth in how often transplant labs used flow cytometry in crossmatch proficiency tests for class one antibodies, and a 20.6 percent jump in their use for class two antibodies (McGowan. Increased utilization of flow cytometry in HLA crossmatching reported in CAP proficiency testing. NewsPath. Nov. 12, 2008).

Of more than 130 labs participating in HLA antibody proficiency testing conducted by the American Society for Histocompatibility and Immunogenetics in 2013, about 70 percent performed the flow cytometric crossmatch. With the flow method becoming mainstream, there is growing interest in getting the assay completed more quickly so that physicians and patients get the answers they need to take action in the time-sensitive world of transplant medicine.


Dr. Liwski

According to the procedure outlined in the ASHI lab manual, the assay should be done by incubating 5.0 × 105 donor cells (30 µL) with 30 µL of the potential recipient’s serum at 4°C for 30 minutes. This is followed by three five-minute wash spins. The next step is to incubate for another 30 minutes with 100 mL of a cocktail of flourescein isothiocyanate, anti-immunoglobulin G, along with peridinin chlorophyll protein-conjugated monoclonal antibody to detect T cells, and the phycoerythrin-labeled monoclonal antibody that spots B cells. That is followed by another two five-minute wash spins. Following these steps adds up to a total assay time of 85 minutes.

“This is the technique that a majority of HLA labs are using,” Robert Liwski, MD, PhD, said during an October 2013 presentation at ASHI’s annual meeting in Chicago. “But there’s no evidence that any of these incubation times or temperatures or washing steps are actually valid.”
For Dr. Liwski, medical director of the HLA Typing Laboratory at Dalhousie University in Halifax, Nova Scotia, there was strong reason to believe that this three-color flow cytometric crossmatch method could be optimized.

“I’m a hematopathologist by trade, and I know that in the blood transfusion world we can perform these crossmatches within 30 minutes,” Dr. Liwski tells CAP TODAY. “I knew that in this different type of scenario, the incubation times are cut and we are still getting very nice reactivity. I knew that it was possible.”

So Dr. Liwski—along with his laboratory colleagues and collaborators from Emory University (Howard M. Gebel, PhD, and Robert A. Bray, PhD)—set out to test whether they could speed up the process without sacrificing the assay’s sensitivity.

First, they conducted time-course experiments, incubating with different dilutions of serum for three, five, 10, 15, or 30 minutes. They found that incubation time beyond 15 minutes had only a marginal effect on the intensity of median channel fluorescence for the B-cell and T-cell crossmatches, which is how the test’s results are determined. They also evaluated the effects of changing the time for the anti-IgG-FITC incubation step at five, 10, 15, or 30 minutes and found a negligible effect of incubation longer than five minutes.

Next, the Halifax team looked at temperature. Did the incubation steps have to be done at 4°C? No, it turned out. Actually, incubating at room temperature led to a slight increase in median channel fluorescence. They found similar improvements in fluorescence by lowering the number of donor cells to 2.5 × 105 (25 µL) and upping the serum volume to 50 µL.

The ASHI manual also recommends that the assay be performed using test tubes. By using a 96-well tray instead—reducing centrifugation time as well as reagent and buffer delivery time—the Halifax team was able to cut another 40 to 50 precious minutes from the process. A critical, potentially life-saving assay that had taken two hours to do, they now had slashed to a tidy 40-minute process.

To validate the optimized flow cytometric crossmatch protocol, Dr. Liw-ski compared it with the more time-consuming procedure in hundreds of predicted negative patient sera, predicted positive patient sera, positive control sera, and negative control sera.

“The results were virtually the same” across the board, Dr. Liwski said in his ASHI talk.

“It’s pretty clear,” he added, “that we can reduce the time it takes to perform the crossmatch without compromising the sensitivity of the assay, and that translates to significant time and cost savings and may have an impact—especially with deceased donors—on clinical care due to reduced cold ischemia time.”

But the work has not stopped there. This rapid flow cytometric crossmatch, dubbed the Halifax protocol, has spread around the world. About 50 labs in Canada, Europe, Brazil, and Australia have implemented or are testing the speedier procedure. A national proficiency testing collaboration supported by Canadian Blood Services involved 13 Canadian labs and further validated the reliability of the faster protocol and its high degree of correlation with results found through the traditional crossmatch procedure.

At the time of Dr. Liwski’s presentation last fall, 13 U.S. HLA labs were exploring the protocol, though he says leaders at several more American labs have since asked for details on how to implement the faster flow crossmatch. He shares the protocol freely. Amy B. Hahn, PhD, program planning chair of the 2013 ASHI annual meeting and director of the Albany Medical College Transplantation Immunology Laboratory, says she hopes to test the Halifax protocol at her lab soon.
Among the U.S. labs already using the faster flow crossmatch is the histocompatibility laboratory at Medstar Georgetown University Hospital. The lab’s director, Sandra Rosen-Bronson, PhD, is a past president of ASHI and founded a series of online continuing education lectures called Current Topics in Histocompatibility and Transplantation. She invited Dr. Liwski to give a talk about the optimized flow crossmatch in November 2012. Professionals from more than 80 labs signed up for Dr. Liwski’s talk. That level of interest in the Halifax protocol was manifest at the Georgetown lab.

“All the techs in our lab got very excited about it,” Dr. Rosen-Bronson says. The following month, the team at the Georgetown lab started doing the Halifax protocol alongside the traditional flow crossmatch procedure. They did parallel testing in 50 cases.

“We had 100 percent concordance between our old protocol and the new protocol,” says Olga Timofeeva, PhD, a director-in-training at the lab. “Based on patients’ antibody profiles, we expected to have 21 positive T-cell crossmatches, and both protocols resulted in 21 positive crossmatches. In the B-cell tests, both protocols gave us the same results.”

And the team shaved an hour off the time it took to run the flow crossmatch assay, says Dong Li, MD, also a director-in-training at the lab. Those hours add up. The Georgetown lab does, on average, about 90 crossmatches every month, Dr. Li says.

“If you asked anybody, ‘Do you want to go back to the old crossmatch?’ Nobody would say yes.”

What is especially nice about the Halifax protocol, adds Dr. Rosen-Bronson, is that adoption is fairly painless.

“The beauty of it is, it’s not a different instrument,” she says. “It’s the same reagents, the same number of well plates. It’s just adjustments to the incubation time and temperature, centrifuges, and things like that.”

This kind of assay-optimization work is important, yet often goes undone, Dr. Rosen-Bronson says.

“In a clinical lab setting, most labs don’t have the time to do those optimization studies. They don’t have the time and the manpower to tweak all those assays,” she says.
That was the situation for Leslie Husbands, CHT(ABHI), and her colleagues at the Oklahoma University Medical Center HLA lab.

“We’d had our flow validation done for years,” Husbands says. “It worked, and we were so overwhelmed with all the other stuff we needed to do that we didn’t have time to bring on and do the validation for the new protocol. It’s just one of those things we never got to.”

In April 2013, Husbands signed on as group lead technologist at Diagnostic Laboratory of Oklahoma, a joint venture between Quest Diagnostics and Integris Health. With a new lab came the opportunity to start from scratch in many areas, including the optimized flow crossmatch.

“We didn’t want to do everything the same. We wanted to do it better and faster,” Husbands says. The Halifax protocol is a “huge timesaver,” she adds. The lab’s HLA testing section is not yet open, but the plan, Husbands says, is to use the optimized flow crossmatch.

“It’s going to be our gold standard,” she says.

The Halifax protocol is likely to be particularly attractive to higher-volume HLA labs, Georgetown’s Dr. Rosen-Bronson says.

“The big labs especially should like this protocol, because they’re running a lot of crossmatches every day,” she says. “It’s convenient for them. It’s those busy labs that should definitely adopt this protocol.”

Kevin B. O’Reilly is CAP TODAY senior editor.