College of American Pathologists
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Chill out-a new idea on platelet storage

August 2003
Anne Paxton

More than 40 years ago, researchers described the shape change that blood platelets undergo when refrigerated, and they observed that platelets—unlike other transplantable tissue—don’t survive long in recipients’ bodies once they have been chilled.

Consequently, platelets must be stored at room temperature, which gives them a notoriously short shelf life. In contrast to red blood cells, which are transfusable for 42 days, platelets are good for only five days. But why chilled platelets have this clearance response has remained a hematological mystery until now. A new study by researchers at Brigham and Women’s Hospital, Boston, suggests an answer, and potentially some ways that scientists might prevent clearance.

Platelets’ five-day term limit is the leading reason U.S. blood services routinely discard 25 percent or more of donated platelets. The wastage aggravates blood shortages already worsened by a declining donor pool. That leaves many patients at risk, and, by some estimates, it costs the health care system more than $1 billion a year.

In an article published in Cell last January (Hoffmeister KM, et al. 2003; 112:87–97), cardiologist Karin M. Hoffmeister, MD, and colleagues proposed that platelets are thermosensors that become more susceptible to activation by thrombotic stimuli when they are cooled.

This “priming,” they speculate, may be an adaptation to limit bleeding at lower temperatures of body surfaces, where most injuries occurred throughout evolution. But they find evidence that the liver may be programmed to clear repeatedly primed platelets—which would explain why chilled platelets do not last after transfusion.

Chilling is desirable because controlling bacterial contamination is arguably the No. 1 priority in blood services today, says Edward L. Snyder, MD, professor and associate chair for clinical affairs in the Department of Laboratory Medicine, Yale University, New Haven, Conn.

“We now have most known transfusion-transmitted viruses under heavy surveillance and they’re being screened for,” he says. “New ones pop up, like monkeypox, West Nile virus, and SARS, and those too are being evaluated for their ability to pose a threat to the blood supply. For some, we have PCR-based nucleic acid testing to address them. So what remains as the biggest risk from platelet transfusion? It’s actually bacterial transmission. Between one in 1,500 and one in 2,000 units of platelets will show bacterial contamination.”

“About 10 or 20 years ago,” Dr. Snyder adds, “regulatory agencies stopped approval of platelet storage at 1° to 6°C, because even though the risk of bacterial contamination was about 30 times smaller in the cold, the problem was that the platelets were cleared from the recipient’s body, and nobody understood why.”

So chances are good that a unit of platelets stored at room temperature will be effective, but the chances that it will also be contaminated are not insignificant. “The fact is that right now in most places in North America, we don’t test blood products for bacteria,” says Dana Devine, PhD, executive director of research and development for Canadian Blood Services, Vancouver, British Columbia.

“Under current storage conditions for platelets, we keep bacteria swimming in the platelet soup in the incubator at room temperature,”Dr. Devine says. “If they’re in there and they start to grow and multiply, in five days you can have enough to cause real problems for a recipient.”

Room-temperature storage causes an additional problem—platelet storage lesion. “We don’t really understand what causes it,” Dr. Devine says, “but it’s almost a platelet aging phenomenon that happens in the bag. Even if the platelet product is completely free of bacteria and you could prove it, by the time it’s left hanging around for seven, eight, or 10 days, the platelets don’t really look great. They’re basically dying of old age in the bag.”

Research has shown that platelet aging is significantly less at the in vitro storage temperature of 22°C than it is at the in vivo temperature of 37°C (Holmes S, et al. Br J Haematol. 1995; 91: 212–218). “Simply biologically, most cells are better in storage if they can be chilled, assuming you slow down their metabolism,” explains Laurence A. Sherman, MD, JD, emeritus professor of pathology at Northwestern University, Chicago, and a member and former chair of the CAP Transfusion Medicine Resource Committee. “Additionally,” he says, “there is old but suggestive data that refrigerated platelets may be more functional in vivo, albeit rapidly cleared from the blood.”

If hematology research could lead to a way to effectively chill platelets, he says, it would be a giant step forward in fixing the shelf-life problem.“The shelf life of platelets was cut back from seven days to five days in 1986 to minimize the chance of bacterial contamination. Any way to get around this issue would substantially benefit platelet inventories,” Dr. Sherman says, noting that nucleic acid testing for various viruses means platelet units had their quarantine period lengthened, cutting into the already shortened five-day shelf life.

Like many successful experiments, Dr. Hoffmeister’s sprang from a hypothesis that didn’t pan out. Since five minutes of refrigeration changes platelets’ shape from a smooth, shiny disk to an unsmooth one, Dr. Hoffmeister, an instructor in medicine in the hematology division at Brigham and Women’s Hospital, used a cocktail of chemicals to prevent the shape change. She expected this to halt the clearance mechanism.

But, as she reports in the Cell article, the platelets still cleared rapidly from the circulation after chilling, even though they had the normal discoid shape. “We used the chemical cocktail, but the platelets didn’t survive. They were cleared just as fast as the chilled platelets without the preservatives. It was one of the points in my research career where the theory didn’t work out,” Dr. Hoffmeister told CAPTODAY.

Realizing that some factor other than shape change had to be triggering the clearance, she decided to look for the organs that handled the process. She injected chilled radioactive-labeled platelets in mice, then looked for the organs that recognized the platelets. “One of the striking results was that the chilled platelets’ clearance occurs mostly in the liver, whereas normal platelets’ clearance is divided between the spleen and the liver,” she says.

By injecting platelets tagged with different colored markers, she found hepatic macrophages that ate the platelets. But it was one receptor—complement type 3—that was important for the clearance of chilled platelets. “If the mice are missing this receptor CR3,” she says, “the chilled platelets are not eaten, so room-temperature and chilled platelets survive the same in the mice.”

“We then had a receptor that normally recognizes ‘bad stuff’ as bacteria or yeast in our body and removes it,” Dr. Hoffmeister says. “The question then was what does the receptor recognize on chilled platelets?”

An interesting finding, she adds, was that chilling platelets causes changes in their surface, leading to aggregation of the glycoprotein Ibα—a receptor for von Willebrand factor—in the lipid membrane, which is not reversible by rewarming the platelets to normal temperatures, but the chilled platelets nevertheless function perfectly. “So chilled platelets are still functional,” says Dr. Hoffmeister. “They respond even a little better to normal agonists compared to room-temperature platelets. The main problem is that they are cleared immediately from the circulation.”

“We proposed,” she adds, “that chilling primes platelets for activation at peripheral sites, where most injuries occurred throughout evolution, and if exposed to lower temperatures, they work better. But in order not to cause pathologic thrombi in our bodies, we clear these primed platelets through the liver.”

In a commentary on Dr. Hoffmeister’s research (Snyder EL, et al. N Engl J Med. 2003; 348: 2032–2033), Dr. Snyder explains that the pathways that govern the clearance of chilled platelets differ from those responsible for hemostasis, and if the mechanisms are distinct, there is a better chance for inhibiting the clearance pathways without affecting hemostasis.

That a single receptor on the platelet is responsible for the clearance response is a promising medical finding, Dr. Hoffmeister says. “If you can find a way to prevent engagement of these receptors, without changing the function, so that the platelet survives, then you could transfuse platelets that are refrigerated.”

Theoretically, a surface modification of the carbohydrates on the receptor could prevent engagement of the two receptors. For example, Dr. Snyder suggests, if the same pathway of platelet clearance exists in humans, modifying the platelet glycoprotein Ibα-binding site might decrease the binding of hepatic macrophages and thereby extend the time chilled platelets spend in the circulation, while preserving the distinct glycoprotein Ibα epitope for the binding of von Willebrand factor.

Companies and research institutions in the United States and Canada are pursuing methods to allow cold storage of platelets without damage, though cold storage is much further from clinical use than pathogen reduction. “There are various approaches that have been actively studied for some years on ways to treat cellular blood products, and platelets in particular,” Dr. Sherman notes, citing UV-light-activated materials or chemicals that could inactivate microorganisms. “But each method has its own potential issues as you go down the road.”

Dr. Snyder and many others believe that mechanisms to prevent platelet removal following cold storage will soon begin to be developed. But blood services are “a little ways away” from putting platelets in the refrigerator, Dr. Devine cautions. “The piece we don’t know is what you have to put in the platelets to prevent cold damage and still be able to use the product,” she says. “In the laboratory there are all kinds of things we can add that prevent deterioration, but most of them are lovely little toxic things that would prevent every other cell in your body from working as well.”

Partly for that reason, blood policy in North America is focusing on bacterial detection as well as prevention. A voluntary standard of the American Association of Blood Banks, to take effect in April 2004, will require blood services to adopt strict bacterial-detection procedures. The National Institutes of Health and American Association of Blood Banks held a meeting July 31–Aug. 1 to discuss pathogen-reduction technology.

Canadian Blood Services has already moved ahead with a mandate for bacterial detection of platelets, beginning with pheresis platelets in early 2004. Although there are concerns about cost, “I think it will become the standard of care” in the United States as well, Dr. Snyder predicts.

A major benefit of effective detection is that it would make it possible to return to a seven-day shelf life. “The fact is that whatever bacteria are there at day seven are there by day five. If you really want to make a dent in the incidence of bacterial contamination, you should only store platelets for three days,” Dr. Snyder says.

“Under current practices, though, hospitals don’t get the platelets until day two,” he adds. “If platelets were only stored for three days, they have one day of shelf life left, and most of them get wasted.” If, through effective bacterial detection, you could show that the platelets are not contaminated on day one, that might allow storage for seven days.

But Dr. Snyder doesn’t think minimizing bacterial contamination will be enough. “In this day and age, a one in 2 million incidence of infection is enough to make the front page. Will you still need pathogen-reduction agents that will do far more? I believe the answer is yes, because there are too many other pathogens threatening the blood supply.”

“If, however, it turns out that current pathogen-reduction technologies are too toxic,” Dr. Snyder says, “then cold storage of platelets may actually be much more beneficial, because it would be better than going back to room-temperature storage.”

“What we don’t know is the effect of cold storage on pathogen-reduced platelets,” he continues. “Are these two technologies synergistic?” For example, researchers might need to assess the effect of storing an inhibition material that allows cold-stored platelets to circulate in a storage bag with other pathogen-reduction technologies.

“No one’s done anything on that,” he says. “There are a lot of unknowns as all of these technologies are coming to the finish line at the same time. The customer, the hospital, and the patient are looking for the most efficacious product at the least expense, but we’re trying to evaluate several things at once, and it will be very difficult to sort out.”

In the meantime, says Dr. Sherman, the College plans to include in its Laboratory Accreditation Program checklist a requirement that laboratories have a means of detecting bacterial contamination of platelets.

“The College’s Transfusion Medicine Committee believes we should be moving toward having good ways of reliably and simply detecting—or preventing—the presence of bacteria in platelets,” he says. The College might consider making recommendations once the FDA defines the systems that are acceptable.
Anne Paxton is a writer in Seattle.