College of American Pathologists
Printable Version

  Bringing wider meaning to a rare disease


CAP Today




January 2009
Feature Story

Karen Titus

The rare disease PNH is emerging from a long slumber. With a new monoclonal antibody treatment available, along with vast improvements in diagnostics, PNH (paroxysmal nocturnal hemoglobinuria) has become an invigorating topic for clinicians and laboratorians.

Granted, PNH remains an unwonted event. Clinical hematologists at small to medium-sized hospitals “may see only two or three patients throughout their entire career,” estimates Stephen J. Richards, PhD, consultant clinical scientist in the Haematological Malignancy Diagnostic Service, St. James’s University Hospital, Leeds, England. Hard numbers are hard to come by. There may be two to six cases per million in Western countries, says D. Robert Sutherland, BSc, MSc, associate professor, Department of Medicine, University of Toronto, and technical director, clinical flow cytometry laboratory, Toronto General Hospital. Reporting in an article he recently coauthored, Dr. Sutherland says in 2004 his institution screened 141 samples for PNH, detecting a mere three new cases.

A colleague in Ontario puts it bluntly. “There’s not that much to say about PNH,” says Ian Chin-Yee, MD. “It’s rare.” Dr. Chin-Yee is chief/chair of the Division of Hematology, Department of Medicine, and professor of medicine, University of Western Ontario, London, Canada.

It’s also been misunderstood, even down to its name. The “nocturnal” appellation sprung from the belief that hemolysis was triggered by acidosis during sleep; in fact, hemolysis occurs during the day. Nor is “paroxysmal” quite accurate, since the hemolysis does not necessarily occur in bursts.

Not surprising for such a rare disease, advances in diagnostics and treatment have, until recently, been sluggish. And, as Dr. Chin-Yee points out, dialogue has tended to dwindle quickly, like Midwesterners discussing their favorite local volcanoes.

For years—decades, really—physicians relied on two diagnostic warhorses for identifying PNH erythrocytes, the acid hemolysin test, or Ham’s test, and the sucrose hemolysis test. Neither one reliably evaluates small PNH clones; moreover, recent transfusions will confound results. They’re insensitive and cumbersome. Yet they’re still in use at some institutions, says Dr. Sutherland. “It’s a very conservative business, medicine,” he says.

In the early 1990s Wendell F. Rosse, MD, now professor emeritus at Duke University, led the way in developing monoclonal antibodies and flow cytometry methods for diagnosing PNH. With this, flow cytometry became the de facto gold standard for diagnosing PNH clones.

Ultimately, as Michael J. Borowitz, MD, PhD, puts it, “PNH relatively rapidly went from kind of an obscure disease to one that’s very important to recognize.” The disease now rouses a high level of clinical suspicion, says Dr. Borowitz, professor of pathology and oncology at Johns Hopkins Medical Institutions, whereas for many years physicians gave it little, if any, thought.

What has stirred PNH from its hibernation? First, there’s now excellent therapy for the disease. Eculizumab, also called Soliris (Alexion Pharmaceuticals), was approved by the FDA in March 2007 and is, in the words of Dr. Borowitz, extraordinarily effective in treating PNH.

The drug works by blocking the complement activation that causes hemolysis in PNH patients. Specifically, eculizumab is a humanized monoclonal antibody that binds to the C5 complement protein and stops it from being cleaved into its active form.

PNH is a hematopoietic stem cell disease, caused by a somatic mutation in a single gene, called PIG A (phosphatidylinositol glycan, class A), ­located on the X chromosome. The PIG-A protein plays a role in the initial synthesis of the GPI (glycosylphosphatidylinositol) anchor; patients with the defective gene have a partial or complete deficiency of all GPI linked proteins/glycoproteins in a clone of he­mato­poietic stem cells, Dr. Sutherland explains.

Physicians long thought there were two culprits responsible for the hemolysis that occurs with PNH: the complement regulatory proteins CD55 and CD59, expressed on red cells. Both are GPI-linked. However, says Dr. Sutherland, CD59 deficiency alone seems to play the major role and is more likely the source of the problem. “There are rare diseases that are not PNH that don’t express CD59, and they look like PNH. So it’s probably CD59 that’s responsible for controlling hemolysis of red cells by activated complement,” he says, explaining that the cleaved portion of C5 is what binds to the CD59 protein; CD59 neutralizes it. When CD59 is absent, the cleaved part of C5 causes cell lysis.

Hemolytic PNH is well-characterized, says Dr. Richards. The syndrome includes hemolytic anemia (usually intravascular hemolysis) and hemoglobinuria. Patients are often cytopenic as well, and many patients present with a significant risk of a major—and unusual—thrombotic event. These typically are venous in nature and can occur somewhat off the beaten path, such as the hepatic vein (causing Budd-Chiari syndrome) or the mesenteric veins (causing mesenteric ischemia). “Very unusual sights,” Dr. Richards says.

Physicians have also begun to recognize that although PNH itself is rare, the underlying biology of the disease appears to have broader clinical implications.

It turns out PIG-A mutations occur not only in PNH but also in aplastic anemia and myelodysplasia, or MDS (myelodysplastic syndromes). Says Dr. Borowitz, “You can see fundamentally the same mutation, but just in a smaller number of cells, in aplastic anemia for sure, and probably in some patients with myelodysplasia.”

Adds Dr. Sutherland: “Because we now have good tests for PNH, we find almost as many cases of aplastic anemia and MDS as we do bona fide PNH.”

Some 50 percent of aplastic anemia patients have an expansion of PNH clones, reports Dr. Sutherland; some of these reach the stage of clinical PNH. “It’s a bit of a gray area trying to differentiate between PNH and aplastic anemia,” he says, “depending on what you know of the history of the patient.” With MDS, he says, about 20 percent of patients have PNH clones. “You can also find PNH clones in normal people, but it’s very, very, very rare—and then not in the stem cells,” he says. With this population, one lineage in the blood system may be affected; with PNH, all lineages are affected (being a stem cell disease).

Current thinking about PNH also suggests that the PNH cell is the mark of an injured bone marrow, says Dr. Chin-Yee. Small clones of cells, missing the GPI-linked proteins, are present in patients who’ve had almost any marrow insult, he says, from aplastic anemia to radiation. “I don’t think the clone is causing it—I think there’s been an injury to the marrow, and these cells just happen to survive.”

In PNH, Dr. Chin-Yee continues, larger clones of GPI-linked negative cells are present. In addition to hemolysis, patients are subject to thrombosis with an unknown mechanism.

“It’s a complicated picture,” Dr. Chin-Yee says. “It’s a fascinating disease.” Thanks to recent improvements in flow cytometry, “We’re finding very small clones of these cells in patients with other bone marrow diseases who don’t have clinical PNH. I’m sure they’ve always been there—we just never had a sufficiently sensitive test to detect them.”

In the past, making the diagnosis of PNH was a tiresome exercise. Flow cytometry changed that about 15 years ago, improving matters greatly, if not completely. The diagnosis requires looking for the absence of the relevant antigens, rather than detecting their presence—a fundamental challenge that can louse up interpretations with both false-positives and false-negatives, says Dr. Richards.

“The real key to being able to do good diagnostic flow cytometry is understanding the biology of the disease,” says Dr. Richards.

In this unfolding story, the first basic principle is, it’s better to look at white cells rather than red cells alone.

The absence or partial expression of GPI-linked antigens in two lineages is diagnostic for PNH, Dr. Sutherland says. Red cells traditionally have been considered a good diagnostic target, for a couple reasons. Red cell lineages turn over extraordinarily quickly; moreover, the hemolysis angle led many to initially assume it was a red cell disease. Relying on red cells alone will mean plenty of false-negatives, however. “Red cells, because they’re sensitive to lysis, will, in patients with PNH, sometimes disappear,” says Dr. Borowitz. “If there’s brisk hemolysis, they may be gone completely.”

Laboratories thus look for expression of GPI-linked antigens on a second lineage: granulocytes, which also have rapid cell turnover.

The second principle: CD55 and CD59 may not be the last word in markers. The reasons for this are not entirely clear, says Dr. Borowitz. “But there’s a whole host of proteins that one could look at on granulocytes that are GPI-linked,” including CD16 and CD24.

In patients who are hemolyzing, adds Dr. Chin-Yee, it’s impossible to estimate clone size of PNH cells by looking at red cells. Knowing clone size is important, however, as some investi­gators have suggested larger clone size may increase the risk of thrombosis.

While classical flow cytometry methods are light years ahead of the Ham’s and sucrose hemolysis tests in terms of sensitivity and specificity, they too have their drawbacks. When Dr. Sutherland and his colleagues ran those 141 samples for PNH in 2004, they used a two-part quantitative immunocytometric approach, staining for CD55 and CD59 on red cells and neutrophils.

“I can say this—it’s not a fun test,” Dr. Sutherland says, noting that it requires seven tubes and two hours and 40 minutes to run one assay. “It was a burdensome, expensive, and inefficient way to screen for such a rare disease.” Anything else? “The data generated were extremely difficult to interpret,” he says.

Clearly there was room for improvement. It has now come, in the form of a test based on FLAER (fluorescent aerolysin).

The bacterium Aeromonas secretes a protein called proaerolysin, which is protolytically cleaved at the cell surface to which it is binding; proaerolysin generates aerolysin, which binds to the GPI part of the GPI-linked molecule on the cell’s surface. “That’s how this was worked out—it binds to normal cells, but not PNH cells,” Dr. Sutherland says. “The major difference is a lack of GPI-linked molecules on the cell surface, so fairly quickly it was shown that that’s what its binding site was.” Researchers modified aerolysin so that it would continue to bind to cells but not lyse them, then affixed a fluorescent label—hence FLAER. Researchers at Johns Hopkins, including Dr. Borowitz, published a paper on FLAER in 2000, demonstrating the technique’s success (Brodsky RA, et al. Am J Clin Pathol. 2000;114: 459–466).

Dr. Borowitz explains further: Aerolysin is a toxin and can bind to the GPI anchors in all cells. “In fact, that’s how the toxin works—it binds to cells and lyses them. So it’s targeting exactly the GPI anchor. It kind of recapitulates what happens in PNH itself—but without the complement and so forth. It just sort of punches holes in the membrane.”

FLAER “is a very nice reagent,” Dr. Borowitz continues. “You don’t have to worry about its expression on different types of cells, or worry about a particular GPI anchor—you can look at the whole family of GPI anchors.” It’s better than CD55 and CD59 on white cells, though it’s not necessarily better than a broad panel of markers. “But for a simple assay to look across the board at all white cell populations, [this] is a nice way of doing it.”

Dr. Sutherland and colleagues have developed a four-color assay based on the new reagent. FLAER is one color. In addition, the test uses CD33, an antibody to myeloid cells. It stains monocytes brightly, neutrophils weakly, and lymphocytes not at all. It also uses CD45, a common white cell antigen that does not stain red cells, platelets, or debris. “Flow cytometers measure all those things, especially debris,” says Dr. Sutherland. They added CD14, a GPI-linked molecule that also identifies monocytes.

Within a single tube, then, they can simultaneously analyze FLAER and CD14 on neutrophil and monocyte lineages. The FLAER reagent sees all GPI molecules, “and misses all of them if they’re not there in a PNH case,” Dr. Sutherland says. The CD14 addition creates “two ways of skinning the cat in the same tube, basically. And the CD33 helps us to identify different subsets of white blood cells, so we know which lineages we’re looking at.”

Dr. Sutherland and his colleagues published on this method in Clinical Cytometry (Sutherland DR, et al. 2007;72B:167–177). “We found that it’s an extremely good test,” Dr. Sutherland says. “It’s easy to use, it takes only 45 minutes to set up and analyze, and you cannot miss PNH clones.”

The new assay showed excellent agreement with the standard CD55 and CD59 method, they reported, with increased sensitivity, thanks to the higher signal-to-noise ratio. They detected as few as one percent PNH monocytes and neutrophils in aplastic anemia (which were undetectable using CD55 and CD59 on red blood cells).

They found abnormal FLAER staining of blast populations in acute leukemia as well. These cases contained neutrophils that stained normally with FLAER, and monocyte-like cells that turned out to be blasts. Acute myeloid leukemic blasts stain with CD33, somewhat resembling monocytes, Dr. Sutherland explains. These cells don’t express CD14 well, and are neither FLAER negative nor FLAER positive, but somewhere in between. CD45 reveals them to be blasts. “We’ve detected four or five acute leukemias with this test—in fact, more,” he says.

“There are a whole range of situations where we’ve been able to say it’s not PNH, but there may be other dysplastic changes or early signs of leukemia occurring,” he continues. “And it’s often turned out to be the case. That’s kind of an unexpected benefit of this particular cocktail.” It’s not the primary reason to use FLAER, he acknowledges. “But if you find three or four cases of acute leukemia that had previously not been detected, it’s a useful benefit.”

The FLAER assay also allows physicians to get a better estimate of the size of the PNH clone, Dr. Chin-Yee says, which may prove useful in the thrombotic arena.

Dr. Sutherland is writing another paper on FLAER, reporting on his lab’s workup of 500-plus samples over a three-year period, in which they found 61 samples (not all unique cases) containing PNH clones.

As an aside, he notes that he developed a simple CD59 red cell assay, which mimicked the red cell portion of the older PNH assay his lab had been using. Only about 55 percent of the 61 PNH clone-containing samples were CD59-negative with this test.

Based on what he’s heard from colleagues at other institutions that do some variant of the FLAER assay, many clinicians still insist on running a red cell assay on every sample. “They’re used to seeing red cells and white cells assessed. Then this new FLAER assay comes along, and they don’t immediately fully understand it. So they want a safety net.” Or the illusion of one, anyway. However, testing for PNH red cells doesn’t supply a safety net—indeed, as noted, the red cell assay is much less sensitive than the FLAER test, Dr. Sutherland says.

There’s no shortage of FLAER-based assays. Dr. Sutherland and his colleagues developed their particular version because most labs have the ability to do four-color assays. “In some respects it’s a bit minimalistic, if that’s a word.” (It’s not, though it pops up frequently in online forums.)

Dr. Borowitz’s lab recently switched from a four-color, FLAER-type assay to a six-color FLAER assay. The four-color test, which used reagents primarily targeted at ensuring users gated the right populations, had a sensitivity in the one to five percent range; the newer assay, he reports, has a sensitivity between .01 and .1 percent. Achieving this sensitivity, he says, requires at least two markers against GPI-linked proteins, looked at simultaneously. “Otherwise you don’t have a way of separating out a little bit of low-level noise from a specific signal.”

Six-color flow is becoming more popular, Dr. Borowitz says, and its challenges are not unique to PNH. He devotes much of his time to minimal residual disease analysis in leukemia, where the principles are the same—looking for a very small population in a big background. But sometimes it’s not as easy to separate the normal and the abnormal signals, whereas in PNH, it’s quite easy. In fact, he says, labs wanting to set up high-sensitivity flow analysis could do worse than to start with PNH.

Likewise, FLAER has a much bigger application than simply PNH. In addition to being useful in identifying patients with aplastic anemia, says Dr. Borowitz, there are data to suggest that among patients with aplastic anemia or diseases that are clinically similar to aplastic anemia, those with PNH clones are much more likely to respond to immunosuppressive therapy. “So there’s clearly a tremendous need to develop these assays and be able to identify these patients, because it has real clinical relevance.”

The rarity of PNH created a rich-poor gap of sorts among medical institutions—the majority of testing has been concentrated in a relatively small number of centers filled with resources and specialists. Dr. Richards, whose own institution is renowned for its PNH expertise, predicts more labs will be asked to do PNH testing as word of eculizumab’s success spreads (along with news of its cost, estimated to be about $389,000 a year).

Now that FLAER and eculizumab have brought PNH out of its torpor, researchers are tackling some more basic questions. Is the FLAER-based assay alone the best approach? Or should it be combined with another diagnostic method? How many markers should be used? “There’s really no standardized approach to this,” Dr. Borowitz says.

They also continue to wrestle with PNH doppelgangers.

Dr. Chin-Yee notes that with cases of acute leukemia, very immature cells can be missing GPI-linked proteins. “You need to make sure, when you look through the microscope, that they’re not blasts,” he says, adding, “I’d hate to think that someone would make that mistake.” He recalls one case at his institution that took his lab colleagues by surprise: a sample that had been sent down for a PNH assay, which revealed a huge FLAER-negative population. “They were all leukemic blasts.”

Aplastic anemia may be less rare than PNH, Dr. Borowitz says, but the diagnostic challenges may be greater since the clone sizes are much smaller. Patients with frank PNH have large clones, often over 50 percent, which are easy to detect. With aplastic anemia, one percent may be clones. With myelodysplasia, the number may be .01 percent to 0.1 percent. “So developing challenges and showing that people could achieve that level of sensitivity, reproducibly, is a difficult problem.”

“We need more people to use it, so we can get some proper quality control data,” agrees Dr. Richards. “There’s just a few people using it at the moment, so it makes it difficult to score its effectiveness against a reagent that every lab is using.”

At the most recent meeting of the Clinical Cytometry Society (in October, in Portland, Ore.), a half-day companion meeting devoted to PNH led to widespread agreement for the need to standardize testing. Dr. Borowitz sees this leading to the development of consensus recommendations; the first step will be to form a working group. He also plans to reach out to members of the International PNH Interest Group (which convenes during the American Society of Hematology annual meeting), made up primarily of clinicians but also some lab folks.

PNH’s time in the limelight is likely to be short. “PNH makes up a relatively minor business for most of us,” says Dr. Chin-Yee. But the FLAER-based assay should give physicians plenty of opportunities to take a larger look at PNH clones. “If it’s applied more frequently, you might start finding these clones are not that uncommon, and they may have some prognostic significance,” he suggests.

FLAER, in other words, doesn’t look like it will flare out anytime soon.

Karen Titus is CAP TODAY contributing editor and co-managing editor.