Paroxysmal nocturnal hemoglobinuria (PNH) is an opportunity to expound upon three major topics: hemolytic anemia, autoimmune mediated bone marrow failure, and thrombosis. For the physician explaining a new diagnosis to the patient, PNH is now a cause for cautious optimism, as we have seen a tremendous improvement in the outcome over the past decades. Indeed, we can now alleviate symptoms and prevent complications in almost all patients, and life expectancy of a newly diagnosed patient is now close to that of age-matched controls. The complement-mediated hemolysis in PNH has inspired the development of a new class of drugs that target complement proteins, and C5 blockade has been shown to not only ameliorate hemolysis but also reduce the risk of thrombosis in PNH. A novel C3 inhibitor was approved recently for PNH and may be particularly useful for those with persistent hemolytic anemia despite C5 inhibition. Anti-complement therapy has also revolutionized the treatment of another hematologic disorder (atypical hemolytic anemic syndrome, aHUS), as well as two neurologic disorders, with more drugs and additional indications likely to come.

In considering the earliest descriptions of patients who might have had PNH, we must keep in mind that most patients throughout history with dark urine, for sure, have not had this rare disorder. However, William Gull reported on a patient in 1866 with an intermittent bloody-appearing urine that did not contain red cells, with the color of the morning samples more pronounced than later in the day—possibly the first reported case. Paul Strübing in 1882 described a young man with urine that appeared black or dark red (Fig. 1A), with associated fatigue and abdominal pain. He made the critical observation that the attacks were not precipitated by expo sure to cold, differentiating this from paroxysmal cold hemoglobinuria (PCH), which, due to its association with untreated syphilis, was then much more common. Strübing deduced that the hemolysis was intravascular, as the patient’s plasma turned red after severe attacks (see Fig. 1B). Van den Bergh later characterized the disorder in a patient, having ruled out PCH by the Donath Landsteiner test, and demonstrated that red cells under conditions of acidification with CO2 were lysed by autologous and ABO blood type-matched donor serum. Unlike antibody-mediated hemolysis, hemolysis in vitro could be abrogated by heat treatment of the serum, which was known to inactivate complement. Soon after, it was determined that urine hemosiderin staining could be seen between attacks, and we now know that in spite of the paroxysms that give the disease its name, hemolysis is, in fact, an ongoing process. The disorder was briefly named for the authors Marchiafava and Micheli who recognized this point and was eventually termed paroxysmal nocturnal hemoglobinuria by Enneking in 1928.

Fig1. (A) Two urine samples from a patient with PNH with a large PNH clone, taken several days apart, prior to treatment. The urine color then normalized soon after starting ravulizumab. Note that since the darkening of the urine is “paroxysmal,” the normalization of the urine color is not sufficient to demonstrate an effect of any therapy– but rather the response is most rapidly demonstrated by the decrease in the lactate dehydrogenase (LDH). (B) Blood samples in a serum separator tube from a patient with PNH compared with the normal donor. Note the red color of the serum above the separator plug and the decreased size of the clotted red cells below the plug, consistent with anemia and intravascular hemolysis. (C) Symptomatic bilirubin gallstones from a cholecystectomy specimen in a patient with PNH on long-term treatment with a C5 inhibitor. Stone formation is a consequence of chronic extravascular hemolysis of various etiologies and is seen in some patients on eculizumab or ravulizumab. This complication was not seen historically in untreated patients with PNH in whom the hemolysis is largely intravascular. The reason for this is that in intravascular hemolysis, free hemoglobin is largely excreted in the urine and is not broken down to bilirubin, whereas for extravascular hemolysis, hemoglobin is broken down in the cells of the reticuloendothelial system, resulting in hyperbilirubinemia. Likewise, patients with PNH who are treated with C5 inhibition can develop iron overload, as seen in other forms of extravascular hemolysis, but this is not typically seen in intravascular hemolysis where iron is lost in the urine.

A major breakthrough occurred in the late 1930s with the devel opment of the Ham test, which provides an estimate of the percentage of red cells that are susceptible to lysis by activation of the alternative pathway, after the acidification of serum in conditions of optimized magnesium concentration. Of note, there are always some red cells that will not lyse in the Ham test even upon repeat exposure to acidified serum, and the severity of the condition roughly correlated with the percentage of cells that were susceptible. Rosse and Dacie in the 1960s delineated these two populations and showed that the abnormal cells were exquisitely sensitive to low levels of human complement in an assay that activated the classical pathway. In 1970, Oni, Osunkoya, and Luzzatto made the critical observation, using G6PD allozymes to infer X-inactivation patterns, that the abnormal red cells in PNH belong to a clone derived from a single cell.

By that time, it had already been recognized that there was a cellular defect in acetylcholinesterase in PNH erythrocytes and alkaline phosphatase in neutrophils, but the lack of these particular enzymes on a clonal population did not yet explain complement sensitivity. That came with the discovery of a regulator of early complement proteins, CD55 (decay accelerating factor)—and the observation by Anne Nicholson-Weller and her colleagues that this protein is missing on PNH red cells and leucocytes. It was soon appreciated that there is a second regulator, membrane inhibitor of reactive lysis (MIRL, CD59), which inhibits the late complement proteins that can form a membrane attack complex, and it was found that like CD55, CD59 is also missing on blood cells in PNH (Fig. 2).

Fig2. FLOW CYTOMETRY ANALYSES. After staining of red cells with a mouse anti-human antibody specific for CD59 conjugated to fluorescein isothiocyanate (FITC), cells from the normal donor (A) pick up high levels of the antibody and fluoresce brightly in a unimodal distribution, as seen on this histogram plot. In contrast, in a patient with PNH (B), abnormal red cells that do not express CD59 co-exist with a normal cell population, producing a bimodal distribution. Some patients with PNH will have a trimodal population (C) with a population of red cells with intermediate levels of expression (the middle peak), known as PNH II cell populations. The PNH abnormality can be readily demonstrated in granulocytes as well. Here, in this illustration, cells are stained with a phycoerythrin (PE) conjugated mouse antibody specific for human CD24 (a GPI-linked antigen) giving red fluorescence and are simultaneously stained with FLAER (fluorescent aerolysin), which binds to cells in a GPI-dependent manner, giving green fluorescence. Normal granulocytes (D) fluoresce brightly in the red and green channels simultaneously and are illustrated as a single population on this pseudocolor density plot. Patients with PNH (E) have a normal population of granulocytes as well as a population that binds to neither FLAER nor to the anti-CD24 antibody, registering in the lower left quadrant. RBC, red blood cell. (Illustration after Borowitz MJ, Craig FE, Digiuseppe JA, et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010;78:211–230.)

The missing membrane proteins (Table 1) have in common the glycosylphosphatidylinositol (GPI) anchor, which is covalently bound to the carboxy terminus of certain proteins and tethers them to the membrane. Taroh Kinoshita’s group and colleagues in Osaka first showed in the early 1990s that PNH cells are defective in the first step in the biosynthesis of GPI32 and then went on to isolate the gene responsible for this step by complementation cloning in cell lines. They named this gene PIGA (phosphatidylinositol glycan A), and demonstrated that there is an acquired somatic mutation in this gene in all patients with PNH they studied. While there are over 20 genes necessary for the production and trafficking of GPI (Table 2), it was understood that PIGA was the only gene found to be mutated in PNH because it maps to the X-chromosome, whereas all the other genes in the pathway are autosomal. Thus, the clonal GPI-negative population would be much more likely to be produced by a single event on the X-chromosome, rather than two separate events involving any of the other genes in the pathway. Identifying the membrane defect in PNH led to a simple flow cytometry test per formed on peripheral blood (see Fig. 2), which by the 1990s was replacing the Ham test. The identification of abnormal leucocyte populations with the same surface defect as red cells and harboring the PIGA mutation confirmed that PNH is indeed not just a red cell disease, but actually is a stem cell disorder.

Table1. Glycosylphosphatidylinositol-Anchored Surface Proteins Absent on Paroxysmal Nocturnal Hemoglobinuria Blood Cells

Table2. Enzymatic Steps in the Synthesis of Glycosylphosphatidylinositol

The link between the mutation, GPI-anchor synthesis defect, and loss of complement inhibition now closed the circle on the most prominent clinical phenotype—hemolytic anemia. But this failed to explain the paradox of why this stem cell clone—whose mature red cell progeny, at least, seem to be weakened—would have a growth advantage. To explain this, in 1989 Rotoli and Luzzatto considered the critical earlier observation of Dacie and Lewis that PNH, a rare disease, is highly associated with another rare and seemingly very different hematologic disorder—immune-mediated aplastic anemia (Fig. 3). This had been commented upon by Dameshek in 1967 and then revisited by Young in 1992. The working hypothesis for the past 32 years has thus been the “immune escape model,” whereby the PNH stem cell has a selective growth advantage in an aplastic marrow environment, because it is less sensitive to immune attack than normal stem cells. This model has not been proven, but it explains many key features of PNH and there is experimental evidence supporting it.

Fig3. THE MARROW CAN HAVE A WIDELY VARIABLE APPEARANCE IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA. (A) Hypercellularity with erythroid hyperplasia (Wright stain, aspirate), as might be seen in any hemolytic anemia; (B) H&E-stained section showing overall nor mocellularity; (C) marked hypocellularity, as would be seen in aplastic anemia. In this case there is a patchy distribution to the marrow with some cellular areas with increased erythropoiesis interspersed with acellular areas. The immune escape hypothesis proposes that it is the aplastic marrow environment that favors the PIGA mutant clones.

The other major piece of the puzzle has been to understand the pro-thrombotic tendency in PNH, noted 70 years ago by Crosby to be a frequent cause of death, often involving unusual sites such as the cerebral dural venous sinuses and intra-abdominal veins (Fig. 4). While there are a few proposed mechanisms for this, the group in the United Kingdom made two major breakthroughs. First, using historical controls, they demonstrated that thrombotic complications in PNH could be largely prevented by primary prophylaxis with warfarin. Then, as reported by Hillmen et al., they pioneered in 11 patients the use of eculizumab, a monoclonal antibody targeting the C5 complement protein. This led to the first randomized trial in PNH (the TRIUMPH Study), and the drug’s approval in 2007 as a treatment for hemolysis in PNH, for which it can be highly effective. Most importantly, it was subsequently shown that eculizumab reduces thrombotic risk by more than an order of magnitude—from 14 per 100 observation-years in untreated patients to approximately 1 per 100 observation-years in treated patients. Probably as a result of this effect, the UK group has now demonstrated a near normal life-expectancy in treated patients.

Fig4. (A) Hepatic MRI images indicative of portal vein thrombosis in a patient with paroxysmal nocturnal hemoglobinuria. (B) Diminutive appearance of the anterior sagittal sinus seen on CT angiogram (left) and MR venogram (right).

Eculizumab has now been engineered in a clever manner with a set of substitutions that increase its half-life about fourfold. However, unfortunately, some patients respond only partially to C5 inhibition. In 2009, Risitano and colleagues made the critical observation that despite C5 blockade, C3 degradation products can still opsonize PNH red cells (due to their lack of CD55), resulting in their uptake in the reticuloendothelial system—a milder form of extravascular hemolysis that had previously been masked by the severe intravascular hemolysis seen in untreated patients (see Fig. 1). These partial responders represented an unmet clinical need, which has led to the development of the first-ever inhibitor of the C3 complement protein—pegcetacoplan. This drug was designed to prevent both forms of hemolysis and has recently been approved by the Food and Drug Administration (FDA) for the treatment of PNH, after it compared favorably with eculizumab in a randomized trial with an endpoint of hemolytic anemia.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم صناعة الشبابيك يباشر أعمال إدامة شباك مقام الإمام المهدي (عجّل الله فرجه)

المجمَع العلمي ينظم ندوته القرآنية الأسبوعية في جامعة الكوفة

جامعة العميد تقيم الملتقى العلمي الثاني لخريجي كلية طب الأسنان

ضمن دورتها العلمية (في رحاب العقيدة) الهيأة العليا لإحياء التراث تقدم محاضرة عن مفهوم الأسبقية في الإسلام

المزيد

الآخبار الصحية

اللحظات الأخيرة قبل الموت.. هذا هو تسلسل "فقدان الحواس"

وخز الأطراف وضعف الذاكرة.. علامات على نقص فيتامين هام

عادة صباحية بسيطة.. تأثيرها قد يحسن ضغط الدم

عادات يومية تدمر قلبك بصمت.. خبراء يطلقون التحذير

المزيد

الآخبار التكنلوجية

الذكاء الاصطناعي يفك لغز آثار أقدام الديناصورات

القمر أولا.."سبيس إكس" تؤجل خططها الخاصة بالرحلات إلى المريخ

الذكور أم الإناث؟ دراسة "تنسف الشائع" بشأن التوحد

خبير يكشف عن اقتراب العلماء من فك شفرات "لغات الحيوانات" !

المزيد

مواضيع ذات صلة

Diseases of The Central Nervous System

Diseases of The Oral Cavity

Diseases of The Upper Respiratory Tract

Hyperthyroidism: Etiology and Treatment

Clinical Features of Hyper- and Hypothyroidism

Clinical Associations of Aplastic Anemia

Typical and Atypical Presentations of Aplastic Anemia

Pathophysiologic Pathways Leading to Aplastic anemia

Epidemiology of Aplastic anemia

Pneumonia

Bronchitis

Pathogenesis of the respiratory tract: Microorganism Factors