Continuing Education Activity

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease that presents clinically with a variety of nonspecific symptoms. It causes complications mainly through intravascular hemolysis, thrombosis, and bone marrow failure. This activity reviews the evaluation and treatment of paroxysmal nocturnal hemoglobinuria and highlights the role of the interprofessional team in evaluating and treating patients with this condition.

Objectives:

• Identify the etiology of paroxysmal nocturnal hemoglobinuria.

• Describe the evaluation of paroxysmal nocturnal hemoglobinuria.

• Outline the management options available for paroxysmal nocturnal hemoglobinuria.

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease that presents clinically with a variety of symptoms, the most prevalent of which are hemolytic anemia, hemoglobinuria, and somatic symptoms including fatigue and shortness of breath. Other findings associated with PNH include thrombosis, renal insufficiency, and in the later course of the disease, even bone marrow failure. The condition is genetic, with the mutations occurring on the X linked gene.[1][2]

While the term paroxysmal nocturnal hemoglobinuria was introduced by Enneking in 1925, case reports dating back to the 1880s can be found. One of the earliest was that of Strubing, who documented the case of a young adult man with fatigue, abdominal pain, and intermittent hemoglobinuria. Strubing also noted that the patient's plasma was red following the most severe episodes, and he deduced that intravascular hemolysis was the cause. Later in 1937, Ham was able to discover that erythrocytes of individuals with PNH hemolyzed when incubated with normal acidified urine. This resulted in the first diagnostic test for PNH, known as the Ham test (acidified serum test). While complement activation was suspected as the etiology for hemolysis, the theory was not formally proven until 1954. Over the following years, the nature of protein deficiencies affecting PNH erythrocytes was identified, and this paved the way for the identification of the responsible genetic mutation.[1]

Although PNH is a rare condition, it has a significant impact on the quality of life of a patient. About 4 or 5 decades ago 10-year survival for this condition was only 50 percent. However, in the last 15 years, advances in treatment such as the development of eculizumab have improved survival to more than 75 percent.[2]

Etiology

Paroxysmal nocturnal hemoglobinuria occurs due to the development of a genetic mutation in hematopoietic stem cells. This mutation of the X-linked gene phosphatidylinositol glycan class A (PIGA), produces a deficiency in the glycosylphosphatidylinositol (GPI) protein, which is responsible for anchoring other protein moieties to the surface of erythrocytes.[3] 

Proteins responsible for the regulation of complement activity, specifically CD55 and CD59, are thereby prevented from attaching to PNH affected cells. The resultant loss of complement inhibition produces chronic complement-mediated hemolysis of PNH cells. This chronic state of hemolysis can be exacerbated if the complement system is activated by stress due to surgery, trauma, or other triggers for inflammation.[1]

Epidemiology

PNH is rare, with occurrence estimated as high as 15.9 individuals per million worldwide.[4] Some authors indicate that this number is probably low as the disease remains undiagnosed in individuals with limited symptomatology, or with comorbid conditions that obscure the PNH diagnosis.[5] Typically most patients fall in the age range of 30 years to 40 years. Children can be affected by PNH as well, but it is uncommon. For instance, according to an analysis of 1610 patients registered in the International PNH Registry in 2012, the median age of all registered patients was 42 years, with the disease duration of 4.6 years. The age range of patients in the registry was 3 to 99 years.[2]

Although the condition is due to an X-linked chromosome mutation, women are affected at a slightly higher rate than men. This is because the acquired defect occurs in somatic, or clone, hematopoietic stem cells, rather than germ cells. The phenotype can be created due to a single somatic mutation. Men only have one X chromosome, and women only express a single X chromosome due to lyonization, or inactivation of the duplicate X chromosome. Therefore, once the mutation occurs, the cell line perpetuates the abnormality until clonal superiority is achieved, and the phenotype is expressed. 

The PIGA mutation that is responsible for PNH can also occur in germline cells; however, this generally results in embryonic death. There has been a variant of the mutation, a hypomorphic PIGA mutation that is responsible for a syndrome known as multiple congenital anomalies-hypotonia-seizures syndromes. These children have severe congenital manifestations, including intellectual disability, abnormal facial features, seizures, and death at an early age. The difference in this expression of PIGA mutation is that it affects granulocytes rather than erythrocytes, and hemolysis does not occur.[6][7]

While the occurrence of PNH has no apparent ethnic or geographic distribution, there is an increased risk of thrombosis in the United States and Europe. For instance, about 30 to 40% of PNH cases are reported in the United States(US) and Europe, whereas less than 10% of PNH cases are reported from Asia. Consequently, the incidence of thromboembolism due to PNH is higher in the US and Europe compared to Japan.[8]

Patients affected by PNH in the US demonstrate differences in complications according to ethnic groups. African-Americans with PNH have a 73% rate of thromboembolism (TE) and Latin Americans, about 50%. White race and Asian Americans have a 36% rate of TE complications. They have also demonstrated bone marrow failure to vary with ethnicity or geography. It is more common in residents of Asia, the Pacific Islands, and Latin America. The reasons for these variations are not clear.[9]

Pathophysiology

In PNH, most symptoms that the patients experience results due to intravascular hemolysis. On lab work, anemia is a common finding and leads to nonspecific complaints such as fatigue and shortness of breath.[1] Some patients may also show inadequate bone marrow response, and conditions like aplastic anemia can be seen with PNH.[10] 

Under normal circumstances, free hemoglobin released by hemolysis is removed by haptoglobin and other clearing mechanisms. However, in PNH, these cleaning mechanism pathways are overburdened resulting in free hemoglobin in the intravascular system. The body tries to compensate by irreversibly binding nitric oxide to produce methemoglobin and nitrate, but this ends up depleting the nitric oxide supply.[11] 

Nitric oxide has multiple essential functions, including vasodilation and relaxation of smooth muscle. When nitric oxide is depleted, smooth muscle dystonia can occur. In PNH, dystonia of the gastrointestinal tract is most common. This is why, in PNH, many patients will present with complaints of dysphagia, esophageal spasm, and abdominal pain. Another consequence of depleted nitric oxide is the inability to vasodilate. For instance, nitric oxide plays a role in the vascular dilatation of the corpora cavernosa. As a result, many male patients present with erectile dysfunction.[12] 

Nitric oxide depletion may also play a role in producing shortness of breath by promoting pulmonary hypertension, though this is not a common occurrence. Echocardiographic studies have indicated that pulmonary artery pressures are elevated in some patients with PNH.[13] Right ventricular dysfunction, as demonstrated by elevated levels of N-terminal pro-brain natriuretic peptide, is also seen.[14] Acute and chronic renal disease can also result. Patients with PNH are six times more likely to develop chronic kidney disease than the unaffected population.[14] Free heme released during periods of hemolysis has a toxic effect on the kidney and can produce an acute kidney injury. [15] Another side effect of chronic hemolysis is iron deposition in the kidney that leads to scarring, cortical infarcts, and proximal tubule dysfunction. This is why hemosiderosis is often seen in patients with hemoglobinuria.[16]

About 40% of PNH patients experience thrombosis during the course of their disease. Thrombosis is the leading cause of significant morbidity and the most common cause of death in this condition. Venous thromboembolism is more common than arterial thrombosis. Cerebral and intraabdominal venous thrombosis is commonly seen. The hepatic vein is the most common site for thrombosis in PNH patients.[17] 

Multiple factors play a role in thrombosis. The GPI anchor protein defect created by a PIGA gene mutation enhances platelet activation and aggregation in addition to causing hemolysis of red cells. Nitric oxide depletion and complement activation also promote thrombosis by enhancing platelet aggregation and inflammatory cytokine release, respectively. Finally, the thrombophilia found in PNH is enhanced by defective fibrinolysis. Deficiency or absence of a GPI anchored protein responsible for plasminogen activation is suspected.[18][19]

History and Physical

Traditionally PNH was characterized by recurrent episodes of intravascular hemolysis, venous thrombosis, and cytopenias associated with bone marrow failure. However, after extensive research on PNH, we know that most patients present with nonspecific and variable signs that do not fit any specific syndrome. Most commonly, patients present with constitutional symptoms like fatigue, generalized malaise, dyspnea, dark urine due to marked hemoglobinuria, renal insufficiency from hemosiderin deposition leading to tubulointerstitial inflammation, dysphagia or esophageal spasms, abdominal pain, back pain and erectile dysfunction which all occur due to smooth muscle dystonia.[20] Because signs and symptoms are so variable, it is often difficult to diagnose this condition, and hence, diagnosis is often delayed.[21][22][23][24]

Evaluation

Diagnostic flow cytometry is considered the gold standard test for PNH diagnosis. It utilizes various monoclonal antibodies, and special reagent called fluorescent aerolysin reagent (FLAER) that binds directly to glycosylphosphatidylinositol (GPI) anchored protein, specifically their glycan portion. This test is capable of evaluating a variety of GPI-anchored proteins, most notably as CD55 and CD59, with high sensitivity and specificity.[22] 

Aerolysin is the principal virulence factor found in Aeromonas hydrophila. Its inert form is secreted as proaerolysin, which selectively binds to GPI anchor protein with high affinity.[1] This is why when FLAER assay is used, whether there is a deficiency of GPI anchoring protein in cell lineage or not can be determined. One caveat to this test is that at least two GPI linked proteins must be studied because this ensures that the test is not false negative due to rare congenital deficiencies of single antigens like CD55 or CD59 or polymorphisms with individual antigens such as seen in CD16 that will cause these antigens undetectable by some monoclonal antibody[25]. In flow cytometry, all GPI negative erythrocytes, monocytes, and granulocytes are identified. PNH red blood cells are further labeled into type 1,2, or 3, where type 1 cells have a normal expression of GPI anchor proteins, type 2 has a partial deficiency, and type 3 lacks all GPI anchor proteins[26]. 

There are two types of flow cytometry tests available: low sensitivity and high sensitivity. Although low sensitivity flow cytometry tests are adequate for the diagnosis of PNH, a high sensitivity test is better at picking up PNH with another bone marrow disorder. Based on the clinical picture and laboratory test results, PNH can be categorized into three types. 1. Classic PNH. 2. PNH with another bone marrow (BM) disorder 3. Subclinical PNH. In PNH with BM disorders, there is evidence of hemolysis as well as primary bone marrow disorder. The most common BM disorders that occur with PNH include aplastic anemia (AA), myelodysplastic syndrome (MDS), and primary myelofibrosis (PMF). In Subclinical PNH, there is no clinical or laboratory evidence of hemolysis. It is crucial to reassess patients at 6 to 12 months interval for the size of the PNH clone to keep an eye on disease evolution. Similarly, if there is a significant change in clinical condition or laboratory exams, patients should be reassessed.[21]

Even though these are not diagnostic tests per se, there is valuable information to be gained by ordering lab work such as complete blood count with differential (CBC w/ diff), basic metabolic panel (BMP), and urinalysis (UA). As mentioned above, recurrent episodes of intravascular hemolysis and cytopenias, and hemoglobinuria or hemosiderosis can be seen in PNH. Thus markedly increased LDH, low haptoglobin, and unconjugated bilirubinemia would be seen due to intravascular hemolysis. On a CBC w/ diff, reticulocyte count will be increased as a compensatory response. Evidence of anemia, leukopenia, and thrombocytopenia will be seen. Evidence of renal dysfunction with an increase in serum creatinine and blood urea nitrogen (BUN) as well as electrolyte abnormalities seen in chronic kidney disease (CKD) can be expected in a BMP. Urinalysis can show evidence of hemoglobinuria and hemosiderosis. Hence, clues from routine CBC w/ diff, BMP, and UA are a further indication for ordering a more specific diagnostic test for PNH, such as flow cytometry.[21][23][27][24]

Generally, when flow cytometry testing shows granulocyte clone of greater than 20%, patients should undergo a thorough assessment of hemolysis, silent thrombosis, and end-organ damage. In this case, the additional test such as D-dimer, brain natriuretic peptide, liver function panel, iron panel, bone marrow aspirate or biopsy, and cytogenetics are helpful. Bone marrow aspirate and biopsy aids in the setting of another bone marrow abnormality to further characterize PNH.[28]

Imaging also plays an important role. Echocardiography can help assess pulmonary hypertension. Doppler abdominal ultrasound can be helpful to assess hepatic blood flow or identify any thrombosis. If there is a concern for silent pulmonary embolism, then computerized tomography pulmonary angiography (CTPA) is useful. Computerized tomography (CT) of the abdomen can be helpful is the suspected cases of Budd Chiari syndrome. MRI of the head can be helpful if there is a concern for central nervous system thrombosis.[24][28]

Treatment / Management

In the past, PNH treatment was mostly supportive. Patients were given a blood transfusion and iron supplementation for recurrent hemolysis and anemia. They were given anti-thrombosis prophylaxis to prevent thrombosis. For bone marrow complications, an allogeneic bone marrow transplant was offered.[29][30][31]

In PNH, complement-mediated hemolysis and chronic dysregulation of the alternative complement pathway are the main culprits. Commonly there is a loss of anchoring proteins such as CD55 and CD59, which causes cells to hemolyze and lead to complications like thrombosis, which causes morbidity and mortality. Hence the mainstay of current therapy for PNH includes drugs to block alternative complement pathways such as eculizumab, ravulizumab, and allogeneic hematopoietic stem cell transplantation.[30][32][33][34]

Eculizumab is a lifesaving therapy that is associated with a greater than 50% reduction in transfusion requirements and a close to 70% reduction in risk of thrombotic events and significant adverse vascular complications.[35][36] Eculizumab works by causing inhibition of factor C5. All complement pathways cause the formation of membrane attack complex (MAC). MAC is formed by C3b, C5b, and other complement factors. Eculizumab prevents C5 to convert into C5a and C5b factors; thus, effectively inhibiting MAC formation and complement-mediated lysis. In some cases, the treatment is refractory and associated with increased risk of encapsulated bacterial infections such as meningococcal meningitis. Hence, patients need to be vaccinated against Neisseria in addition to receiving daily oral antibiotic prophylaxis before starting eculizumab.[37] 

In addition, about 11 to 27% of patients on eculizumab undergo fatigue and breakthrough hemolysis (BTH) on approved treatment doses due to insufficient complement inhibition.[38] High cost and short half-life requiring frequent dosing were the main issues with this drug causing reduced quality of life for patients.

In an attempt to overcome those limitations, new drug ravulizumab was developed. In December 2018, the US Food and Drug administration approved ravulizumab for the treatment of PNH.[31] Ravulizumab has 3 to 4 times longer half-life and requires dosing every eight weeks. It is shown to be more cost-effective compared to eculizumab. It has fewer breakthrough hemolysis episodes, and it is non-inferior to eculizumab in terms of efficacy and safety profile. However, since the drug is new, there are no long term data available. In the future, ravulizumab will likely replace eculizumab as the first-line treatment for PNH.[29][39][40][41] There are many other anti-C5 monoclonal antibodies therapies under investigation. Other novel therapy development projects are focusing on targets upstream in the complement pathway, such as C1 inhibitors, C3 inhibitors, and Factor D inhibition therapy.[42][43][44]

Another possible option is allogeneic hematopoietic stem cell transplantation (allo-HSCT). This is the only curative therapy for PNH. Limited data suggest that risks such as graft versus host disease are at acceptable levels for high-risk patients when risk versus benefit is considered for this treatment.[33]

Other interventions of PNH are based on the potential complications related to PNH. For instance, PNH related to acute kidney injury is best treated with continuous renal replacement therapy (CRRT).[30]

Differential Diagnosis

Differential diagnosis includes other hemolytic anemias, other causes of atypical thrombosis, and conditions that cause bone marrow failure. Differential diagnosis considerations should include conditions such as paroxysmal cold hemoglobinuria, autoimmune hemolytic anemia, primary BM disorders such as aplastic anemia, myelodysplastic syndrome, primary myelofibrosis, microangiopathic hemolytic anemia, disseminated intravascular coagulation, and other hereditary anemias.[45][46][47]

Prognosis

Before complement inhibitors such as eculizumab were developed, PNH patients usually survived for 10 to 22 years. The primary cause of death in these patients used to be thrombotic events.  After the development of eculizumab and ravulizumab, the survival of PNH patients is close to those without the disease.[30]

Complications

As mentioned above common complications of PNH include thrombosis including hepatic, cerebral, and abdominal and both venous and arterial thrombosis, acute or chronic renal disease, pulmonary hypertension, erectile dysfunction, and dysphagia.

Deterrence and Patient Education

The importance of medication compliance, laboratory tests or imaging work up compliance, and regular follow up at the appointments, especially primary care physician and hematology follow-up should be stressed. Time should be taken to clearly explain to the patient the risk versus benefits of the indicated treatment. The patient should be informed about the prognosis, and discussion about the code status and advance directives should take place at some point.

Enhancing Healthcare Team Outcomes

An interprofessional team that provides a holistic and integrated approach to care can help achieve the best possible outcomes. Based on the type of patient population involved in PNH, different specialist involvement can improve the outcome for the patient. If children are affected, the care should be coordinated by a hematologist and pediatrician. Similarly, if a pregnant patient is involved, then a primary care doctor, gynecologist, and hematologist should have excellent internal communication to provide optimal care. Also, surgery can precipitate PNH, and if a patient ever requires any elective or emergent surgery, the patient’s primary hematologist should be consulted and be involved in the care when the patient is hospitalized.

If a patient is receiving eculizumab treatment, then frequent dosing requirements and breakthrough hemolysis can cause poor quality of life. Patient and family education can help improve the quality of life of the patients. If the family is more supportive and understanding of what the patient is going through, and if the patient can rationalize the difficulties they face during treatment, it can reduce the mental stress on the patient. In a situation where a patient is unable to afford the treatment, then a social worker should be involved in the treatment to help with finding ways to reduce the cost burden.

The patient’s hematologist and primary care physician should aid the patient in a discussion about different options such as continuing with less effective and less costly symptomatic management or allogeneic bone marrow transplant finally, if the patient has multiple comorbidities and expresses a desire to focus on comfort care then palliative care should be involved. The palliative care team, patient, and the family should have a good discussion about the options of care going forward considering the mortality burden on a patient on a case by case basis.