Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell clonal disease caused by mutations in the phosphotidyl inositol glycan complementation group A (PIG-A) gene in somatic cells. The disease is an acquired clonal disorder of hematopoietic stem cells. The occurrence of intravascular hemolysis in patients shows a temporal correlation with sleep, hence the name. I. Etiology and pathogenesis PNH is a disease in which the synthesis of glycosyl phosphatidylinositol (GPI) I is blocked by mutations in the PIG-A gene on chromosome X in one or more hematopoietic stem cells. This results in the loss of a group of membrane proteins anchored by GPI on the cell membrane. GPI anchor-linked proteins include: (i) complement regulatory proteins, such as decay accelerator (CD55), membrane attack complex inhibitor (CD59), complement C8 binding protein and membrane cofactor protein (MCP); (ii) adhesion molecules, such as CD58, CD48, CD67, CD67; (iii) enzymes, such as CD73; (iv) receptor classes, such as CD16, CD14; ⑤ blood group antigens. The variety of GPI-attached antigens also contributes to the complexity of interpreting the biological behavior of PNH cells, but two GPI anchor proteins, CD55 and CD59, have always been closely followed in the pathogenesis, clinical manifestations, diagnosis and treatment of PNH because of their important role in complement regulation. CD55 and CD59 are CD55 is a C3 convertase decay accelerator on the cell membrane, which regulates the early complement cascade by modulating C3 and C5 complement protein convertases. CD59 is also known as a reactive membrane attack complex inhibitor, which prevents the admixture of complement C9 into the C5b-8 complex, and prevents the formation of membrane attack units to inhibit the complement terminal attack response. The complete or partial absence of CD55 and CD59 in the cell membrane prevents the activation of the complement system from being effectively inhibited, triggering erythrocyte damage by complement, intravascular hemolysis, release of free hemoglobin, thrombosis and impairment of organ function. Dacie’s so-called dual pathogenesis theory (DPT) of PNH pathogenesis is a generally accepted hypothesis: firstly, PNH clonal expansion is associated with immune abnormalities and hematopoietic failure. First, hematopoietic stem cells mutate under certain conditions to produce GPI-deficient PNH clones; second, due to some factor (nowadays mostly considered as immune factor), hematopoietic damage or hematopoietic failure occurs and PNH clones gain proliferative advantage. Most PNH combined with impaired hematopoiesis can also be preceded by aplastic anemia, which gradually reveals PNH clones. Small PNH clones can also be seen in lower risk myelodysplastic syndromes. The organ damage and clinical symptoms caused by PNH are associated with intravascular hemolysis, increased free hemoglobin, continuous activation of the complement system, nitric oxide depletion, and dysfunction of the coagulation system, among others, resulting in malaise, shortness of breath, abdominal pain, and hemoglobinuria, with a high risk of complicating thrombosis, renal failure, and pulmonary hypertension. Thrombosis in PNH patients is the result of a combination of multiple factors. Complement activation and hemolysis in PNH patients release some substances, such as complement C5a, which can put the body in a potentially inflammatory state. Inflammation can cause monocytes, vascular endothelial cells to overexpress and release tissue factors, and initiate the coagulation process. Inflammatory mediators can destroy endothelial cells, endothelial cell activation increases, and a large amount of tissue factor is released into the blood to initiate the exogenous coagulation pathway. CD59 deficient platelets are more susceptible to activation or NO depletion, which increases platelet activation and aggregation and adhesion to form thrombi. Intravascular hemolysis causes the accumulation of free hemoglobin and increases blood viscosity, and may also affect platelet function due to the deposition of hemoglobin on platelets, keeping the blood in a hypercoagulable or pre-thrombotic state. Intravascular hemolysis releases free hemoglobin into the blood, and the binding capacity of free hemoglobin to NO is a hundred times higher than that of oxygen, making the NO content in the blood decrease; or hemolysis makes the endothelial function of blood vessels disrupted, and the endothelial synthesis of an important class of vasodilatory substances is NO, and the endothelial function disorder reduces the chemical synthesis of NO. This leads to spasm of small vessel smooth muscle in PNH, which causes pulmonary hypertension, abdominal pain, dysphagia, and erectile dysfunction in men. Chronic kidney disease is also the result of a combination of factors, repeated hemolysis, hemoglobin reabsorption in the renal proximal tubule and deposition in the renal tubular epithelium after decomposition into iron-containing heme in the proximal tubular epithelium to damage the renal tubules; NO depletion, decreased bioavailability of the kidney vasoconstriction, increased resistance, reduced renal blood flow, renal ischemia and hypoxia; thrombus formation in the kidney will affect the renal function causing acute and chronic kidney injury. After sleep, the accumulation of acidic metabolites in the body leads to an acidic body fluid environment, and the acidic pH 6.8-7.0 environment is most suitable for the action of complement, which facilitates the destruction of red blood cells by complement, as well as the concentration of urine, so patients often find that the urine color is thick tea or soy sauce color after sleep, and then gradually reduced. II. Clinical manifestations According to the clinical manifestations of patients and the scale of PNH clone, it can be divided into classic type (hemolytic type, thrombotic type), bone marrow failure type (combined with aplastic anemia, or myelodysplastic syndrome, PNH clone <10%), and subclinical type (PNH clone <1%). (A) Hemoglobinuria Paroxysmal hemoglobinuria is a typical symptom of the disease, and about 1/4 patients have the first episode of hemoglobinuria. In typical PNH patients, due to intravascular hemolysis, free hemoglobin is excreted through the urine in a soy sauce color or thick tea color. Episodes of hemoglobinuria are often associated with sleep. They usually last 2-3 days in mild cases and 1-2 weeks or even longer in severe cases, accompanied by weakness, fatigue, chest tightness, shortness of breath, dysphagia, abdominal pain, etc. The abdominal pain is mostly spasmodic, and about 50% of male patients have erectile dysfunction. Those with mild chronic intravascular hemolysis, which is episodic or non-episodic, often have only positive urine occult blood and iron-containing hemoglobin tests. Hemolysis can be induced by fatigue, colds, infections, menstruation, surgery, and the use of iron, aspirin, ammonium chloride, luminal, and other conditions that activate the complement system and make the body's environment acidic. (ii) Hematopoietic failure Many patients have hematopoietic decreases, or gradually develop into hematopoietic decreases. PNH cells expand into the dominant clone after abnormal immune damage to the normal clone, and the normal clone is suppressed to cause hematopoietic failure, which is manifested as PNH-anaplastic anemia syndrome, or some patients have aplastic anemia first, and then appear as PNH. Hemolysis leads to different degrees of anemia. Long-term chronic intravascular hemolysis with iron loss via urine leads to iron deficiency anemia. Neutropenia and dysfunctional susceptibility to infection, and thrombocytopenia leading to bleeding tendency. (iii) Thrombosis PNH is a high-risk group for thrombosis, which used to be the primary factor of death in the West.PNH thrombosis can appear at any site, but is mostly seen in the abdomen and brain. Deep vein thrombosis is the most common type of thrombosis, and multisite thrombosis occurs in about 20% of patients with PNH. In PNH, atypical sites include thrombosis of hepatic veins (Budd-Chiari syndrome), mesenteric veins, cerebral veins, and venous sinuses, with a higher incidence than in the general population. clinical manifestations of thrombosis in PNH are diverse, such as stasis of tissues and organs, hypoxia, pulmonary hypertension, dyspnea, and Budd-Chiari syndrome. The proportion of arterial thrombosis is also not low, often occurring in cerebral arteries and coronary arteries, with cerebral ischemia and acute myocardial infarction as the main clinical manifestations. Thrombotic events may occur in 40% of PNH patients, and approximately 40% to 67% of these patients die from thrombotic events; thrombosis in PNH patients often has a poor prognosis, and a single thrombotic event will increase the mortality of PNH by 5 to 10 times. The incidence of thrombosis in Asian populations was previously thought to be low, but studies have found that the median time to thrombosis is more than 2 years after the diagnosis of PNH, and the short follow-up period may not be observed enough. The current data from Asia shows that the incidence of thrombosis is found to be similar in the East and West. (iv) Organ damage There are many organs that are functionally impaired in PNH due to the continuous activation of the complement system, intravascular hemolysis, and the long-term interaction of the coagulation system. Impaired hepatic function can be seen with increased glutamate transaminase, increased indirect bilirubin as the predominant bilirubin, long-term hemolysis leading to stone formation in the hepatobiliary system, and cholecystitis. Chronic renal impairment is seen with renal tubular epithelial cell detachment with urinary excretion to form iron-containing hemoglobinuria. Thrombosis in the kidney triggers lateral lumbar and rib pain or abdominal pain, and imaging may show enlarged diseased kidneys. If bilateral renal vein trunk thrombosis can lead to acute renal failure, manifested by oliguria, anuria, and progressive increase in blood creatinine and urea nitrogen. Abnormal renal tubular function can also lead to nephrogenic glycosuria, proteinuria, and glomerulosclerosis with severe hypertension associated with hemolysis. A series of metabolites from hemolysis cannot be excreted from the body in time, forming a vicious cycle that eventually progresses to renal failure. Dyspnea and shortness of breath are often thought to be due to anemia, but may be triggered by pulmonary hypertension in some patients. Cardiac color Doppler and brain natriuretic peptide precursors often indicate pulmonary hypertension and right heart insufficiency. Laboratory tests (a) Blood picture Orthocytic anemia is common, and microcytic hypochromic anemia is also seen in iron deficiency. Reticulocytes are mostly increased, but they are decreased by the combination of reocclusion. Granulocytes and platelets are also often reduced. Nucleated erythrocytes, polychromatic erythrocytes and erythrocyte fragments can be seen on peripheral blood smears during hemolytic episodes. (b) Bone marrow picture Bone marrow proliferation is obviously active or active, and the red lineage is mostly increased. In the case of remittent disease, the proliferation is reduced. In case of iron deficiency, the bone marrow iron staining shows reduced or even negative iron inside and outside the bone marrow. (C) intravascular hemolysis See section 1 of this chapter for details. (iv) Diagnostic tests 1. Flow cytometry test The aeromonas lysin precursor variant (FLARE) distinguishes PNH cells (GPI-) from normal cells (GPI+) by binding specifically to the GPI protein on the cell membrane. Diagnosis of PNH by flow cytometry combined with FLARE is both sensitive and specific, and the size of PNH clones can be accurately determined by analysis of granulocytes and monocytes, independent of hemolysis and transfusion. Detection of CD59-erythrocytes by flow cytometry distinguishes between populations of erythrocytes with different degrees of complement sensitivity depending on the degree of CD59 deficiency: type I, normal; type II, partially deficient; and type III, completely deficient. The current flow cytometry technique is the gold standard for the diagnosis of PNH. The expression of some complement regulatory proteins such as CD55 and CD59 can also be affected by other factors, such as cellular dysplasia, inflammatory response and abnormal globulins in the blood that may lead to the absence or obscuration of membrane proteins, resulting in a negative test. It is recommended to screen for PNH in hemolytic anemia, hemoglobinuria, aplastic anemia, refractory anemia, unexplained thrombosis or rare site thrombosis, coexistence of thrombosis and hemolysis, unexplained hematocrit especially in young patients with negative Coombs test or with iron deficiency. 2, serological test The main detection method in the early years, acid hemolysis test (Ham test) used to be the classic confirmatory test, with good specificity but insufficient sensitivity. Sugar water test is highly positive, but poor specificity, often used as a screening test. There are also the snake venom factor hemolysis test and the heat hemolysis test. The diagnosis and differential diagnosis of classic PNH, often with clinical and laboratory changes of intravascular hemolysis, can be diagnosed by combining the Ham test, snake venom factor hemolysis test or urine ferric hemoglobin test with any two positive tests in the past. The presence of PNH clones can be confirmed by the detection of GPI-granulocytes or monocytes by flow cytometry, and the combined hematopoietic failure or subclinical type of PNH can be detected, and each type of PNH cells can be distinguished by the degree of CD59 deficiency in erythrocytes. PNH needs to be differentiated from aplastic anemia, myelodysplastic syndrome and other hemolytic anemias. V. Treatment (a) Supportive therapy In severe anemia, transfusion of concentrated red blood cells is required. It was thought that PNH should be transfused with washed red blood cells to avoid bringing in complement in the plasma. But in fact, the plasma volume in concentrated red blood cells is about 30%, while the blood volume of normal adults accounts for about 7%-8% of the whole body weight, so a few tens of milliliters of plasma input will be rapidly diluted by the organism and will not trigger hemolysis in PNH. On the other hand, the washed red blood cells are washed by saline, although almost all the plasma proteins are removed, there is also a certain loss (about 20%) and damage to the red blood cells, and because the original confinement system is destroyed during the washing process, the red blood cells should be stored at 4℃~6℃, and must be transfused within 24 hours. Therefore, PNH infusion of washed red blood cells is no longer recommended. Iron can cause the generation of reactive oxygen species, some oxygen radicals and intermediate products released, PNH cells are sensitive to oxidative damage and easily induce hemoglobinuria. For sure PNH combined with iron deficiency, treatment should start with a small dose of 1/3 to 1/10 of the conventional dose, and should be discontinued for those who have a reaction. In addition to raising hemoglobin and maintaining tissue oxygen demand, blood transfusion can also inhibit PNH erythropoiesis and indirectly reduce complement-sensitive erythrocytes. Therefore, PNH combined with severe iron deficiency anemia can be directly transfused with red blood cells. Androgens have a stimulating effect on red lineage hematopoiesis and have an ameliorating effect on anemia in some patients. Patients with combined immune abnormalities can use immunosuppressants, such as cyclosporine, as appropriate. Severe aplastic anemia combined with small PNH clone can also be treated with anti-human thymocyte immunoglobulin. (ii) Hemolytic attack control Firstly, avoid factors that induce hemolysis, such as cold, diarrhea, certain drugs, etc. Glucocorticosteroids can reduce or alleviate hemoglobinuric episodes, starting with prednisone 0.5-1mg/kg/d, halving the amount after the episodes stop, and then gradually continue to reduce the amount until the minimum amount, or maintenance amount. Oral or intravenous sodium bicarbonate alkalinization of blood and urine can assist in controlling hemolysis and reducing the burden and damage to liver and kidney organs. Antioxidant drugs were previously thought to have a protective effect on cell membranes, such as vitamin E, sodium ferulate p selenite, but the efficacy is not exact. (iii) Thrombosis Those with thrombosis should be treated with thrombolysis and thrombectomy. Considering that PNH is often combined with thrombocytopenia, there is a trade-off between anticoagulation, thrombolysis and bleeding after thrombosis. In the acute phase of thrombosis, heparin or low-molecular-weight heparin is considered first, followed by antagonists of vitamin K-dependent coagulation factors. Early heparin therapy is associated with increased hemolysis due to low concentrations of heparin, which activates the complement replacement pathway, but as concentrations increase, high concentrations of heparin inhibit complement activation, with the likely inhibitory link being C5b-9. Therefore, low molecular weight heparin is more appropriate. Warfarin reduces the risk of PNH thrombosis and is recommended to be considered for prophylactic use in PNH without contraindications to warfarin when the neutrophil PNH clone exceeds 50% and platelets are greater than 100,000/dL. However, even with prophylaxis with warfarin, thrombosis can still occur. Antiplatelet agents such as aspirin and clopidogrel are not effective in reducing the risk of thrombosis, and are not recommended for PNH with thrombocytopenia as they tend to increase the risk of bleeding. Eculizumab is effective in reducing thrombosis. (iv) Anti-complement C5 monoclonal antibody Complement C5 is the last enzymatic substrate in the complement cascade reaction, and C5 is cleaved into C5a and C5b by C5 convertase, and C5b is involved in the formation of membrane attack complex (MAC) C5b-9. Eculizumab is a recombinant human monoclonal antibody that specifically binds to human terminal complement protein C5 and blocks the release of inflammatory factor C5a and the formation of membrane attack complex C5b-9 by inhibiting the cleavage of complement C5 to C5a and C5b. Eculizumab treatment returns serum lactate dehydrogenase to normal or near-normal values, effectively controlling hemolysis and reducing the need for blood transfusions. Eculizumab inhibits well the activation of the complement system in PNH, controls hemolysis and thrombosis, and leads to a decrease in the incidence of thrombosis, with most patients no longer experiencing thrombosis. Eulizumab inhibits hemolysis, reduces NO consumption, reduces thrombotic events, regulates blood pressure, improves renal and pulmonary blood flow, and is effective in reducing the incidence and extent of renal damage and pulmonary hypertension. In patients with renal insufficiency, Eculizumab treatment can be supplemented with diuretics and correction of electrolyte disturbances, and hemodialysis can be considered for severe renal impairment. Follow-up data showed that the survival time of PNH treated with eculizumab in a standardized manner was comparable to that of the normal population and could improve the natural course of PNH. There is a risk of meningococcal infection after eculizumab treatment, and vaccination is advisable prior to treatment. Penicillin should be given to prevent the occurrence of meningitis in those who do not have time to administer the vaccine in emergency situations. (v) Chemotherapy PNH is a clonal disease, so combination chemotherapy has also been explored for refractory PNH. regimens are erythromycin or hypertrigonelline combined with cytarabine in low dose chemotherapy. Most patients see increased hemoglobin levels, decreased transfusions and clonal suppression of PNH. However, this method is highly myelosuppressive, has a long recovery period, and requires good isolation protection and supportive therapy. (vi) Allogeneic hematopoietic stem cell transplantation PNH is a clonal disease, so only allogeneic hematopoietic stem cell transplantation can cure PNH, but PNH has a benign clinical course and the risk of transplantation needs to be considered. In foreign countries, with the widespread use of eculizumab, PNH has achieved survival comparable to that of the normal population, and with the higher mortality associated with transplantation, the need for allogeneic HSCT for PNH in the eculizumab era has been questioned. allogeneic HSCT for PNH is usually delayed until disease progression, life-threatening complications such as complications of severe Allogeneic HSCT for PNH is usually delayed until disease progression, life-threatening complications such as complicated by severe hematopoietic failure, or recurrent uncontrolled hemolysis. Prognosis The survival period for PNH is long, with a median time of 10 to 15 years, and the main causes of death are thrombosis, infection and bleeding. Many patients have combined aplastic anemia. Transformation to myelodysplastic syndrome and acute leukemia is rare and the prognosis is poor.