Paroxysmal nocturnal hemoglobinuria

Introduction to paroxysmal nocturnal hemoglobinuria Paroxysmal nocturnal hemoglobinuria (PNH), a chronic intravascular hemolysis caused by acquired erythrocyte membrane defects, often worsened during sleep, may be associated with paroxysmal hemoglobinuria and pancytopenia, although this disease is rare, but In recent years, there has been an increasing trend. More than half of the north of China is in the south, and more than half of them are young adults aged 20-40, and some are under 10 years old and over 70 years old. More men than women, PNH has always been classified as hemolytic disease, but in addition to anemia is often accompanied by a decrease in neutrophils and (or) platelets, and molecular lesions of PNH involve various blood cells, so in recent years some authors regard PNH as Hematopoietic stem cell disease. The main cause of death is infection in the country and vascular embolism in foreign countries. basic knowledge The proportion of illness: 0.003% Susceptible people: no specific population Mode of infection: non-infectious Complications: thrombosis, cholelithiasis, leukemia

Paroxysmal nocturnal hemoglobinuria

Cellular gene mutation (30%):

PNH is an acquired disease, there has never been a report of congenital onset (except for congenital CD59 deficiency), and there is no tendency for familial aggregation. The exact cause of hematopoietic stem cell lesions is unclear. This disease is an acquired pluripotent hematopoiesis. Stem cell diseases, pathogenic factors may be chemical, radiation or viral infections, pathogenic chromosome mutations, abnormal stem cell lines, proliferation, differentiation of red blood cells, granulocytes and platelets have common defects.

Abnormal clones (20%):

Therefore, it is speculated that some PNH may have more than one abnormal clone. PNH patients' red blood cells, neutrophils, monocytes, lymphocytes, and platelets all have membrane protein deletions. It is conceivable that gene mutations must occur in very early hematopoietic stem cells. However, it is unclear why there are genetic mutations in PNH patients and what are the foreign mutagens. In addition, in view of the fact that PNH patients often have more than one abnormal clone, there is more than one PIG-A gene mutation; In 2002, Horikawa K et al also reported that a Hypoxanthine-Guanine phosphoribosyl transferase (HPRT) gene, which is not associated with the pathogenesis of PNH, is also susceptible to mutations in PNH patients, in other words, PNH patients. There were more PIG-A mutations and HPRT mutations than those without normal PIG-A mutations, suggesting that there is also intrinsic gene instability in the exogenous mutagen, but Purow DB et al. also observed two unrelated genes in PNH patients, HPRT and TcR genes, in 1999. The results showed that individual PNH patients also had the above two genes. One mutation, and the majority of these two genes did not change, so whether PNH patients have widespread genetic instability remains to be confirmed.

Hematopoietic cells are inhibited (25%):

Abnormal cloning of PNH patients does not have the nature of autonomous infinite amplification, but after all, there must be a certain expansion ability to increase the number of abnormal cells enough to produce disease manifestations, in view of the fact that PNH often interacts with aplastic anemia (AA) or At the same time, it is thought that whether the two are related to the etiology, that is, the PNH clone can only be expanded when the normal hematopoietic cells are inhibited. Therefore, in addition to the mutagen, it is necessary to think of many causes of AA. For example, due to certain factors such as viruses, drugs, etc., which can inhibit autoimmune diseases of hematopoietic cells, this is the dual cause of PNH or the two-step pathogenesis.

Pathogenesis

The pathogenesis of PNH involves more than one factor.

1. Gene mutation causes the appearance of abnormal cell clones. PNH abnormal blood cells have the common feature that the surface of the cell membrane lacks a group of membrane proteins. These membrane proteins are linked to the membrane by glycositol phospholipids (GPI), collectively called glycositol phospholipid connexin (GPI connexin), the protein involved and GPI are formed in the endoplasmic reticulum. Once a protein is formed, it is immediately linked to the GPI, and then the outer layer of the cell membrane is transferred. Since PNH cells can detect the free protein and the corresponding mRNA, It can be inferred that PNH abnormal cells lack GPI connexin, not because they cannot produce protein, but because they cannot produce GPI, so this protein cannot exist on the membrane. GPI consists of lipid part and core structure, and lipid part of different kinds of GPI. The difference is very large, but the core structure is very conservative, consisting of 1 inositol phospholipid, 1 glucosamine, 3 mannose and 1 ethanolamine in sequence, one fatty acid on the inositol phospholipid (some are Three) are inserted into the outer layer of the lipid membrane of the cell membrane, and the other end is connected to the protein by ethanolamine. One key enzyme is required for each step of GPI production.

In recent years, abnormal lymphocytes infected with PNH (such as B lymphocytes infected with Epstein-Barr virus) have been used to establish cell lines, and different thymoma cell lines that are known to lack GPI in mice and have a clear GPI production disorder occurred in that step [Thy] -1(-) cell line is fused, and if expressed, GPI connexin can be expressed, indicating that the defects of the two are different and can be complementary; if the GPI connexin is not expressed after fusion, the defects are the same, so they are not complementary. With this method of cell fusion, it has been demonstrated in 40 patients that the defects of PNH abnormal cells are all the same as those of type A Thy-1(-) cells. The lack of GPI is due to the first step in the production of GPI, namely N- Acetylglucosamine cannot be added to phosphoinositide (PI), so it is no longer possible to add 3 mannose and 1 ethanolamine to form a complete GPI decatenin. Label the different components of GPI with radionuclide and observe GPI. In the process of production, it can also be found that PNH abnormal cells cannot produce GPI due to the above-mentioned obstacles. It is known that PNH abnormal cells lack one protein and have great homology with mouse acetylglucosamine transferase. The cDNA and gene nucleotide sequences of this protein have been clarified, and the PIG-A gene was expressed by fluorescence in situ hybridization at the p22.1 site of the X chromosome. Studies have shown that it is found in the blood cells of all tested PNH patients. Mutation of the PIG-A gene results in the partial or total deletion of the GPI connexin, indicating that the PIG-A gene mutation plays an important role in the pathogenesis of PNH.

The cDNA of PIG-A is 1452 bp, encoding 484 amino acids. The nucleotide sequence of PIG-A gene is more than 17 kbp in length, and there are 6 exons: the first exon is only 23 bp, encoding the 5' untranslated region; The second exon has 777 bp, encoding the other part of the 5' untranslated region and about half of the protein; the third exon has 133 bp, encoding a portion of the protein; the fourth exon is 133 bp, encoding a portion of the protein; The 5th exon is 207 bp, encoding a part of the protein; the 6th exon is 2316 bp long, encoding the rest of the protein and the 3' untranslated region, and the 5' flanking 583 bp region of the PIG-A gene has promoter activity. There is no TATA-like sequence in this region, there are 4 CAAT boxes, 2 AP-2 sequences, 1 CRE sequence, and the PIG.A gene has 2 alternative splicing products, which are 347 and 658 nucleotides, respectively, so it is normal. The RT-PCR product of PIG-A mRNA also has three bands, 1500 bp, 1250 bp, and 850 bp, respectively, and the latter products have no function.

PNH-A gene mutation of PNH is heterogeneous. More than 100 gene mutations have been reported so far, widely distributed in multiple coding regions and splicing sites, no mutation clusters or hot spots, and mainly small mutations, large The mutation is rare, and the PNH-A mutation has no significant difference in the genetic map of PNH compared with the AA-PNH syndrome. According to Rosse et al. (1995), the results of 72 patients in 11 laboratories have been reported. Of the mutations, 53 were nucleotide deletions or insertions, of which 42 were deletions; about 1/3 (24 of 84 mutations) had only 1 nucleotide deletion, and only 2 mutations had large segments ( Deletion of more than 100 nucleotides; relatively few nucleotide insertions, 5 cases of deletions and insertions at the same time, the main consequence of deletion or insertion is frameshift, 45 of 51 small deletions or (and) insertion mutations Premature termination code causes protein to decrease, 1 crosses the termination code to increase the protein by 32 amino acids, and 4 deletion mutations change the exon/intron splicing site, affecting the size and stability of PIG-A mRNA. Sex, there is another deletion in the box, the lack of 3 nucleotides makes the protein lack the 151st ammonia Base acid (phenylalanine), 1/3 of all mutations (31 of 84 mutations) are point mutations, and 1 nucleotide is replaced by another nucleotide, in these 31 point mutations: 18 are missense mutations, so that one amino acid in the amino acid sequence of the protein is replaced by another amino acid; 6 are nonsense mutations, resulting in an immediate termination code; 7 are splicing point mutations, affecting the size of PIG-A mRNA And stability.

No point mutations in the 5' promoter region or the 3' untranslated region of the PIG-A gene have been found so far, and one large deletion involves the promoter region, the first exon and part of the first intron, Kinoshita Et al. (1995) obtained the same understanding from 62 cases. All PNH patients had PIG-A gene mutations, and the mutation sites were randomly distributed. Of the 62 cases, 56 mutations involved only 1 or 2 base changes, and the consequences of mutation were shifted. The most common code (63%), different types of PIG-A gene mutations in different patients, only 5 of the 62 cases were found in 2, 3 patients, if the same patient has 2 abnormal cells (GPI connexin no Or missing, it may be caused by two abnormal clones produced by two mutations, but in fact, some patients can only detect one mutation although there are two abnormal cells, and some patients have only one GPI connexin completely missing. Abnormal cells, but can detect two mutations, these cases need to be carefully examined, Luzzatto in 2000 analyzed 28 of the 146 PNH patients reported in the world 174 PIG-A mutations, of which 135 (including large sections will It is the complete inactivation of the PIG-A gene product.

In the other group, 35 were missense mutations and 4 were small in-frame deletions. As a result, part of the PIG-A gene product was deleted. The cells formed in the former group completely lacked GPI connexin (equivalent to PNH III type cells). ), the latter group of cells partially deleted GPI connexin (equivalent to PNHII type cells), and Norris et al. initially studied the effects of different PIG-A gene mutation sites on the structure and function of PIG-A protein. The authors analyzed 18 For example, the missense mutation of the PIG-A gene was found to be located in the coding sequences 128-129 and 15l-156. These coding sequences are located in highly conserved regions of mouse and yeast PIG-A homologous genes. Therefore, conjecture These coding sequences may encode a key part of the PIG-A protein. The authors obtained the PIG-A cDNA with these coding mutations by point mutagenesis and transferred them to prokaryotic and eukaryotic expression systems to measure their PIG-A protein. The structure and function of the gene indicate that a missense mutation in the coding for histidine 128 (H128), serine 129 (S129) and serine 155 (S155) results in partial loss of PIG-A protein function and occurs in coding. Encoding of side chain amino acid residues Sense mutations in PIG-A had no effect on protein function, cue coding H128, S129 and S155 is the key part of the encoded sub-PIG-A affect protein function.

In conclusion, the PIG-A mutation site of PNH patients is spread over multiple sites in the entire coding region of exon 2-6, and among some introns, there is no mutation hotspot, the second exon is longer, and the mutation site is also There are many types of mutations, and the PIG-A mutations of different patients are often different. At present, only a few of the same mutations are found in different patients, but it is not known whether patients in different countries may have differences. According to reports, in Japan 20 Among the mutations, 9 were base substitutions, and only 1 was a polybase deletion; while in Thailand, only 2 of them were base substitutions, but 4 were multibase deletions; large fragments were missing in European and American countries. More common than in Japan, I wonder if the mutagens in different places will be different.

Transfer of normal PIG-A cDNA into PNH abnormal cells can correct the defect that the latter does not express GPI connexin. PNH can be confirmed by mutation of PIG-A gene. However, at least 12 genes involved in the whole process of GPI production. In addition to PIG-A, there are PIG-C, PIG-H, etc., but except for PIG-A, other genes are located on the autosome, and two alleles are simultaneously inactivated. The probability is very small, and the PIG-A gene is located on the X chromosome. Even in women, the X chromosome that is mutated is not randomly inactivated, so no mutations other than the PIG-A gene mutation have been found. PNH.

2. Maintenance and amplification of abnormal cell clones How to maintain and continue to expand after cell clone formation, especially in the presence of normal hematopoietic cells, how the abnormal cells compete, and the number increases enough to cause disease manifestation, It is not clear at present, there are two aspects that can be thought of:

(1) Abnormal cells are not easy to be apoptotic and have strong vitality: in 1997, Brodsky RA et al. and Horikawa K reported that abnormal blood cells of PNH patients have the ability to resist apoptosis, so as to explain the increase in the proportion of abnormal cells, but Ware RE et al. (1998) reported that neutrophils with GPI connexin are not different from neutrophil apoptotic cells lacking GPI connexin; PIG-A cDNA is introduced into PNH phenotype B-lymphocyte strain There was no difference in Fas ligand or X-ray induced apoptosis between pre- and post-introduction cells, indicating that PIG-A gene deletion does not affect apoptosis. In view of this different observation, PNH abnormal cells are not easy to apoptosis. Further research is needed. In the future, we should focus on the early hematopoietic cells and compare the abnormalities of PNH patients with normal and normal human cells, and under normal conditions or under the conditions of in vivo.

(2) Abnormal cells have stronger proliferative capacity: the mice with severe combined immunodeficiency disease that are sublethally irradiated are infused with bone marrow of PNH patients or normal people. After 7 months, the normal bone marrow disappears, and the bone marrow of PNH patients remains. Existence, Han Bing et al., 2000, used flow cytometry to sort out CD34 CD59 cells from PNH patients compared with their own CD34+ CD59- cells and normal human CD34+ CD59 cells. The results were either single cell culture or population cell culture. In liquid medium, it was shown that the CD34 CD59- cells in PNH patients had more division, colony formation and total number of amplification than CD34+ CD59 cells, and both were worse than normal human CD34+ cells. It is suggested that the abnormal phenotype of early hematopoietic cells in PNH patients is stronger than that of normal phenotypes, but neither is the corresponding cells of normal humans. In other words, abnormal cells of PNH have certain effects on their normal cells. Proliferative advantage, and the patient's so-called normal cells are actually abnormal in terms of proliferative capacity.

However, Xiao Juan et al. used the immunomagnetic strain method to sort cells in the same period, and failed to show the proliferation advantage of PNH abnormal phenotype cells in both liquid culture and semi-solid culture. However, the patient's normal phenotype and abnormal phenotype The proliferative capacity of early hematopoietic cells is far worse than that of normal human cells, and it has been confirmed again. It is also reported that the same gene bone marrow transplantation is performed for PNH patients. If the proper pretreatment is not performed, the disease will relapse after it has been relieved. It is also shown that PNH clones have a proliferation advantage. However, scholars who believe that PIG-A gene-inactivated hematopoietic cells do not have intrinsic proliferation advantages can also cite some examples: for example, embryonic stem cells inactivated by PIG-A gene cannot survive and grow. If the PIG-A gene is inactivated during early embryonic development and the chimera is successfully produced, only 5% of the red blood cells and neutrophils lack GPI-linked protein when he starts hematopoiesis, and then the percentage is gradually reduced and finally stabilized, indicating that the cells There is no tendency to continue to proliferate; as another example, Araten DJ et al. (1999) found that most normal humans have red blood cells and neutrophils lacking CD55 and CD59. They are 22/1 million and 8/1 million, respectively, and can also detect PIG-A gene mutations, but do not develop into diseases. In short, whether PNH abnormal clones have intrinsic growth advantages is inconclusive, but overall PNH patients The ability of hematopoietic cells to proliferate (whether normal or abnormal) is below normal and can be determined.

(3) PNH abnormal cloning is based on the attenuation of normal hematopoietic function of bone marrow to obtain relative growth advantages: Li Qiang et al (1997), Xiao Juan et al (2000), Han Bing et al (2000) in their own work. The same phenomenon was found, that the CD34+ hematopoietic stem/progenitor cells in the bone marrow of PNH patients were less than normal, and CD59- cells were significantly more than CD59+, suggesting that the number of normal hematopoietic stem/progenitor cells in PNH patients is small, abnormal hematopoietic stem/ Progenitor cells have a comparative advantage in quantity. It can be speculated that PNH abnormal clones are expanded on the basis of normal hematopoietic failure. In 2001, Pakdeesuwan K of Thailand also had similar observations. As early as 1961, Dacie and Lewis proposed Clinically, PNH is closely related to aplastic anemia. Later, many scholars confirmed and believed that the two are also related to the pathogenesis. In recent years, one view is that PIG-A gene mutation can occur in normal people and in many cases. However, only when the normal hematopoietic function is depleted, it is possible to develop into a disease. Even PNH is considered to be necessary for aplastic anemia, and attention will be focused on autoimmune re On the other hand, it is suggested that there are some GPI connexins on the surface of normal hematopoietic cells, which can be killed by antigens or costimulatory factors that can stimulate cell killing cells (such as T lymphocytes), and cells with PIG-A gene mutations are missing GPI. Connexins can thus evade killing. Karadimitris A et al. (2000, 2001) suggested that all components of T cells may be found to be abnormal, and GPI connexins are present in the targeting of autoreactive T cells.

(4) Others: The effect of serum of PNH patients on normal hematopoietic stem/progenitor cells, the observation results are different. According to Wang Yuzhou's observation in 2001, patients' CD59+ or lymphocytes and their culture supernatants have no effect on the proliferation of CD34+ cells. According to Han Bing in 2000, the colony forming ability of bone marrow fibroblasts in PNH patients was normal, and the mRNA expression of TNF and IL-6 in fibroblasts was also normal; others observed that EPO and G-CSF in serum increased. IFN is normal, and Nishimura et al. proposed that the FGF receptor is also a GPI connexin. The hematopoietic cells of PNH are not inhibited by the proliferation of TGF due to the lack of TGF receptor.

In conclusion, although the pathogenesis of PNH has been studied in recent years, PIG-A gene mutation and attenuation of normal hematopoietic cell proliferation in bone marrow are two important factors, but how to form an imbalance between abnormal hematopoietic cells and normal hematopoietic cell formation and proliferation. How to determine the number of abnormal cells, the factors affecting the number of abnormal cells and the development and changes of PNH disease, how to obtain natural relief of PNH, and whether the hematopoietic microenvironment has changed, etc., further research is needed.

Paroxysmal nocturnal hemoglobinuria prevention

The pathogenesis of paroxysmal nocturnal hemoglobinuria is not completely clear, so it is not known how to prevent the occurrence of this disease, but it is clear that some factors can induce or aggravate hemoglobinuria, so patients should pay attention to avoid infection, especially upper respiratory tract infection; avoid Excessive fatigue or mental stress; avoid abuse of drugs, etc., because the disease is imaginary, it is necessary to reconcile the spirit and nurse the body, "just in the air, evil can not be done", can properly practice qigong, Taijiquan, to improve resistance.

Paroxysmal nocturnal hemoglobinuria complications Complications thrombosis cholelithiasis leukemia

Common complications:

Infection

PNH patients are susceptible to various infections, especially respiratory and urinary tract infections, which can induce hemoglobinuria. In China, severe infections are often the leading cause of death in PNH patients.

2. Thrombosis

Thrombosis in different parts accounts for 23% to 50% of PNH cases in Europe and America, which is the main cause of death in PNH patients in these areas. In China, thrombosis is much less than in Europe and America. China, Thailand, and Japan do not report. More than 10%, domestic cases are characterized by more thrombosis in a single site, more frequent, more shallow blood vessels, less involving important organs, lower extremity venous thrombosis, followed by cerebral thrombosis, very few portal vein or mesentery Thrombosis, and according to Hillmen (1995), 39% of 80 patients had venous thromboembolism; in 41 venous thrombosis, hepatic vein, mesenteric vein, cerebral vein were the most common, pulmonary embolism was also quite common, and there were also Vena cava, spleen, renal vein embolization, 1/4 in deep veins or superficial veins of the limbs, in addition to 6 cases of myocardial infarction, 2 cases of cerebral infarction, the overall appearance is more venous thrombosis than arteries, involving more severe embolism of organs .

3. Cholelithiasis

PNH as a long-term hemolytic disease with cholelithiasis is not as much as imagined, according to domestic reports, but 4%, may be due to asymptomatic, the actual case will be more.

4. Renal failure

PNH patients have hemosiderin in the kidney, but clinically, renal function damage is rare. A small number of cases have mild proteinuria and/or increased blood urea nitrogen. Some people think that if you observe it for a long time, you can find it. The renal function of patients with this disease is gradually reduced. Infection or severe hemolysis can cause acute renal failure, but it can be recovered after treatment. In recent years, magnetic resonance imaging analysis has found that the renal cortical signal intensity of most patients with PNH is weakened, suggesting that there is Hemosiderin is a result of long-term intravascular hemolysis, but not in patients with autoimmune hemolytic anemia.

5. Other

Long-term anemia can cause anemia of heart disease, severe cases can cause heart failure, individual patients have severe bleeding such as cerebral hemorrhage, gastrointestinal bleeding, in addition, long-term use of adrenal cortical hormone secondary diabetes is not uncommon.

Transformation: About 20% of PNH patients are transformed with aplastic anemia (Aplastic anemia), most of which are converted to PNH after a period of aplastic anemia or after recovery. In recent years, many patients with aplastic anemia have been reported to have anti-lymphatic After treatment with cell (or thymocyte) globulin (ALG, ATG) or other immunotherapy, 10% to 31% converted to PNH. Recently, about 30% of patients with aplastic anemia were found by detecting GPI-linked protein on the surface of blood cells. Cells with PNH characteristics can be found in peripheral blood or bone marrow cells, suggesting that patients with aplastic anemia may have the possibility of switching to PNH. Whether the transformation can depend on the number of remaining normal hematopoietic cells and whether PNH clones can grow or survive. Advantages, a small number (about 5%) of PNH patients change to aplastic anemia after a period of time, and some patients have both PNH and aplastic anemia. These conditions are collectively called PNH aplastic anemia syndrome, which is 479 in different regions of China. In the case of PNH, 79 cases (16.5%) belong to this. In general, the conversion of aplastic anemia to PNH is more, PNH is less likely to be aplastic anemia, and there are not many characteristics of both. In addition, individual PNH patients Can be converted to leukemia, which is acute Myeloid leukemia is predominant.

Paroxysmal nocturnal hemoglobinuria symptoms common symptoms fatigue heart palpitations, appetite loss, hemoglobinuria, nausea, pale protein, urinary jaundice, low molecular proteinuria

First, the symptoms:

The onset is slow, the first symptom is anemia for early performance accounted for 60.3%, and a small number of patients are more acute onset, due to acute hemolysis, and suddenly appeared in the urine, the most common symptoms of chronic anemia, fatigue, dizziness, pale, palpitations, shortness of breath , tinnitus, eye hair, etc., paroxysmal aggravation or paroxysmal hemoglobinuria is a typical symptom of this disease, 35% of patients with hemoglobinuria and sleep, hemorrhagic episodes can be seen after sleep brown urine and soy sauce, a few patients may have a serious attack Backache, sore limbs, loss of appetite, fever, nausea and vomiting, urinary insufficiency, urethral pain.

Second, the characteristics:

1. The vast majority of patients with anemia have varying degrees of anemia, often moderate, severe, due to anemia can be pale, pale lips, and pale pale and nail bed light, because anemia is mostly slow, patients often have Good adaptability, so hemoglobin is still low but still active, and even work. In addition, due to long-term intravascular hemolysis, the skin has hemosiderin deposition, so the face and skin often have dark brown.

2. Hemoglobinuria The typical hemoglobinuria is soy sauce or rich brown color, which lasts for 2 to 3 days. It does not need to be treated to resolve itself. It is severe for 1 to 2 weeks, and even lasts longer. Some patients have frequent hemoglobinuria, and some have frequent attacks. The patient has an episode or a seizure once in a few months. In some patients, although the urine color is not deep, the urinary occult blood continues to be positive, and about 25% of the patients have no seizures during a long course or observation period, which can cause hemoglobinuria. The factors of the attack include colds or other infections, blood transfusions, taking iron, tiredness, etc. Hemoglobinuria may have cold fever, low back pain, abdominal pain and other symptoms. As for why some patients have hemoglobinuria during sleep, there is no good Explain that it was suggested that due to the absorption of bacterial lipopolysaccharide in the intestine during sleep, the activation of complement was caused; Wang Weizhou's observation in 2000 showed that there was no significant change in blood pH value during sleep, and the C3d of complement 3 activation product increased slightly. Same as normal people.

3. About one-third of patients with PNH have mild hemorrhagic manifestations, such as gingival bleeding, nasal oozing and skin bleeding, etc., and female patients can also show more menorrhagia. Individual patients can have a large number of Bowel, non-local causes can explain postoperative hemorrhage, post-abortion hemorrhage, tar-like blood in the stool and fundus hemorrhage.

4. Because of hemolysis, 47% of patients have jaundice in the course of the disease, while those with jaundice as the first performer account for 4%, and jaundice is mostly mild or moderate.

5. Most patients with hepatosplenomegaly have no hepatosplenomegaly. About 1/4 of PNH patients have only mild hepatomegaly, and less than 15% have mild splenomegaly.

6. Other long-term anemia hearts can be seen compensatory expansion.

Paroxysmal nocturnal hemoglobinuria

1. Peripheral blood complete blood cells are reduced, most patients have different degrees of anemia, only a small number of hemoglobin is normal, reticulocytes often increase, but often not as obvious as other hemolytic diseases, bone marrow is mostly active or significantly active, red The system is prosperous, and very few patients have some degree of pathological hematopoiesis.

Bone marrow: from hyperplasia to hyperplasia, with obvious proliferation of young red blood cells, normal granulocyte, megakaryocyte cell line, intracellular, extracellular iron is reduced or absent.

Indirect blood erythropoietin increased, serum haptoglobin decreased or disappeared, plasma free hemoglobin increased, and urinary hemosiderin was positive.

2. Acidified serum hemolysis test (Ham test) PNH pathological red blood cells are easily lysed by alternative pathway activated at pH 6.4, and normal red blood cells are not. This test has strong specificity and is regarded as a diagnosis of PNH at home and abroad. The main basis is to use the photoelectric colorimetric method to see the degree of hemolysis. PNH is mostly above 10%, and about 79% of patients with this disease are positive.

3. Sugar water hemolysis test (sucrose hemolysis test) This test is highly sensitive, and PNH patients are about 88% positive. Japanese scholars believe that it is the best screening test for the diagnosis of this disease. The shortcoming of the sugar water test is that it is prone to false positive reaction.

4. Snuff venom factor (CoF) hemolysis test This test also has strong specificity, sensitivity is stronger than Ham test, slightly worse than syrup test, and PNH patients are about 81% positive.

5. Complement hemolysis sensitivity test This test can divide PNH red blood cells into three types I, II and III. The clinical hemolysis weight depends on the number of type III cells.

6. Detection and quantification of abnormal blood cells of PNH can detect abnormal reticulocytes, which is the most specific, sensitive and quantifiable method for establishing diagnosis. The detection of bone marrow cells is more meaningful than peripheral blood cells.

7. Direct anti-human globulin test, indirect anti-human globulin test was negative.

1. Bone marrow cell culture often finds that CFU-E, CFU-GM and other colonies are less than normal bone marrow.

2. Brodsky et al. reported a new diagnostic method using a bacterium (Aerolysin) produced by Aeromonas genus bacteria, which can form a channel on the cell membrane by linking with GPI protein, thereby rupturing normal cells. Kill it, and PNH cells are not affected by this toxin due to the lack of GPI protein. PNH cells remain intact. This method is simple, easy, inexpensive and specific, and can detect flow cytometry. PNH cells that cannot be detected have broad application prospects in clinical practice.

3. Scanning electron microscopy showed that red blood cells mostly lost double concave disc shape, which showed different sizes, uneven edges and uneven bumps.

4. According to clinical manifestations, symptoms and signs are selected for X-ray, B-ultrasound, electrocardiogram, biochemistry, liver and kidney function tests.

Diagnosis and diagnosis of paroxysmal nocturnal hemoglobinuria

Diagnostic criteria

First, the diagnostic conditions of PNH

1. Clinical manifestations are consistent with PNH.

2, laboratory tests: acidified serum hemolysis test (Ham test), sugar water test, snake venom factor hemolysis test, urinary occult blood (or urine hemosiderin) and other experiments, where the following conditions:

a, more than two positive;

b, a positive but with the following conditions:

(1) More than two positives, or one positive, but the operation is normal, there is a negative control, the results are reliable, and the immediate repeat is still positive.

(2) Other indirect evidence of hemolysis, or a positive hemoglobinuria.

(3) Can exclude other hemolysis, especially hereditary spherocytosis, autoimmune anemia.

Second, the diagnosis of aplastic anemia-PNH syndrome

Where aplastic anemia is converted to PNH or PNH into aplastic anemia, or both, is aplastic anemia-PNH syndrome.

Third, although this disease is called paroxysmal nocturnal hemoglobinuria, but not all hemoglobinuria, even if it is not necessarily episode, it is not necessarily in the sleep, and only a few patients with hemoglobinuria as the first performance, According to the comprehensive data of 651 cases in China, 54.9% is anemia, 18.3% is anemia and bleeding as the initial performance. After a considerable period of time, hemoglobinuria appears, even from hemoglobinuria without visible eyes, only 22.5% with hemoglobinuria as the first performance. In addition, the clinical manifestations of comorbidities and disease transformation, resulting in PNH patients often can not get timely diagnosis, and even missed diagnosis, misdiagnosis, in patients with hemoglobinuria or long-term chronic anemia, especially accompanied by Leukocytes and (or) thrombocytopenia and bone marrow hyperplasia, should be considered in the differential diagnosis of the disease, the diagnosis of this disease requires some laboratory diagnostic methods.

Diagnostic evaluation

(1) The Ham test is still considered as the main basis for the diagnosis of paroxysmal nocturnal hemoglobinuria at home and abroad: the positive rate of the Ham test is higher in the onset of hemolysis, and the negative result in the hemolysis interval is often negative. At the time, the sulphur water hemolysis test is prone to false positive results. The snake venom hemolysis test is more sensitive than the Ham test. If the conditional laboratory should do 3 tests at the same time, it is helpful for clinical diagnosis.

(2) Because PNH is intermittently episode: one must meet one of the following criteria for diagnostics when analyzing laboratory results:

More than 12 positive,

21 positive, but more than 2 positive, or only 1 positive, the test operation and results are very reliable, however, the results must be combined with clinical manifestations, pay attention to the time of sending specimens, and have a positive hemoglobinuria Or direct or indirect basis of intravascular hemolysis, and can exclude other hemolytic diseases.

(3) Using flow cytometry to detect blood cell-specific antibodies CD55, CD59, which are used to determine abnormal cells lacking membrane proteins, and can calculate the percentage of abnormal cells. This method can detect red blood cells, neutrophils, single Nuclear cells, lymphocytes and other cells, abnormal neutrophils are the earliest detected, can appear before the Ham test is positive, so suggest that even if there are abnormal cells, it is easy to diagnose in combination with clinical and other laboratory tests.

(4) In order to find the hemoglobinuria which is not easily noticeable to the naked eye: urine occult blood should be examined every day for several days. For example, after hemolysis has just occurred or after a large amount of blood transfusion, laboratory tests are prone to negative results.

(5) 20% of patients with PNH can be transformed with aplastic anemia: called aplastic anemia-PNH syndrome. Both diseases can occur simultaneously or simultaneously. Both diseases are often misdiagnosed as aplastic anemia due to complete hematocytopenia. For such patients, multiple investigations of PNH laboratory tests and bone marrow puncture should be conducted.

(6) The hemosiderin in the urine Rous test comes from renal tubular cells: so it can be negative in acute hemolysis, usually positive after a few days of hemolysis, and lasts for a while, so it is helpful to judge whether there is any in the near future. Reliable evidence of hemoglobinemia.

PNH-Aplastic Barrier Syndrome includes the following four conditions:

1 Aplastic anemia - PNH: refers to the original affirmative aplastic anemia (not the early manifestation of PNH that was not diagnosed) and then converted to PNH, and the performance of aplastic anemia is no longer present;

2PNH-re-barrier: refers to the original positive PNH (instead of the fourth category below) and then converted to aplastic anemia, and the performance of PNH (including laboratory tests) no longer exists;

3PNH with aplastic anemia characteristics: refers to clinical and laboratory tests that indicate that the condition is still PNH-based but accompanied by one or more hyperplasia of bone marrow, megakaryocytes decreased, and the number of reticulocytes is not high, such as aplastic anemia;

4 Aplastic anemia with PNH characteristics: Both clinical and laboratory tests indicate that the condition is still dominated by aplastic anemia, but PNH abnormal blood cells appear (positive tests for detecting complement sensitivity, or PNH abnormal cells can be detected by other methods).

According to recent research, the classification of PNH can be simplified to:

1 hemolytic PNH: mainly characterized by frequent or sustained hemolysis, and the number of cells lacking GPI connexin is increased;

2 hypoplastic PNH: mainly manifested by significant whole blood cell reduction or hypoplasia, normal hematopoietic cell hyperplasia, using flow cytometry combined with clinical and bone marrow examination can be typed, this simple typing method It has certain guiding significance for diagnosis and treatment.

The early missed diagnosis of this disease, the rate of misdiagnosis is high, about half of the whole blood cell reduction is misdiagnosed as aplastic anemia, and secondly misdiagnosed as other proliferative anemia, or misdiagnosed as hepatitis, nephritis due to jaundice, abnormal urine, etc., the key to timely diagnosis is:

1 think about the disease and recognize the diversity of clinical manifestations of the disease;

2 pay close attention to the appearance of hemoglobinuria, check urine occult blood every day for several days, sometimes help to find hemoglobinuria that is not easily noticeable to the naked eye;

3 The correctness of the test results for determining PNH should be judged correctly. The positive test depends on the number of abnormal blood cells. Immediately after hemolysis, the test may be negative because the abnormal red blood cells have been destroyed, and the normal cells after a large number of blood transfusions. Increased, abnormal cells are relatively reduced, it will also affect the results, so can not be negative because of a negative result, should be repeated and multiple tests at the same time, in recent years using specific antibodies and flow cytometry technology may find some Early or advanced cases of PNH, and can detect abnormal neutrophils, etc., thereby reducing the impact of blood transfusion, but all these tests only indicate the presence of abnormal cells, whether the main symptom is PNH still need comprehensive analysis and close Follow-up observation can draw conclusions, because in some other diseases such as myelodysplastic syndrome, a small amount of abnormal red blood cells similar to PNH can also occur. A small number of abnormal cells in the abnormity process may also be transient and not necessarily develop into PNH.

Differential diagnosis

1. Aplastic anemia is easily confused with PNH. 47.3% of cases also have complete cytopenia. The main difference between the two is that aplastic anemia should be reduced by myeloproliferation, while PNH is active in myeloproliferation (especially erythroid). Reduced and can detect PNH-like abnormal red blood cells, or clinical and laboratory findings of PNH, but low myeloproliferative, should be suspected of disease conversion or both diseases (aplastic anemia-PNH syndrome) ).

2. Iron deficiency anemia PNH loses iron due to long-term repeated hemoglobinuria, which may be associated with iron deficiency, but unlike iron deficiency anemia, it is impossible to completely correct anemia after iron supplementation.

3. Nutritional megaloblastic anemia due to hemolysis promotes bone marrow compensatory hyperproliferation, folic acid may be relatively insufficient, resulting in megaloblastic anemia, but after supplementation with folic acid can not completely correct the anemia caused by this disease.

4. Myelodysplastic syndrome (MDS) Individual PNH patients can see pathological hematopoiesis, even a slight increase in granulocytes or a small amount of blasts in peripheral blood. Some scholars even consider PNH as MDS. One, but according to our observation, the pathological hematopoietic or blast-producing phenomenon of PNH is transient and can disappear. Very few patients can completely become MDS. On the other hand, some patients with MDS can also have abnormal blood cells similar to PNH. However, its basic characteristics and disease development are still dominated by MDS, and typical hemoglobinuria or PNH performance rarely occurs.

5. Autoimmune hemolytic anemia Individual PNH patients can be positive for direct anti-human globulin test. On the other hand, individual hemolytic hemolytic anemia patients can be positive for saccharification and hemolytic test, but after tracing these tests can be negative, more important The two diseases have their own clinical and experimental features, and the identification is not difficult. In addition, in most cases, the effect of corticosteroids on autoimmune hemolytic anemia is much better than PNH.

6. Hereditary spherocytosis: For identification points, see autoimmune hemolytic anemia.

7. Glucose-6-phosphate dehydrogenase deficiency: For identification points, see autoimmune hemolytic anemia.

8. Paroxysmal cold hemoglobinuria: After the cold, the hand and foot cyanosis appeared, and it improved after warming; the hot and cold hemolysis test was positive.

The material in this site is intended to be of general informational use and is not intended to constitute medical advice, probable diagnosis, or recommended treatments.