Introduction
Paroxysmal nocturnal hemoglobinuria(Paroxysmal nocturnal hemoglobinuria, PNH), 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, it has an increasing trend in recent years. 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 often accompanied by anemiaNeutrophilAnd (or) the reduction of platelets, and the molecular lesions of PNH involve various blood cells, so in recent years some authors regard PNH as a hematopoietic stem cell disease. The main cause of death is infection in the country and vascular embolism in foreign countries.
Cause
(1) Causes of the disease
PNH is an acquired disease that has never been reported for congenital onset (except for congenital CD59 deficiency) and has no tendency to familial aggregation. The exact cause of the pathogenesis of hematopoietic stem cells is unclear. The disease is an acquired pluripotent hematopoietic stem cell disease. The pathogenic factors may be chemical, radiological or viral infections, pathogenic chromosomal mutations, abnormal stem cell lines, and red blood cells, granulocytes and platelets produced by proliferation and differentiation. Common defects.
PNH is caused by a mutation in the hematopoietic stem cell geneCrohn's diseaseThe diseased cells and normal cells exist at the same time, and the pathological blood cells are all from the same clone. The evidence is: 1 In the female PNH patients with heterozygous G6PD isoenzymes, the abnormal red blood cells all have the same isozyme, indicating that the abnormal cells are From the same source; 2 in recent years, molecular biological methods to analyze the type of X chromosome inactivation in female patients, it can also be found that abnormal cells are of the same type; 3 using flow cytometry technology can see the total peripheral blood and bone marrow of PNH patients Abnormal and normal cells exist at the same time, while abnormal cells have the same abnormality (such as lack of CD59 on the cell surface), while the normal blood cells of the patient have no such abnormality, indicating that there are two kinds of cell sources; abnormal cells of 4PNH patients can be investigated. The phosphatidylinositol-A (PIG-A) gene mutation, but the normal cells of the same patient do not have this mutation, further indicating that the two sources are different; 5 a small number of leukemia cells transformed from PNH to acute leukemia may have PNH membrane protein The missing features indicate that leukemia is derived from the original PNH clone. In view of the fact that the degree of abnormal cell-deficient membrane proteins in the same PNH patient may vary, there are some patients with more than one type of PIG-A gene mutation, so it is speculated that some PNH may have more than one abnormal clone. In PNH patients, red blood cells, neutrophils, monocytes, lymphocytes, and platelets are all missing membrane proteins. 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 foreign mutagens are present. 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 addition, in 2002 Horikawa K et al also reported: a hypoxanthine 嘌呤 磷酸 phosphoribose not related to the pathogenesis of PNH The Hypoxanthine-Guanine phosphoribosyl transferase (HPRT) gene is also susceptible to mutations in PNH patients. In other words, PNH patients with PIG-A mutations have more HPRT mutations than those without normal PIG-A mutations. Therefore, it is suggested that whether the patient has intrinsic gene instability or not, in addition to the mutagen in the exclusion zone. However, 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 mutations in one of the above two genes, while most of the two genes did not change. . Therefore, whether PNH patients have widespread genetic instability remains to be confirmed.
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 is often transformed or coexisting with aplastic anemia (AA), it is thought that there is also a correlation between the two, that is, PNH clones can only be expanded when normal hematopoietic cells are inhibited. In this way, in addition to the mutagen, it is necessary to think of many causes of AA, such as autoimmune diseases that can inhibit hematopoietic cells due to certain factors such as viruses and drugs. This is the dual cause of PNH or the two-step pathogenesis.
(two) 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). Both the protein and the GPI are formed in the endoplasmic reticulum, and a protein is immediately linked to the GPI and then transferred to the outer layer of the cell membrane. Since PNH cells can detect free proteins and corresponding mRNAs, it can be assumed that PNH abnormal cells lack GPI connexins because they cannot produce proteins but because GPI cannot be produced, so this protein cannot exist on the membrane. GPI consists of a lipid moiety and a core structure. The lipid fractions of different GPIs vary widely, but the core structure is very conservative, consisting of 1 inositol phospholipid, 1 glucosamine, 3 mannose and 1 ethanolamine. Composed in sequence, two fatty acids (three in some) on the inositol phospholipid are inserted into the outer layer of the lipid bilayer of the cell membrane, and the other end is bound to the protein by ethanolamine. One key enzyme is required for each step of GPI production.
In recent years, cell lines have been established by abnormal lymphocytes infected with PNH (such as B lymphocytes infected with Epstein-Barr virus). Fusion is performed with different thymoma cell lines [Thy-1(-) cell strains] in mice that are known to lack GPI and have a clear GPI production disorder occurring at that step. If the GPI connexin can be expressed after fusion, it indicates that the defects of the two are different and can be complementary; if the GPI connexin cannot be expressed after fusion, the defects of the two are the same, so they are not complementary. This method of cell fusion has been demonstrated in 40 patients to demonstrate that PNH abnormal cells are all defective in the same manner as type A Thy-1(-) cells. The lack of GPI is due to the first step in the production of GPI, that is, N-acetylglucosamine cannot be added to phosphoinositide (PI), so it is impossible to add 3 mannose and 1 ethanolamine to form a complete GPI. Connexin. The different components of GPI were labeled with radionuclides to observe the process of GPI production. It was also found that PNH abnormal cells could not produce GPI due to the above obstacles. It is known that PNH abnormal cells lack one protein and have great homology with mouse acetylglucosamine transferase. At present, the cDNA and gene nucleotide sequences of this protein have been clarified, and the PIG-A gene is called. It was confirmed by fluorescence in situ hybridization to locate the p22.1 site of the X chromosome. Studies have shown that PIG-A gene mutations in all the blood cells of patients with PNH have led to the partial or total loss of GPI connexin, indicating that PIG-A gene mutation plays an important role in the pathogenesis of PNH.
The cDNA of PIG-A has a 1452 bp encoding 484 amino acids. The nucleotide sequence of PIG-A gene is more than 17kbp in length, and there are 6 exons: the first exon is only 23bp, encoding the 5' untranslated region; the second exon has 777bp, encoding 5' end. The other part of the untranslated region and about half of the protein; the third exon has 133 bp, encoding a part of the protein; the fourth exon is 133 bp, encoding a part of the protein; the fifth exon is 207 bp, encoding a part of the protein; The 6 exons are 2316 bp long, encoding the rest of the protein and the 3' untranslated region. The 5' flanking 583 bp region of the PIG-A gene has promoter activity. This region has no TATA-like sequence and has 4 CAAT boxes, 2 AP-2 sequences, and -1 CRE sequences. The PIG.A gene has two alternative splicing products, 347 and 658 nucleotides, respectively. Therefore, the RT-PCR product of normal PIG-A mRNA also has three bands, which are 1500 bp, 1250 bp, and 850 bp, respectively, and the encoded products of the latter two are not functional.
The PNG-A gene mutation of PNH is heterogeneous. More than 100 gene mutations have been reported so far, widely distributed in multiple coding regions and splice junctions, and no mutation clusters or hot spots. Moreover, mainly small mutations are dominant, and large mutations are rare. There is no significant difference in the genetic map of PIG-A mutations in PNH with typical hemolysis symptoms compared with AA-PNH syndrome. According to Rosse et al. (1995), out of the 84 mutations reported in 11 of the 11 laboratories, 53 were nucleotide deletions or insertions, of which 42 were deletions; approximately 1/3 (84 mutations) Of the 24), only 1 nucleotide was deleted, only 2 mutations had large segments (more than 100 nucleotides) deleted; nucleotide insertions were relatively rare, and 5 patients had both deletions and insertions. The main consequence of deletion or insertion is frameshift, with 45 of the 51 small deletions or (and) insertion mutations leading to a premature termination code, which reduces the protein, and 1 crossing the termination code increases the protein by 32 amino acids. There are also four deletion mutations that alter the exon/intron splicing site, affecting the size and stability of PIG-A mRNA. There is another deletion in the box, which lacks the 151th amino acid (phenylalanine) due to the lack of 3 nucleotides. One third of all mutations (31 of 84 mutations) are point mutations, and one nucleotide is replaced by another nucleotide. Among the 31 point mutations: 18 are missense mutations, which make one amino acid sequence in the amino acid sequence of the protein replaced by another amino acid; 6 are nonsense mutations, producing 1 immediate termination code; 7 are splice junctions Mutations affect PIG-A mRNA size 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. One large segment of the deletion involved the promoter region, the first exon and a portion of the first intron. Kinoshita et al (1995) obtained the same understanding from 62 patients. All PNH patients had PIG-A gene mutations, and the mutation sites were randomly distributed. Of the 62 patients, 56 mutations involved only one or two base changes, and the consequences of the mutation were The most important is the frame shift (63%). Different patients had different types of PIG-A gene mutations, and only 5 of the 62 mutations were found in 2 or 3 patients. If the same patient has 2 abnormal cells (GPI connexin is absent or absent), it may be derived from 2 abnormal clones produced by 2 mutations, but in fact, some patients can only find one if there are 2 abnormal cells. Mutations, and some patients have only one abnormal cell with complete deletion of GPI connexin, but can detect two mutations, and these cases need to be carefully examined. In 2000, Luzzatto analyzed 174 PIG-A mutations in 28 of the 146 PNH patients reported worldwide, of which 135 (including large segments will be completely inactivated of the PIG-A gene product, and 35 in the other group) Missense mutations, 4 are small in-frame deletions, the result is a partial loss of function of the PIG-A gene product. The cells formed by the former group completely lack GPI connexin (equivalent to PNH III type cells), and the latter group formed Part of the cell lacks GPI connexin (equivalent to PNHII type cells). Norris et al. preliminarily studied the effects of different PIG-A gene mutation sites on the structure and function of PIG-A protein. The authors analyzed the error of 18 PIG-A genes. For the sense mutation, 6 were 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, it is suspected that these encodings may encode PIG- A key part of protein A. The authors obtained the cDNA of PIG-A with these coding mutations by point mutagenesis, and transferred them into prokaryotic and eukaryotic expression systems to measure the structure and function of PIG-A protein. , encoding histidine 128 (H128), silk A missense mutation in the codons of acid 129 (S129) and serine 155 (S155) results in partial loss of PIG-A protein function, whereas a missense mutation occurs in the coding sequence encoding a side chain amino acid residue against PIG-A protein. No effect on function. It is suggested that the coding for H128, S129 and S155 is a key part of the function of PIG-A protein.
In conclusion, the PIG-A mutation site of PNH patients is spread over multiple locations throughout the coding region of exon 2-6, and among some introns, there are no mutation hotspots. The second exon is longer and the mutation occurs more. There are many types of mutations. Different patients have different PIG-A mutations, and only a few of the same mutations have been found in different patients. However, it is not known whether patients in different countries may differ. It is reported that 9 of the 20 mutations in Japan are base substitutions, and only 1 is a polybase deletion; while in Thailand, only 2 of them are base substitutions. However, there are 4 deletions of multiple bases; the deletion of large fragments in European and American countries is more common than in Japan. I don't know 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, and PNH can be confirmed by mutation of PIG-A gene. However, there are at least 12 genes involved in the whole process of GPI production, and the genes involved in the first step include PIG-C, PIG-H and the like in addition to PIG-A. However, except for PIG-A, other genes are located on the autosome, and the probability of simultaneous inactivation mutations of the two alleles is minimal. However, the PIG-A gene is located on the X chromosome, and even if the X chromosome in which the mutation is generated is not randomly inactivated, the PNH-A gene mutation has not been found to have caused PNH caused by mutation of other genes.
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. It is unclear how the abnormal cells compete and increase in number enough to cause disease manifestations. There are two aspects that can be conceived:
(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, thereby explaining the increase in the proportion of abnormal cells. However, the following year Ware RE et al (1998) reported that neutrophils with GPI connexin did not differ from neutrophil apoptosis in the absence of GPI connexin; PIG-A cDNA was introduced into the B-lymph of the PNH phenotype There was no difference in Fas ligand or X-ray induced apoptosis between the cells before and after introduction, indicating that the loss of PIG-A gene did not affect apoptosis. In view of this different observation, whether PNH abnormal cells are not easy to be apoptotic remains to be further studied. In the future, the early hematopoietic cells should be observed, and the abnormalities of PNH patients and normal and normal human cells should be compared under physiological conditions. Or may occur in apoptosis in vivo conditions.
(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. presence. Our hospital Han Bing et al. used flow cytometry in 2000 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 the liquid medium, it was shown that the CD34 CD59- cells of 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 is confirmed again. It has also been reported that PNH patients undergoing isogenic bone marrow transplantation, if not properly pretreated, will be relapsed after the disease has been relieved once, which seems to indicate that PNH clones have a proliferation advantage. However, scholars who believe that PIG-A gene-inactivated hematopoietic cells do not have an intrinsic proliferation advantage can also cite some examples: for example, embryonic stem cells inactivated by PIG-A gene cannot survive and grow, if inactivated during early embryonic development. The PIG-A gene successfully produced chimeras. Only 5% of red blood cells and neutrophils lacked GPI connexin when he began to hematopoietic, and the percentage decreased gradually, and finally stabilized, indicating that the cells did not continue to proliferate; Araten DJ et al. (1999) found that most normal humans had red blood cells and neutrophils lacking CD55 and CD59, which were 22/1 million and 8/1 million, respectively, and PIG-A gene mutations were also detected. But it does not develop into a disease. In conclusion, whether PNH abnormal clones have intrinsic growth advantages is inconclusive, but in general, the hematopoietic cell proliferation ability (whether normal cells or abnormal cells) of PNH patients is lower than 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, and it can be speculated that PNH abnormal clones are expanded on the basis of normal hematopoietic failure. Similar observations were made in 2001 in Pakdeesuwan K, Thailand. As early as 1961, Dacie and Lewis proposed that PNH is closely related to aplastic anemia in clinical practice. Later, many scholars confirmed that they are also related to the pathogenesis. In recent years, a point of view is that PIG-A gene mutations can occur in normal people and in many cases, but it is only possible to develop diseases when normal hematopoietic failure. Even PNH is considered to be necessary for aplastic anemia, and attention is focused on autoimmune aplastic anemia. It is suggested that some hematopoietic cells have some GPI connexin on the surface, which can be used to stimulate cell killing cells (such as T lymphocytes). The antigen or co-stimulatory factor is thus killed, and the cells with the PIG-A gene mutation can escape the killing due to the deletion of the GPI connexin. Karadimitris A et al. (2000, 2001) suggested that abnormalities may be found in all components of T cells, and GPI connexins are present in the targeting of autoreactive T cells. Our work, Wang Yuzhou and other work from 2000 to 2001, also found that the proportion of T8 cells/T4 cells in peripheral blood of patients with PNH increased, and activated CD8 with HLA-DR marker. Lymphocytosis; gamma interferon and IL-2 have an inhibitory effect on normal hematopoietic stem/progenitor cells of CD34+ CD59+. Supporting PNH has immunological disorders and is not conducive to normal hematopoietic cells. It is believed that the involvement of immune factors may explain that PNH occurs after some acquired aplastic anemia, but rarely occurs after congenital aplastic anemia or chemotherapy-induced bone marrow hematopoietic failure. However, the relationship between PNH and aplastic anemia may not be as simple as PNH disorders do not occur during the most severe period of myelosuppression in aplastic anemia, but often after recovery from aplastic anemia; in addition, PNH often occurs in patients with aplastic anemia who use immunosuppression. Agent. After ATG and the like are relieved, not when autoreactive T lymphocytes are not inhibited. Furthermore, the ups and downs and stability of PNH patients' conditions may require additional explanation.
(4) Others: The effect of serum of PNH patients on normal hematopoietic stem/progenitor cells was observed. According to Wang Yuzhou's observation in 2001, patients' CD59+ or lymphocytes and their culture supernatants had no effect on the proliferation of CD34+ cells; according to Han Bing's observation in 2000: PNH patients' bone marrow fibroblasts have normal colony forming ability, fibroblasts. The mRNA expression of TNFα and IL-6 was also normal; in others, the serum EPO and G-CSF were increased, and IFNγ was normal. In addition, Nishimura et al. proposed that the FGFβ receptor is also a GPI connexin, and that 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.
symptom
[clinical manifestations]
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 appearing color 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 to severe. Because of anemia, the complexion is pale, the lips are pale, and the paleness and the nail bed are pale. Because anemia is mostly slow, patients often have better adaptability, so hemoglobin is often low but still active and even working. In addition, due to long-term intravascular hemolysis, the skin has hemosiderin deposition, so the face and skin often have dark brown.
2. Hemoglobin The typical hemoglobinuria is soy sauce or rich brown. It usually lasts for 2 to 3 days and disappears without treatment. It is heavy for 1 to 2 weeks, and even lasts longer. Some patients have frequent episodes of hemoglobinuria, and some patients have occasional episodes or episodes of several episodes. In some patients, although urine color is not deep, urine occult blood continues to be positive. Approximately 25% of patients have never had an episode over a long course of disease or observation period. Factors that can cause hemoglobinuria are colds or other infections, blood transfusions, iron medications, and fatigue. 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 explanation. It has been suggested that due to the absorption of bacterial lipopolysaccharide in the intestine during sleep, the activation of complement is caused; Wang Weizhou's observation in 2000 showed that there was no significant change in blood pH value during sleep, and a slight increase in complement 3 activation product C3d, and normal. Like people.
3. About 1/3 of patients with PNH have mild hemorrhagic manifestations, which are mild to moderate bleeding such as gingival bleeding, nasal oozing and skin bleeding. Female patients can also show more menorrhagia. Individual patients may have a large number of epistaxis, postoperative hemorrhage that can be explained by non-local causes, post-abortion hemorrhage, tar-like blood in the stool, and fundus hemorrhage.
4. Astragalus Due to 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. Hepatosplenomegaly Most patients 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.
【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. In the experiments of venom factor hemolysis test, urinary occult blood (or urinary hemosiderin), the following conditions are met:
a, more than two positive;
b, a positive but with the following conditions:
(1) More than two positives, or one positive. However, the operation is normal, there is a negative control, the result is reliable, and the immediate repeat is still positive.
(2) Other indirect evidence of hemolysis, or a positive hemoglobinuria.
(3) Except for other hemolysis, especially hereditary spherePolycythemia, autoimmune anemia.
two,Aplastic anemia -PNH diagnosis of comprehensive symptoms
Where aplastic anemia is converted to PNH or PNH into aplastic anemia, or both, is aplastic anemia-PNH syndrome.
Third, although the disease is calledParoxysmal nocturnal hemoglobinuria, but not all hemoglobinuria, even if it is not necessarily episodes, it is not necessarily in the sleep. And only a small number of patients with hemoglobinuria as the first performance. According to the comprehensive data of 651 cases in China, 54.9% was anemia, 18.3% was anemia and bleeding was the initial performance. After a considerable period of time, hemoglobinuria appeared, even from hemoglobinuria, which was not visible to the naked eye. Only 22.5% showed hemoglobinuria as the first performance. Coupled with the complication and the transformation of the disease, the clinical manifestations are diverse, which makes PNH patients often unable to get timely diagnosis, and even missed diagnosis and misdiagnosis for a long time. In patients with hemoglobinuria or long-term chronic anemia, especially those with white blood cells and/or thrombocytopenia and bone marrow hyperplasia, the disease should be considered in differential diagnosis. It is necessary to have some laboratory diagnostic methods to diagnose this disease.
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. time. The syrup 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 will help the clinical diagnosis.
(2) Since PNH is intermittently episode: it is necessary to meet one of the following criteria for the diagnosis of laboratory tests: more than 12 positive. 21 positive, but more than 2 positive, or only 1 positive, the test operation and results are very reliable. Despite this, the results must be combined with clinical performance, paying attention to the time of sending the specimen. At the same time, there are affirmative hemoglobinuria or direct or indirect basis of intravascular hemolysis, and can exclude other hemolytic diseases.
(3) Detection of blood cell-specific antibodies CD55 and CD59 by flow cytometry is used to determine abnormal cells lacking membrane proteins and to calculate the percentage of abnormal cells. This method can detect red blood cells, neutrophils, monocytes, lymphocytes and other cells. The abnormal neutrophils are detected at the earliest and can appear before the Ham test is positive, so it is suggested that even if abnormal cells exist, it is necessary to combine clinical And other laboratory tests are easy to diagnose.
(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 one after another, or both diseases. In the early stage, due to the complete diagnosis of whole blood cells, it is misdiagnosed as aplastic anemia. For such patients, the laboratory examination items related to PNH and bone marrow puncture should be traced several times.
(6) Hemosiderin in the urine Rous test comes from renal tubular cells: so it can be negative in acute hemolysis. It usually appears positive after a few days of hemolysis and lasts for a while, so it is reliable evidence to help determine whether there is hemoglobinemia in the near future.
PNH-Aplastic Residue Syndrome includes the following four conditions: 1 Aplastic anemia - PNH: refers to the original positive aplastic anemia (not the early manifestation of PNH that is not diagnosed) and then converted to PNH, and the performance of aplastic anemia is no longer present. 2PNH-Aplastic anemia: refers to the original positive PNH (instead of the fourth category below) and then converted to aplastic anemia, while the performance of PNH (including laboratory tests) no longer exists; 3PNH is associated with aplastic anemia characteristics: Clinical and laboratory tests indicated that the condition was still PNH-based but accompanied by one or more hyperplasia of bone marrow, megakaryocytes decreased, reticulocyte counts were not high, and other aplastic anemia manifestations; 4 aplastic anemia with PNH characteristics: refers to 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 by frequent or continuous hemolysis, and more cells lacking GPI connexin; 2 hypoproliferative PNH: with significant whole blood cells Reduction or low myeloproliferation is the main manifestation, and normal hematopoietic cells are poorly proliferated. Classification can be done by flow cytometry combined with clinical and bone marrow examination. This simple typing method has certain guiding significance for diagnosis and treatment.
The rate of missed diagnosis and misdiagnosis is high in the early stage of the disease. About half of the patients are misdiagnosed as aplastic anemia due to complete blood cell reduction, and secondly misdiagnosed as other proliferative anemia, or misdiagnosed as hepatitis and nephritis due to jaundice and abnormal urine. The key to timely diagnosis is: 1 think about the disease, and recognize the diversity of the clinical manifestations of the disease; 2 pay close attention to the appearance of hemoglobinuria, check the urine occult blood every day, for several days, sometimes help to find the naked eye is not easy Perceived hemoglobinuria; 3 have a correct judgment on the test results to determine PNH. The positive test 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. After a large number of blood transfusions, normal cells increase, and abnormal cells decrease relatively, which also affects the results. Therefore, the diagnosis cannot be denied because the result is negative once. It should be checked multiple times and multiple tests should be performed at the same time. In recent years, the application of specific antibodies and flow cytometry technology has the potential to detect some cases of early or progressive development of PNH, and can detect abnormal neutrophils, etc., thereby reducing the impact of blood transfusion. However, all these tests only indicate the presence of abnormal cells. Whether the main symptom is PNH or not, comprehensive analysis and close follow-up observation can be concluded, because a small number of PNH-like abnormalities may occur in other diseases such as myelodysplastic syndrome. Red blood cells, a small number of abnormal cells in the process of aplastic anemia may also be transient and not necessarily develop into PNH.
diagnosis
1. The reason that the aplastic anemia PNH is easily confused with it is that 47.3% of the cases also have a complete blood cell reduction. The main distinguishing point between the two is that aplastic anemia should be reduced by myeloproliferation, while PNH is active in myeloproliferation (especially the red line). If the myeloid hyperplasia is reduced and abnormal red blood cells similar to PNH can be detected, or those with PNH clinical and laboratory findings but low myeloproliferative, it should be suspected whether there is a disease conversion or a combination of two 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 with a slight increase in granulocytes or a small amount of granulocytes in peripheral blood. Some scholars even regard PNH as a kind of MDS. However, according to our observation, the pathological hematopoiesis or primordial increase of PNH is transient and can disappear. Very few patients can completely become MDS. On the other hand, some patients with MDS may also have abnormal blood cells similar to PNH, but their 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 examination 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, there are cyanosis of the hands and feet, and it will improve after warming; the hot and cold hemolysis test is positive.
complication
Common complications:
1. Patients infected with PNH are prone to various infections, especially respiratory and urinary tract infections, which in turn can induce hemoglobinuria. In China, serious infections are often the leading cause of death in PNH patients.
2. Thrombosis Thrombosis in different parts of the PNH cases in Europe and the United States accounted for 23% to 50%, is the main cause of death in PNH patients in these areas. In China, thrombosis is much less than in Europe and America. Reports from China, Thailand, and Japan are no more than 10%. The characteristics of domestic cases are that thrombosis occurs in a single site, with fewer multiples, more blood vessels in the shallow part, and fewer those involving important organs. Lower extremity venous thrombosis is the most common, followed by cerebral thrombosis, and very few portal vein or mesenteric thrombosis. According to Hillmen (1995), 39% of the 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 inferior vena cava. Spleen and renal vein embolization, 1/4 in the deep vein or superficial vein of the limb. In addition, 6 cases were myocardial infarction and 2 cases were cerebral infarction. In general, there are more vein embolizations than arteries, and more severe embolism involving 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 more symptoms due to asymptomatic cases.
4. Renal failure PNH patients with 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 that the kidney function of patients with this disease is gradually reduced. Infection or severe hemolysis can cause acute renal failure, but it can often 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 hemosiderin is deposited as 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, and severe cases can cause heart failure. Individual patients have severe bleeding such as cerebral hemorrhage and gastrointestinal bleeding. In addition, it is not uncommon to have secondary diabetes due to long-term use of adrenocortical hormone.
Transformation: About 20% of PNH patients are converted to aplastic anemia (Aplastic Anemia), most of which are converted to PNH over a period of time after or after recovery. In recent years, many patients with aplastic anemia have been converted to PNH by 10% to 31% after treatment with anti-lymphocyte (or thymocyte) globulin (ALG, ATG) or other immunotherapy. Recently, the method of detecting GPI-linked protein on the surface of blood cells has found that cells with PNH characteristics can be found in peripheral blood or bone marrow cells of about 30% of patients with aplastic anemia, suggesting that the possibility of conversion to PNH is obvious in patients with aplastic anemia. Transformation depends on the number of remaining normal hematopoietic cells and whether PNH clones can gain growth or survival advantages. A small percentage (about 5%) of PNH patients have been converted to aplastic anemia over time. Other patients have both PNH and aplastic anemia. These conditions are collectively referred to as PNH aplastic anemia syndrome. Of the 479 PNHs in different parts of the country, 79 (16.5%) belonged to this. In general, the conversion of aplastic anemia to PNH is more, and PNH is less likely to be aplastic anemia. In addition, individual PNH patients can be converted to leukemia, which is mainly acute myeloid leukemia.
treatment
(a) treatment
1. Radical cure is to rebuild normal hematopoietic tissue function and eliminate abnormal hematopoietic stem/progenitor cells. It is currently believed that bone marrow transplantation is the only way to cure this disease. However, PNH is a benign clonal disease, and some patients may still The more, the bone marrow transplantation carries a certain risk. Therefore, whether or not a bone marrow transplant is performed in a PNH patient requires consideration of various factors. As bone marrow transplantation technology matures, it is hoped to be a safe, effective and acceptable treatment for most people. Most of the patients who have undergone transplantation in recent years are patients with low myeloproliferative and recurrent severe vascular embolism. Most of the early reports did not properly pre-treat patients with the implantation of isogenic or allogeneic bone marrow, and most of the results were ineffective or relapsed. Recently, there have been many reports of successful pretreatment and allogeneic bone marrow transplantation. Saso et al analyzed 57 patients with PNH collected at the International Bone Marrow Transplant Registration Center from 1978 to 1995, and 48 patients received HLA-matched bone marrow from siblings. The disease-free survival rate after transplantation for 2 years was 56%; The bone marrow of the twin brothers survived for 8 or 10 years, respectively; 7 patients received bone marrow from other sources, and 1 patient survived for 5 years after transplantation. What is the mechanism for treating this disease? Araten DJ et al believe that the main aspects are as follows: 1 clearing PNH clones, 2 providing normal hematopoietic stem cells, 3 providing strong immunosuppression
Treatment (such as ATG, CsA, etc.), and, in PNH patients, the last point is more important, it is considered to relieve the inhibition of normal cells, so that PNH clones lost the proliferation advantage. Common pretreatment regimens are cyclophosphamide/TBI, oxyfluorene (melphalan)/CyclophosphamideWait. Because of the risk of bone marrow transplantation, from the survival curve, the survival rate of patients who have undergone bone marrow transplantation is lower than that of simple supportive care in the earliest stage, but after 6 years, the long-term survival chance of transplanted patients is greater.
Although bone marrow transplantation has achieved certain effects, it is risky and the source of the donor is difficult. Therefore, other workarounds need to be studied: 1 use their own hematopoietic stem/progenitor cells. In recent years, the techniques and methods for isolating early hematopoietic stem cells from peripheral blood have been continuously improved. The application of autologous hematopoietic stem cells and the research of purification technology have prompted us to envisage the isolation of hematopoietic stem cells from autologous peripheral blood of PNH patients and to remove abnormal hematopoietic stem cells by appropriate methods. Progenitor cells, amplifying normal hematopoietic stem/progenitor cells, and then returning, hope to be a low-risk method for the treatment of this disease. Our study by Xiao Juan et al. in 2000 showed that the autologous normal phenotype of CD34+ cells can be expanded 22.5 times under appropriate conditions for 7 days, and can maintain its multi-directional differentiation potential. However, as mentioned above, in addition to the small number of normal hematopoietic stem/progenitor cells in PNH, whether they are truly normal in terms of survival, proliferation and other biological characteristics remains to be further studied before they can be put into practice. In addition, it has been found that CD34 cells mobilized into peripheral blood by G-CSF are mainly CD59-, but not CD59. It is speculated that abnormal cells lack some GPI-linked adhesion factor, so they are more likely to leave the matrix and enter the periphery. blood. If this is the case, it will add a new problem to the implementation of this law. Prince GM et al. (1995) also used the idea of using PNH patients' own hematopoietic stem/progenitor cells to treat this disease. Stiff P et al. reported in 2000 that in vitro expansion of autologous bone marrow cells, reconstitution of blood function after high-dose chemotherapy, and successful treatment of one case of breast cancer, suggesting that the above ideas are possible. 2 with small transplant or non-myeloablative hematopoietic stem cell transplantation. To avoid transplant-related deaths, Suenaga et al. reported a case of PNH in 2001, using Cladribine,Busulfan(Maliland), rabbit ATG for pretreatment, and then given HLA-matched brothers' peripheral blood hematopoietic stem cells, with cyclosporine A to prevent graft-versus-host disease, no obvious toxicity, after 14 days of transplantation, donor cells accounted for 90% to 100% % and maintain this level, observed no recurrence for 6 months. If non-myeloablative hematopoietic stem cell transplantation can be used, it is ideal because: 1 the risk of pre-transplantation pretreatment is small; 2 the immunosuppressive agents for the treatment of aplastic anemia are applied before and after transplantation to solve the pathogenic factors of immune disorders; For PNH, it is not necessary to completely eliminate abnormal cells, because according to the observation of patients with complete clinical remission, there are still 15% of abnormal cells in the peripheral blood, but no disease manifestations. Of course, the use of non-myeloablative hematopoiesis for cell transplantation requires more cases and longer observations.
2. Immunosuppressive therapy according to foreign and domestic experience, alone or in combination with anti-thymocyte globulin, anti-lymphocyte globulin, cyclosporine A and other immunosuppressive agents, may have a certain effect on patients with myeloproliferative disorders It is ineffective or less effective for PNH, which is mainly hemolysis. However, the use of the aforementioned immunosuppressive agents is reasonable based on the assumption of dual pathogenesis. South Korea Cho SG et al reported in 2001 that one patient with PNH was pretreated with high-dose cyclophosphamide, and then hematopoietic stem cells were given. The results relapsed 12 months after remission. The authors believe that anti-lymphocyte globulin should be added at the beginning. In addition, PNH clones were temporarily reduced after treatment with anti-thymocyte globulin. It is thought that anti-thymocyte globulin may activate the classical pathway of complement, which causes hemolysis of PNH cells and leads to a decrease in PNH clones.
3. Ways to reduce the onset of hemolysis should pay attention to avoid the causes of hemorrhagic episodes such as colds, certain drugs, etc. The most common treatment for hemolysis that has occurred is to useAdrenocorticotropic hormoneTo reduce or alleviate the hemoglobinuria that is occurring. Available at the beginningPrednisone30 ~ 40mg, after the onset of the cessation of the dose, and then gradually continue to reduce the amount until the minimum amount. Many patients do not need to maintain the amount, if prednisone is used as maintenance therapy, the minimum amount is applied. In addition, vitamin E is usually used, 300mg/d, divided into 3 times, but the effect is not certain. Some people in China have tried anti-solvent (Arbutus root bark), ligustrazine (sodium ferulate) anisodamine (654-2), and procaine intravenous drip. Some people in foreign countries have tried dapsone and metronidazole. However, most of them do not have sufficient theoretical basis and multiple verifications, and there is no recognized efficacy. In our hospital, PNH patients with severe hemolysis and persistent bone marrow hyperplasia have been given small doses of chlorambucil (tumorine) while frequently transducing red blood cells (the former Soviet Union used cyclophosphamide, others) With 巯嘌呤), it is envisaged that the abnormal hematopoietic cells can be reduced, and some patients are effective; a very small dose of chloroquine has also been tried, but the number of trials is very small, and it is difficult to confirm the effect. Some people have tried to use combined chemotherapy to remove abnormal hematopoietic cells. As a result, the condition can be improved for a period of time. However, abnormal cloning cannot be completely eliminated, and recurrence is still inevitable. At the same time, because chemotherapy has an effect on normal hematopoietic cells, it can cause severe bone marrow suppression. Fatal infections should be taken with caution. At present, new drugs for inhibiting complement activation are also being studied. It is pointed out that one of the mechanisms of action of adrenocortical hormone is also inhibition of complement. It has also been mentioned that heparin can inhibit both complement and thrombus, and has a double-edged effect. However, it is necessary to further confirm its true effect. Others have used cell engineering to transfer CD59 on normal cells to abnormal cells lacking CD59, which can correct their sensitivity to complement, but after all, this can only have a temporary effect, and the new abnormal cells still have no CD59.
4. Treatment of anemia for the diagnosis of myeloproliferationStetanol(Kanglilong), testosterone propionate (testosterone propionate), danazol and so on. If there is laboratory evidence of iron deficiency, a small amount of iron can be given (1/5 to 1/10 of the normal dose, a large amount can induce hemoglobinuria). Those who lack folic acid should be supplemented. Severe or rapidly developing anemia can be red blood cells or red blood cells washed with saline to avoid the onset of hemoglobinuria. In recent years, some people have tried high-dose erythropoietin (500 U/kg body weight, 3 times a week). It is said that some patients are effective after half a year of treatment, but there are not many cases, and there is no certain conclusion.
5. Gene therapy uses a retroviral vector to transfer a gene encoding a transmembrane CD59 into PNH pathological cells. As a result, transmembrane CD59 can be expressed, which can replace the missing CD59 that needs GPI to attach to the membrane, so that the cells are alleviated. The sensitivity of complement. Nishimura J et al. reported in 2001 that retroviral-based vectors can efficiently and stably transfer PIG-A-containing genes into a variety of cell lines derived from PNH patients that lack the PIG-A gene and a single nucleus of peripheral blood and bone marrow. The cells are allowed to restore the expression of GPI connexin and can also be transferred to CD34 cells in peripheral blood. It is suggested that it is possible to restore pathological cells by gene therapy.
6. Treatment of complications, vascular embolism, acute renal failure, etc. should be treated accordingly.
(two) prognosis
The disease is a benign chronic disease. Most patients have moderate to severe anemia for a long time, but half of them can still engage in daily activities or participate in appropriate work. About 10% of patients get relief or achieve recovery after repeated episodes. Most of them are considered natural relief because they cannot be attributed to special treatment. These patients did not have any PNH performance, no clinical symptoms, and the complement sensitivity test was negative. A few of the patients in the test were still able to find a small amount of PNH abnormal cells by flow cytometry. PNH itself is rarely fatal, mainly due to complications, the first infection in China, followed by vascular embolism, and a small number of deaths from anemia, cerebral hemorrhage. In Europe and the United States, the first cause of death is venous thrombosis of important organs, followed by central nervous system or gastrointestinal bleeding; another 15% of patients died of myocardial infarction or cerebrovascular accident, which is considered to be unrelated to this disease. In the past, PNH was also classified as pre-leukemia, and in fact there was very little conversion from PNH to acute leukemia. Only 25% of the 160 cases reported in Japan, Hillmen (1995) reported that none of the 80 cases of long-term follow-up were converted to leukemia, and none of the 182 cases of long-term follow-up in our hospital were converted to acute leukemia. Therefore, although there have been reports of conversion from PNH to leukemia, the overall chances are small. In fact, PNH should not be classified as “pre-leukemia”. According to the results of long-term follow-up: 80 patients in the UK had an estimated median survival of 10 years, 28% survived 25 years after diagnosis, and 34% of those who survived for more than 10 years were finally relieved; 182 patients were followed up for a long time in our hospital. The estimated median survival period is 15 years, and the survival rate is still more than 50% after 25 to 30 years of diagnosis; the situation in Japan is similar. Thus, PNH actually belongs to long-term chronic benign diseases. According to the domestic PNH report, about one-fourth of the patients died during the course of the disease, mainly due to infection, anemia, central nervous system bleeding, and excessive infection, further reducing the deaths caused by complications, strengthening the symptomatic and Supporting treatment and improving the quality of life may lead to long-term survival.
About 80% of domestic PNH is converted from aplastic anemia.
PNH combined with pregnancy is prone to worsening the condition and fetal death, miscarriage, etc. Most foreign countries advocate early termination of pregnancy. However, according to our own experience, pregnancy can be continued with caution, not necessarily failure of pregnancy or significant impact on patients.
prevention
The pathogenesis of paroxysmal nocturnal hemoglobinuria is not fully understood, so it is not known how to prevent the occurrence of this disease. However, it has been clarified that some factors can induce or aggravate hemoglobinuria, so patients should pay attention to avoid infection, especially upper respiratory tract infections; avoid excessive fatigue or mental stress; avoid drug abuse. Because the disease is imaginary, it is necessary to reconcile the spirit and nurse the body. "It is right in the air, and the evil can't be done." You can practice qigong and tai chi to improve your resistance.
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