Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disease. Its pathogenesis is mainly due to the mutation of PIG-A gene on somatic X chromosome, which leads to the disorder of glycosylated phosphatidylinositol (GPI) anchor synthesis on the surface of blood cell membrane and the loss of anchor connection protein. Traditional diagnostic methods mainly include sucrose hemolysis test, acid hemolysis test and Routh test, but these methods have poor sensitivity and specificity. In recent years, the detection of CD55 and CD59 by flow cytometry has become a routine method for the diagnosis of PNH, especially CD59 is more sensitive than CD55, and is considered as a better index for the diagnosis of PNH. The fluorescently labeled variant of inactivated Aeromonas hydrophila lysin precursor (FLAER) can specifically bind to GPI ankyrin, and its specificity and sensitivity are superior to the traditional methods of flow cytometry. PNH, aplastic anemia (AA) and myelodysplastic syndrome (MDS) are all bone marrow failure diseases with similar pathogenesis and clinical manifestations, so it is often difficult to make early differential diagnosis. In this paper, FLAER, CD45, CD 15, CD24 were combined to detect granulocyte PNH clone, and CD59 was combined to detect erythrocyte PNH clone, which effectively improved the sensitivity and specificity of PNH clone detection and contributed to the early differential diagnosis of bone marrow failure diseases. The report is as follows.

1 data and methods

1. 1 General data All cases came from 48 consecutive inpatients in hematology department of our hospital. Among them, there were 9 patients with PNH and 3 patients with AA/KLOC-0. The diagnosis met the criteria of diagnosis and curative effect of hematological diseases. There were 1 1 MDS patients, which met the diagnostic classification standard of WHO in 2008. The control group 15 cases were all megaloblastic anemia patients.

1.2 instrument and reagent FACSAria flow cytometer, hemolysin, phosphate buffer (PBS) and reagents CD59, CD45, CD 15 and CD24 were all purchased from BD company, and FLAER reagent was purchased from Protox Biotechnology Company in Canada.

1.3 method

1.3. 1 Detection of peripheral red blood cell CD59 Take 100μL EDTA anticoagulated whole blood, wash it with PBS, dilute it with 1.5ml PBS, take 100μL and add 20μL CD59-PE monoclonal antibody, at 4℃. Wash twice with 2mL PBS solution, resuspend in 1mL PBS solution, and detect by computer. The expression rate of CD59 cells was detected by flow cytometry.

1.3.2 FlAER detection of peripheral blood granulocytes Take100μ l of EDTA anticoagulated whole blood, add 20μL of antibodies CD45, CD 15, CD24 and FLAER respectively, incubate in the dark for 30min, then add 2mL of hemolysin, and keep it in the dark at room temperature for 10min, after hemolysis is completed. The supernatant was discarded, washed twice with 2mL PBS solution, resuspended in 500μL PBS solution for liquid machine detection, and the expression rate of FLAER was determined by flow cytometry.

SPSS 17.0 software was used for data analysis, and the measurement data were expressed in X S. T test and Mann-Whitney U test were used for comparison between groups, P

Two results

2. 1 clinical features 9 patients with PNH, including 6 males and 3 females, aged 16 ~ 60 years, with a median age of 30 years. There were 13 AA patients, including 5 males and 8 females, aged from 7 to 63, with a median age of 38. MDS patients 1 1, male 5, female 6, aged 4 1 ~ 72 years, with a median age of 53 years, including RCMD 1, RCUD 6, RAEB-I 2, RAEB-II 1, 5q syndrome 65. Control group 15 cases, including 6 males and 9 females, aged from19 to 61year, with a median age of 34 years.

2.2 Compared with different detection methods for PNH patients, the deletion rates of FLAER and CD59 in PNH patients were (70.07 28.77)% and (38.5 1.29. 1.05)%, respectively, which were significantly higher than those in control group, AA group and MDS group (P

2.3 Comparison of different methods for detecting non-PNH patients The average deletion rate of FLAER in each group was higher than that of CD59, but the difference was not statistically significant (P & gt0.05), indicating that there was no significant difference between FLAER and CD59 in non-PNH groups. The deletion rate of FLAER in control group was 0.0%, and the specificity was 100%. CD59 was deleted in 3 cases, and the deletion rate was less than 65438 0%. In 13 AA patients, FLAER deletion was detected in 5 cases, CD59 deletion was detected in 4 cases, and 1 case was not detected. In 1 1 MDS patients, there were 3 cases with FLAER deletion, including 2 cases with CD59 deletion, and the CD59 deletion rate in 1 patients was 0. The results showed that FLAER was more sensitive and specific than CD59.

3 discussion

Up to now, it has been found that there are more than 20 kinds of protein expression deletions on the surface of blood cells in patients with PNH, among which the deletion of decay accelerating factor (CD55) and reactive hemolysis membrane inhibitory factor (CD59) on erythrocyte membrane is considered as the main reason for the pathophysiology of PNH. Red blood cells are the best target cells for sensitivity detection because of their large number and strong antibody expression. Especially in some neutropenia diseases, such as AA and MDS, red blood cell detection is particularly important. At the same time, by comparing the values of red blood cells and white blood cells, more information can be provided for clinic. The expression of CD55 on red blood cells is low, while the fluorescent staining of CD59 is strong and uniform, which has become the "gold standard" for the diagnosis of PNH in recent years. However, more and more studies have found that detecting the expression of CD59 in red blood cells has certain limitations in the diagnosis of PNH.

This study found that among the two patients in PNH group, the deletion rate of CD59 was 5.5% in one patient and 2.9% in the other, while the deletion rate of FLAER was 23.2% and 47.7% respectively. It may be because the patient was in the active stage of the disease and received blood transfusion treatment, which affected the expression of CD59 in red blood cells and led to the false positive expression of CD59. Studies have shown that in small cell hypopigmentation anemia, the expression of CD59 on the surface of patients' red blood cells is decreased, but the granulocytes are normal. If only CD59 on red blood cells is detected, its expression will be false negative. In addition, during the aging process of normal cells, the surface molecule CD59 may be lost spontaneously, which leads to the deviation between the detection value and the fact. With the continuous development of science and technology, people have developed FLAER technology. FLAER is a variant of Alexa-488-labeled inactive Aeromonas hydrophila lysin precursor, which can specifically bind to GPI anchor chain protein and express it on all granulocytes with GPI anchor chain protein, and will not cause errors due to different numbers and types of GPI anchor chain proteins expressed by different cells. Because FLAER can directly detect GPI protein, it is helpful to distinguish real PNH from immune cytopenia and real GPI negative cells. In this study, the deletion rate of FLAER in the control group was 0, and the detection results were more specific than CD59. In PNH group, the deletion rate of FLAER was significantly higher than that of CD59, and the sensitivity was higher. FLAER is also the best antibody to prevent contaminated cells from appearing in the door. If a small number of cells with negative expression of CD24 are contaminated in the door, they may be mistaken for small PNH clones. The combination of FLAER and CD24 can improve the accuracy of PNH detection, and CD24/FLAER double negative cell population is the real PNH clone cell population. FLAER multi-parameter detection requires that peripheral blood samples must be sent for inspection in time after collection, otherwise the survival rate of granulocytes will decrease and the nonspecific staining of cells will increase, which will affect the detection effect. This affects the clinical application of FLAER to some extent. The detection of PNH clone is of great significance to the clinical manifestations, treatment and prognosis of MDS and aplastic anemia. Some AA patients with PNH clones have better immunotherapy effect, even if the clone is less than 0. 1%, it will affect the therapeutic effect.

For AA patients with a small number of PNH clones, it is necessary to monitor the changes of PNH clones, because patients may have hemolytic anemia. In this study, the relationship between PNH and MDS is limited to low-risk MDS, which is characterized by cytopenia and low myeloproliferation. The incidence of genetic abnormalities is low, and the clinical process is lazy, which is more manifested as bone marrow failure. The deletion rate of FLAER in AA group and MDS group was higher than that in CD59. Because it is difficult to distinguish AA from low proliferative MDS, so far, there is no report about MDS patients progressing to PNH. Monitoring PNH clone has certain significance for the differential diagnosis of these two diseases. PNH clones detected in AA and MDS patients are usually very small, and CD59 detection is not easy to detect. Both groups were 1, and the deletion rate of CD59 was 0, while the expression of FLAER was missing, with the deletion rates of 4.2% and 2.9% respectively. FLAER detection is more sensitive than CD59. AA, hypoplastic MDS and PNH are all bone marrow failure diseases, and their pathogenesis, clinical manifestations and biological characteristics are similar, so it is difficult to distinguish them. FLAER detection is more sensitive and specific than CD59. It can provide a more sensitive and specific basis for PNH cloning in the early stage. For suspected PNH patients with atypical clinical symptoms and unclear diagnosis, FLAER detection is helpful for early diagnosis or exclusion. Therefore, the high-sensitivity detection of PNH clones by FLAER, the diagnosis and differentiation of these three diseases, is of certain significance for understanding the clinical process, prognosis and outcome.

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