Objective To establish a rapid, accurate and low-cost screening method for the detection of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN).Methods Seventy cases diagnosed with MPN were collected from 2012 to 2016. PCR combined with high resolution melting (HRM) analysis were used to screen the CALR mutations, and Sanger sequencing and T-A sequencing were applied to verify the HRM positive samples. CALR wild type DNA, type 1 and type 2 mutant DNA samples were selected and analyzed 4 times/day for 5 days to detected the CVs of Tm (melting temperature) respectively. JAK2 mutations were also analyzed in MPN patients to compare the association between JAK2 and CALR mutations.Results PCR-HRM analysis showed 7 cases (26.9%) and 5 cases (20.8%) patients with CALR mutations were screened out from 26 essential thrombocythaemia (ET) cases and 24 primary myelofibrosis (PMF) cases, but no CALR mutations were found in cases with polycythaemia vera (PV). All mutations were confirmed by direct sequencing or cloning sequencing. The CVs for HRM analysis of CALR wild type DNA, type 1 and type 2 mutant DNA samples were 1.91%,1.59% and 1.43%, respectively.There were 47 cases with JAK2 V617F and 1 case with exon12 mutation. No coexistence of JAK2 mutation and CALR mutations were found in a single sample.Conclusion PCR-HRM can be used for rapid screening of CALR mutation. Subsequent sequencing can be applied for rapid diagnosis of MPN patients in clinical practice.

Objective To compare the efficacy and safety of vitrectomy combined with internal limiting membrane flap and vitrectomy combined with internal limiting membrane peeling for the treatment of idiopathic macular hole with different sizes.Methods A total of 127 consective patients (127 eyes)were divided into two groups according to the size of the hole diameter of the smallest split points by less than or equal to 500 μm (small diameter macular hole group) and more than 500 μm (huge diameter macular hole group).According to different surgical methods the patients were divided into non ILM flap coverage group (peeling 1 group and peeling 2 group) and ILM flap cover group (covering 1 group and covering 2 group).All the patients underwent vitrectomy combined with internal limiting membrane peeling or vitrectomy combined with limiting membrane flap.Preoperative and postoperative best correct visual acuity,closure ratio of macular hole and postoperative major complications were observed and followed up.Results The postoperative best correct visual acuity improved in all the groups,there was no significance difference between small diameter macular hole group and huge diameter macuiar hole group (t =0.112 2,0.750 8;all P ＞0.05).The closure ratio of peeling 1 group and covering 2 group at postoperative 6 months were all 100％,there was no statistical difference (P ＞ 0.05),which in peeling 2 group and covering 2 group were 84.85％ and 100.00％,there was statistical difference (x2 =13.292,P ＜ 0.05).There was no statistical difference in preoperative defect diameter of the inner and outer junction between peeling 2 group and covering 2 groups (P ＞0.05),there was also no statistical difference between peeling 2 group and covering 2 groups at postoperative 1 months (P ＞ 0.05),but there were statistical differences at postoperative 3 months,6 months and 12 months (all P ＜ 0.05),the covering 2 group were less than the peeling 2 group.Conclusion ILM flap coverage helps to heal macular holes greater than 500 μm diameter,and has no extra effect on healing of diameter less than 500 μm.

Objective To explore the characteristics and surgical managements of recurrent retinal detachment in silicone oil tamponade eyes.Methods The records of consecutive series of 134 patients (134 eyes) with recurrent retinal detachment in silicone oil tamponade eyes from January 2012 to December 2015 in our hospital were reviewed retrospectively,the vitrectomy combined with silicone oil replacement or scleral buckling procedure were performed.The follow-up time was 6 months,the surgical efficient was evaluated.Results 101 eyes underwent vitrectomy combined with silicone oil replacement.Retina was completely reattached in 79 eyes,and vitrectomy was reperformed in the left 22 eyes,the successful rate was 78.2％ (79/101);The visual acuity improved in 16 eyes,unchanged in 56 eyes,decreased in 29 eyes;The intraocular pressure of 31 eyes increased more than 25 mmHg (1 kPa =7.5 mmHg).33 eyes underwent scleral buckling procedure.Retina was completely reattached in 23 eyes,and vitrectomy combined with silicone oil replacement was performed in the left l0 eyes,the successful rate was 69.7％ (23/33);The visual acuity improved in 5 eyes,unchanged in 16 eyes,decreased in 12 eyes;The intraocular pressure of 14 eyes increased more than 25 mmHg.Conelusion For limited retinal detachment caused by inferior or peripheral holes,proliferative vitreoretinopathy in the A or B-class,the refractive medium does not affect the fundus examination,scleral buckling surgery is preferred;For the hole in the posterior pole or extensive retinal detachment caused by giant retinal holes,proliferation or retinal fixed fold formation,vitrectomy combined with silicone oil replacement is a better option.

Objective:To observe the efficacy of platelet-rich fibrin (PRF) membrane tamponade combined with air filling for giant macular hole (MH).Methods:A prospective case-control study. From January 2019 to February 2021, 56 patients (56 eyes) diagnosed with giant MH from Eye Center of Renmin Hospital of Wuhan University were enrolled. Among them, there were 17 males with 17 eyes and 39 females with 39 eyes. The average age of the patients was 64.23±9.30 years old. The average MH minimum diameter was 827.36±83.16 μm. The best corrected visual acuity (BCVA) and optical coherence tomography angiography (OCTA) examination were performed before surgery. The Chinese version of 25-item National Eye Institute visual functioning questionnaire (NEI VFQ-25) was used to investigate patient's visual-related quality of life. There were 28 eyes of 28 cases receiving PRF membrane covering, as PRF group, another 28 eyes of 28 cases receiving inverted internal limiting membrane (ILM) insertion into giant MH, as ILM group. The differences of the age ( t=-1.588), sex ratio ( χ2=0.760), BCVA ( Z=-0.400), macular hole minimum diameter ( t=-0.604), choriocapillary blood flow area (CBFA) ( t=1.331) and NEI VFQ-25 score ( t=0.921) were not statistically significant ( P>0.05). All eyes underwent 23G minimally invasive vitrectomy. In the PRF group, PRF membrane was used to fill the hole, and in the ILM group, the hole was filled with ILM inversion, and filled with sterile air after full gas-liquid exchange. The follow-up time after surgery was ≥6 months. The same equipment and methods as before surgery were used to conduct related examinations, and the changes of BCVA, the shape of hole closure, CBFA and the improvement of vision-related quality of life were compared between the two groups. For comparison between groups, independent samples t-test was used for data with normal distribution, and Mann-Whitney U test was used for data with non-normal distribution. For intra-group comparisons, paired-samples t-test was used for data with normal distribution, and Wilcoxon rank-sum test was used for non-normally distributed data. Results:Six months after surgery, in the eyes of PRF group and ILM group, the hole of 27 (96.4%, 27/28) and 26 (92.6%, 26/28) eyes were closed; the median BCVA was 0.70 and 0.70, respectively; CBFA were 1.99±0.20 and 1.91±0.18 mm 2; NEI VFQ-25 scores were 81.36±12.39 and 78.39±10.12, respectively. Compared with before surgery, the BCVA ( Z=-4.636,-4.550) and CBFA ( t=-27.115,-31.135) of the affected eyes in the PRF group and ILM group were significantly improved after surgery, and the NEI VFQ-25 scores ( t=-15.557, -10.675) was significantly increased, and the difference was statistically significant ( P<0.05). There was no significant difference in BCVA ( Z=-0.167), CBFA ( t=1.554), and NEI VFQ-25 scores ( t=0.980) between the two groups after interocular surgery ( P=0.726, 0.126, 0.331). Conclusion:PRF membrane insertion with air filling has the same efficacy as ILM insertion in the treatment of giant MH, which can improve the closure rate of MH, patients' vision and vision-related quality of life, and increase choroidal blood perfusion.

Objective:To observe and explore the feasibility and effectiveness of platelet-rich fibrin (PRF) membrane packing and air filling in the treatment of refractory macular holes.Methods:A retrospective clinical study. From January 2019 to January 2020, 17 patients with refractory macular hole (17 eyes) who diagnosed in Renmin Hospital of Wuhan University were included in the study. Among them, there were 7 males (7 eyes) and 10 females (10 eyes), with the age of 55.18±7.91 years. All eyes underwent 23G minimally invasive vitrectomy combined with internal limiting membrane stripping and PRF packing, and air filling was performed at the end of the operation. The best corrected visual acuity (BCVA) and optical coherence tomography angiography were performed in all eyes before surgery and at 1 week and 1, 3 months after surgery. The BCVA examination was performed using a international standard logarithmic visual acuity chart, which was converted into logarithm of the minimum angle of resolution visual acuity during statistics. Taking 3 months after surgery was as the time point to judge the efficacy, the changes of BCVA, superficial retinal vascular density (SVD), foveal avascular zone (FAZ) area and central foveal thickness (CFT) before and after surgery were compared. Paired t-test was used to compare the indicators before and after surgery. Results:Among the 17 eyes, there were 6, 7, and 4 eyes with giant macular hole, high myopia macular hole, and recurrent macular hole, respectively; the hole diameter was 723.94±38.30 μm. Three months after surgery, all holes were closed. Compared with before surgery, the BCVA ( t=4.458) and SVD ( t=2.675) increased, and the CFT ( t=6.329) and FAZ area ( t=4.258) decreased at 3 months after surgery, and the differences were statistically significant ( P<0.05). At the last follow-up, there was no complications such as intraocular hypertension and retinal detachment in all eyes. Conclusion:Minimally invasive vitrectomy combined with internal limiting membrane stripping and PRF tamponade in the treatment of refractory macular holes can increase the closure rate, improve visual acuity and retinal blood perfusion.

Objective To establish and verify the microscopic review rules of Dirui GMD-S600 gynecologi-cal secretion analysis system.Methods A total of 2 619 samples of vaginal secretions were collected and test-ed for tangible components and dry chemistry items using the Dirui GMD-S600 gynecological secretions analy-sis system.All samples were subjected to manual microscope examination using a double blind method,and the results of instrument testing,manual image review of instrument testing,and manual microscope examina-tion were recorded separately.Using the results of manual microscope examination as the standard,the false negative rates and the false positive rates of instrument testing results and tangible component results after manual image review of instrument testing were analyzed.At the same time,the false negative rates and the false positive rates of instrument detection dry chemistry items and their corresponding tangible components were analyzed,and the review rules were derived based on this.Subsequently,344 samples were selected to verify the effectiveness of the review rules.Results The false positive rates of white blood cells,fungi,trichomonas and clue cells detected by the instrument were 28.77%,14.71%,20.09%and 2.30%,and the false negative rates were 0.28%,15.06%,4.35%and 10.34%.After manual image review and correction,the false positive rates of white blood cells,fungi,trichomonas and clue cells were 28.77%,7.06%,8.67%,0.66%,and the false negative rates were 0.28%,3.35%,4.35%and 10.34%.The false positive rates of white blood cell esterase(LE)for white blood cells,N-acetylhexylglucosidase(NAG)for fungi,NAG for trichomonas,hydrogen peroxide(H2O2)for bacteria,lactic acid(LA)for bacteria,sialidase(SNA)for clue cells,and proline aminopeptidase(PIP)for clue cells were 13.83%,22.47%,24.10%,95.52%,2.52%,12.92%and 5.54%,and the false negative rates were 28.17%,48.12%,0.00%,23.55%,78.82%,0.00%and 74.14%.The false positive rate of 260 validation samples was 1.68%,the false negative rate was 4.22%,and the review rate was 45.38%.Conclusion The review rules of the Dirui GMD-S600 gynecological secretion analysis system are as follows(1)All specimens need to be reviewed with images,and the following rules are based on this rule.(2)When the result of white blood cell shows positive,then the microscopic exam-ination is required.(3)When the result of fungi shows positivity,then the microscopic examination is re-quired.(4)When the result of trichomonas shows positive,then the microscopic examination is required.(5)When the result of SNA does not match the positive result of clue cells,then the microscopic examination is required.

Objective To investigate the clinical significance of plasma methylated Septin 9 (SEPT9)gene test for colorectal cancer(CRC).Methods Clinical data of this retrospective study were obtained from Huashan Hospital of Fudan University(2016-2017).The subjects were divided into three groups,84 patients in CRC group,50 patients with adenoma in precancerous group,and 20 cases as healthy controls.A fluorescent PCR assay was used to analyze SEPT 9 methylation in DNA extracted from plasma. Chi square test was used for statistical analysis.Results The positive incidence of SEPT9 gene methylation in plasma was 63.1%(53/84)in CRC group,significantly higher than 10%(5/50)in precancerous group (χ2=35.993, P<0.001), and undetectable in healthy group.The sensitivity of the methylated SEPT9 gene test was 63.1%(53/84), and the sensitivity of a joint detection combined with carcinoembryonic antigen(CEA)was 75%(63/84).The receiver operating characteristic curve(ROC)showed that methylated SEPT9 gene test had 0.828 in the area under the curve(AUC),higher than 0.795 in the AUC of CEA test.In CRC patients,51.4%(19/37)in the stage Ⅰ-Ⅱand 72.3%(34/47)in the stage Ⅲ-Ⅳ were positive for methylated SEPT9 gene test(χ2=3.917, P<0.05).There were no significant differences in gender,age and primary tumor site.Conclusion The SEPT9 gene methylation in plasma is helpful for early screening for CRC,and is associated with CRC progression.