Objective:To analyze changes of donor corneal endothelial cell density (ECD) and morphology from eye bank before and after keratolasty and the influencing factors.Methods:An observational case series study was performed.A total of 118 donor corneas, retrieved by the Shandong Province Eye Bank between July 2020 and June 2021 for penetrating keratoplasty (PKP) and endothelial keratoplasty (EK) were included.Among them, 99 corneas (83.90%) were used for PKP, and 19(16.10%) for EK.The basic information of donors and the results of corneal quality tests were analyzed and compared with ECD measured by endothelial microscopy one month after keratolasty.Morphological changes in endothelial cells before and after surgery were observed, and factors influencing corneal ECD and morphology were analyzed.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Shandong Eye Hospital (No.SDSYKYY20170319).Written informed consent was obtained from each subject.Results:Among the 99 donor corneas for PKP, there were statistically significant differences in preoperative donor corneal ECD and 1-month postoperative ECD of implant among different age groups ( F=18.136, 5.936; both P<0.01), which were lower in the 31-60-year-old group and the >60-year-old group than in the 0-30-year-old group and higher in the 31-60-year-old group than in the >60-year-old group, with statistically significant differences (all P<0.01).There were statistically significant differences in the preoperative donor corneal ECD among different donor cause of death groups ( F=4.524, P<0.01), which was higher in the traumatic accident group compared to the cardiovascular and cerebrovascular disease group, chronic organ failure group and malignant tumor group (all P<0.01).The preoperative donor ECD in the death-tissue retrieval time ≤6 hours group was (2 577.66±284.63)cells/mm 2, which was higher than (2 372.46±399.75)cells/mm 2 in the death-tissue retrieval time >6 hours group, with a statistically significant difference ( t=2.289, P<0.05).There were statistically significant differences in 1-month postoperative ECD among the preservation-surgery time ≤3 days, 3-6 days, and >6 days groups ( F=6.201, P<0.01), with higher ECD in preservation-surgery time ≤3 days groups than in 3-6 days and >6 days groups (both P<0.01).The preoperative donor corneal ECD applied to EK was significantly higher than that applied to PKP ( t=-2.660, P<0.01).ECD at 1 month after surgery applied to PKP was significantly higher than that applied to EK ( t=4.286, P<0.01).The ECD reduction rate was 7.14% (0.01%, 17.69%) and 31.07% (22.11%, 45.86%) in PKP group and EK group, respectively, with a statistically significant difference ( Z=4.969, P<0.01).The ECD was lower in the group with dark area than in the non-dark area group before PKP, with a statistically significant difference ( t=6.789, P=0.011).There was no significant difference in ECD at 1 month after keratoplasty between the two groups ( t=0.005, P=0.945).Multivariate logistic regression model results showed that preservation-surgery time >6 days and the cause of donor death being malignant tumor were risk factors for the appearance of dark areas in donor corneal endothelium ( OR=9.038, P=0.030; OR=6.577, P=0.018). Conclusions:The older the donor, the lower the ECD.Prolonged preservation-surgery time (>6 days) is the main factor contributing to the decline in ECD after keratolasty.Compared to PKP, there is a higher endothelial cell loss after EK.The tissue preservation-surgery time >6 days and the cause of donor death being malignant tumor are the main risk factors affecting the appearance of dark areas in the donor corneal endothelium.But the presence of physiological dark areas does not significantly influence the ECD after surgery.

Objective:To analyze changes of donor corneal endothelial cell density (ECD) and morphology from eye bank before and after keratolasty and the influencing factors.Methods:An observational case series study was performed.A total of 118 donor corneas, retrieved by the Shandong Province Eye Bank between July 2020 and June 2021 for penetrating keratoplasty (PKP) and endothelial keratoplasty (EK) were included.Among them, 99 corneas (83.90%) were used for PKP, and 19(16.10%) for EK.The basic information of donors and the results of corneal quality tests were analyzed and compared with ECD measured by endothelial microscopy one month after keratolasty.Morphological changes in endothelial cells before and after surgery were observed, and factors influencing corneal ECD and morphology were analyzed.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Shandong Eye Hospital (No.SDSYKYY20170319).Written informed consent was obtained from each subject.Results:Among the 99 donor corneas for PKP, there were statistically significant differences in preoperative donor corneal ECD and 1-month postoperative ECD of implant among different age groups ( F=18.136, 5.936; both P<0.01), which were lower in the 31-60-year-old group and the >60-year-old group than in the 0-30-year-old group and higher in the 31-60-year-old group than in the >60-year-old group, with statistically significant differences (all P<0.01).There were statistically significant differences in the preoperative donor corneal ECD among different donor cause of death groups ( F=4.524, P<0.01), which was higher in the traumatic accident group compared to the cardiovascular and cerebrovascular disease group, chronic organ failure group and malignant tumor group (all P<0.01).The preoperative donor ECD in the death-tissue retrieval time ≤6 hours group was (2 577.66±284.63)cells/mm 2, which was higher than (2 372.46±399.75)cells/mm 2 in the death-tissue retrieval time >6 hours group, with a statistically significant difference ( t=2.289, P<0.05).There were statistically significant differences in 1-month postoperative ECD among the preservation-surgery time ≤3 days, 3-6 days, and >6 days groups ( F=6.201, P<0.01), with higher ECD in preservation-surgery time ≤3 days groups than in 3-6 days and >6 days groups (both P<0.01).The preoperative donor corneal ECD applied to EK was significantly higher than that applied to PKP ( t=-2.660, P<0.01).ECD at 1 month after surgery applied to PKP was significantly higher than that applied to EK ( t=4.286, P<0.01).The ECD reduction rate was 7.14% (0.01%, 17.69%) and 31.07% (22.11%, 45.86%) in PKP group and EK group, respectively, with a statistically significant difference ( Z=4.969, P<0.01).The ECD was lower in the group with dark area than in the non-dark area group before PKP, with a statistically significant difference ( t=6.789, P=0.011).There was no significant difference in ECD at 1 month after keratoplasty between the two groups ( t=0.005, P=0.945).Multivariate logistic regression model results showed that preservation-surgery time >6 days and the cause of donor death being malignant tumor were risk factors for the appearance of dark areas in donor corneal endothelium ( OR=9.038, P=0.030; OR=6.577, P=0.018). Conclusions:The older the donor, the lower the ECD.Prolonged preservation-surgery time (>6 days) is the main factor contributing to the decline in ECD after keratolasty.Compared to PKP, there is a higher endothelial cell loss after EK.The tissue preservation-surgery time >6 days and the cause of donor death being malignant tumor are the main risk factors affecting the appearance of dark areas in the donor corneal endothelium.But the presence of physiological dark areas does not significantly influence the ECD after surgery.

Objective:To investigate the role of the NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome and its downstream interleukin(IL)-1β, IL-6, and IL-18 in the pathogenesis of antineutrophil cytoplasmic antibody-associated vasculitis(AAV) in children.Methods:A retrospective study was conducted.Specifically, the localization and expression of the NLRP3 inflammasome in renal tissues of 22 children who were diagnosed with primary AAV and underwent renal biopsy in the Department of Pediatric Nephrology and Rheumatology, the First Affiliated Hospital of Sun Yat-Sen University from September 2003 to September 2020 were detected by the immunohistochemical method.The IL-1β, IL-6 and IL-18 levels in serum and urine were measured by enzyme-linked immunosorbent assay.The measurement data conforming to normal distribution were compared by the t test between two groups and by the single factor ANOVA test among multiple groups.The measurement data that did not conform to normal distribution were compared by the Wilcoxon signed rank sum test.Classification variables were examined by the χ2 test. Pearson correlation coefficient or Spearman rank correlation coefficient were used to analyze the correlation among variables. Results:NLRP3 was widely expressed in the tubulointerstitium, and the expression level in the active group was higher than that in the control group, the semi-quantitative scores of NLRP3 in the renal tubule and glomeruli in the active group were higher than those in the control group ( F=0.859, 8.320, all P<0.05). In the active group, the semi-quantitative score of NLRP3 in the renal tubule was higher than that in the glomeruli( F=3.517, P<0.05). The semi-quantitative score of NLRP3 in the renal tubule was positively correlated with the pediatric vasculitis activity score at renal biopsy ( r=0.471, P=0.027)and negatively correlated with the estimated glomerular filtration rate at renal biopsy ( r=-0.548, P=0.008)in the active group.The serum IL-1β, serum IL-18 and urinary IL-6 levels in the active group were higher than those in the remission group and the control group ( F=16.449, 16.449, 0.637, 29.891, 27.612, 7.464, all P<0.05). The serum IL-18 level in the remission group was higher than that in the control group( F=18.671, P<0.05). In the active group, a positive correlation was found between the serum IL-1β level and the semi-quantitative score of NLRP3 in the renal tubule( r=0.805, P=0.002), between the serum IL-6 level and the C-reactive protein level at renal biopsy ( r=0.728, P=0.017), and between the urinary IL-6 level and the crescent proportion at renal biopsy ( r=0.677, P=0.032). The serum IL-18 level in the active group was positively correlated with the semi-quantitative score of NLRP3 in the renal tubule, pediatric vasculitis activity score and glomerular sclerosis proportion at renal biopsy, and negatively correlated with the estimated glomerular filtration rate at renal biopsy ( r=0.644, 0.612, 0.695, -0.577, all P<0.05). The urinary IL-18 level was positively correlated with the complement C 4 level at renal biopsy ( r=0.855, P<0.05). Conclusions:The NLRP3 inflammasome and its downstream IL-1β, IL-6, and IL-18 may be involved in the pathogenesis and progression of AAV, and can be used as one of the reference indicators for disease activity assessment.

Objective: To investigate the expressions of tissue factor (TF) and vascular endothelial growth factor (VEGF) in diffuse large B-cell lymphoma (DLBCL) and their clinical significances. Methods: The clinical data of 80 cases of DLBCL diagnosed at the Second People's Hospital of Lianyungang from January 2010 to December 2017 were collected, and 30 cases of reactive hyperplasia of lymph node (RLN) were selected as the controls. The expressions of TF and VEGF in the two groups were detected by using immunohistochemical staining. Results: The positive rate of TF and VEGF in the DLBCL group was higher than that in the RLN group [TF: 86.3% (69/80) vs. 50.0% (15/30) ; VEGF: 90.0% (72/80) vs. 53.3% (16/30) ; both P < 0.01]. And there was a positive correlation between the expression of TF and VEGF (r = 0.704, P < 0.05). There was no significant difference in the positive rates of TF and VEGF among the patients with different gender, age and Hans subtypes in DLBCL group (all P > 0.05). The positive rate of TF in DLBCL patients with B symptom, increased LDH, physical status grade ≥2, and extranodal lesion number >1 was higher (all P < 0.05). The positive rate of VEGF in patients with Ann Arbor stage Ⅲ-Ⅳ, B symptom, increased LDH, and extranodal lesion number >1 was higher (all P < 0.05). The positive rate of TF in international prognostic index (IPI) high-risk group was higher than that in low-risk group (P < 0.01); the positive rate of VEGF in IPI high-risk group and middle-high-risk group was higher than that in low-risk group (all P < 0.01). The expressions of TF (r = 0.491, P < 0.01) and VEGF (r = 0.529, P < 0.01) were positively correlated with IPI. The overall survival rates of TF and VEGF low-expression group were higher than those of TF and VEGF high-expression group (both P < 0.05). Conclusion The expressions of TF and VEGF are highly expressed in DLBCL, which is associated with the IPI. It can provide a reference value in evaluating prognosis of DLBCL.

Objective:To study the effects of early breastfeeding volume on neonatal necrotizing enterocolitis (NEC) and feeding intolerance in very low birth weight infants.Methods:This study retrospectively analyzed the clinical data of 275 cases of very low birth weight infants (birth weight<1 500 g) born in Shenzhen Maternity & Child Healthcare Hospital from June 2017 to May 2018. Based on whether breastfeeding or not and the ratio of breast milk intake over the total volume of intake within two weeks after birth, they were divided into three groups: breast milk intake>50% group (>50% group, n=199), breast milk intake≤50% group (≤50% group, n=55) and formula group ( n=21). Differences in the incidence of NEC and feeding intolerance among the three groups were analyzed using Chi-square test (or Fisher's exact test). Effects of breast milk intake on the incidence of NEC and feeding intolerance were evaluated using univariate and multivariate logistic regression analysis. Results:The incidence of NEC in the >50% group, ≤50% group and formula group was 1.5% (3/199), 27.3% (15/55) and 9.5% (2/21), respectively ( P<0.01), and the incidence of feeding intolerance was 17.6% (35/199), 56.4% (31/55) and 28.6% (6/21), respectively ( χ2=34.826, P<0.01). Univariate logistic regression analysis showed that compared with the >50% group, the risk of NEC in the≤50% and formula group increased ( OR=24.500, 95% CI: 6.755-85.594; OR=6.877, 95% CI: 1.081-43.744); that of feeding intolerance increased in the≤50% group ( OR=6.316, 95% CI: 3.293-12.113). Multivariate logistic regression analysis showed that compared with the >50% group, the risk of NEC in the≤50% and formula groups increased ( OR=28.452, 95% CI: 7.280-111.195; OR=8.610, 95% CI: 1.262-58.766); that of feeding intolerance increased in the≤50% group ( OR=7.207, 95% CI: 3.601-14.425). Conclusions:Breastfeeding accounting for more than half of the total volume of intake within two weeks after birth may reduce the incidence of feeding intolerance and NEC in very low birth weight infants.

Objective To analyze some related causes of hypocytosis or hematocytosis with bone marrow dry pumping. Methods Bone marrow histology, reticular fiber staining and selective immunohistochemical staining were performed in 34 bone marrow dry pumping patients with hypocytosis or hematocytosis in the Second People's Hospital of Lianyungang from January 2012 to August 2017. Results All the patients showed dizziness, fatigue, splenomegaly, night sweats and bleeding, including primary myelofibrosis (17 cases, 50.0 %), chronic myelocytic leukemia (4 cases, 11.8 %), acute myelocytic leukemia (2 cases, 5.9 %), acute lymphoblastic leukemia (1 case, 2.9 %), myelodysplastic syndrome (2 cases, 5.9 %), multiple myeloma (2 cases, 5.9 %), non-Hodgkin lymphoma with bone marrow infiltration (2 cases, 5.9 %), polycythemia vera (1 case, 2.9 %) and bone marrow metastatic tumor (3 cases, 8.8 %). The bone marrow proliferative degree in primary myelofibrosis group was mainly "grade Ⅱ" to "grade Ⅳ", and the non primary myelofibrosis group was mainly "grade Ⅲ" to "grade Ⅴ", and the differences of proliferative degree component between them were statically significant (χ2＝ 12.900, P＝ 0.004). The fibrosis level in primary myelofibrosis group was mainly "grade 2" to "grade 3", and the non primary myelofibrosis group was mainly "grade 1" to "grade 2", and the differences of myelofibrosis degree component between them were also statistically significant (χ2＝ 12.692, P＝ 0.003). Conclusions Hematological malignancies, especially primary myelofibrosis, are the common causes of bone marrow "dry pumping". Bone marrow histology, reticular fiber staining and selective immunohistochemical staining are of great significance in the etiological diagnosis.

Isotope Dilution Mass Spectrometry (IDMS) is the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key is getting the corresponding fully uniformly(U) ¹³C-labeled metabolites to be measured. The conventional procedure for getting fully U ¹³C-labeled metabolites is through batch cultivation, but intracellular metabolites concentrations by this method are generally low. By applying U ¹³C-labeled glucose pulse, combined with fast sampling and quenching, mixture of fully U ¹³C-labeled intracellular metabolites was successfully extracted with higher concentration from Pichia pastoris G/DSEL fed with fully U ¹³C-labeled glucose as only carbon source. Quantitative results from liquid chromatography tandem mass spectrometry (LC-MS) and gas chromatography tandem mass spectrometry (GC-MS) show that concentrations of organic acids, sugar phosphates, amino acids and nucleotides were 2-10 folds higher than those without glucose pulse. Therefore, the glucose pulse method can efficiently improve the usage of fully U ¹³C-labeled glucose converting to ¹³C-labeled metabolites, and achieve the detection of intracellular metabolites with lower concentrate than the instrument detection limit.

Objective To investigate the clinical and laboratory features of IgG-2κ light chain multiple myeloma. Methods The clinical data and laboratory results of 2 multiple myeloma (MM) patients with IgG-2κ light chain were analyzed and the related literatures were reviewed. Results Two male and 1 female patients were 50-82 years old and mainly suffered with backache, infection, anemia and renal dysfunction. Multiple osteolytic bone destruction was detected in X-ray as well as magnetic resonance imaging (MRI). The level of serum IgG was normal, slight or obviously increased, but the levels of IgA and IgM were decreased. The levels of κ light chain in serum and urine were both increased significantly, and Bence-Jones protein was positive. Double M protein peaks of serum in γ area were detected by protein electrophoresis in 2 patients. A single band of IgG and double bands of light chain κ were revealed by immunofixation electrophoresis. Bone marrow smear showed that abnormal plasma cells were increased obviously. One patient gave up chemotherapy because of lung infection, acute left heart failure and acute renal failure, the others 2 patients achieved partial remission and stable disease by receiving DVD and VAD chemotherapy. Conclusions IgG-2κ light chain MM lacks typical clinical presentation, but some laboratory characteristics may be different from those of IgG-κ light chain. Further researches are needed to confirm whether or not it belongs to biclonal MM.

Objective To establish the animal model of traumatic myositis ossificans through striking the quadriceps muscle of rabbits repeatedly.Methods Nine white adult New Zealand rabbits were selected,as their left lower limbs were used as the model group and the right lower ones served as controls.The left hind limbs were stricken by a 0.25 kg ball falling from a height of 100 cm every 3 days for and then immobilized with the knee in extension,while the right knees were immobilized in the same way without striking.The rabbits were sacrificed at the 4th,6th and 8th weeks respectively.The swelling,local physical signs and pathological changes of the heterotopic ossification had been assessed.Results The swelling of left quadriceps was obvious,with progressively stiffness of the left knees accompanied by distinctly palpable indurations,while in the right hind limbs,the joint was stiff but without indurations.The imaging examination showed the ossification began to appear in the impact sites of the model group at the 4th,6th and 8th weeks.The hematoxylin-eosin staining demonstrated that there was obvious cartilage or bone formation in the muscle tissues of the left quadriceps at the 8th weeks.Conclusion An animal model of post-traumatic myositis ossificans can be successfully established in rabbits through beating their quadriceps repeatedly.

Objective To explore the proliferative changes and clinical significance of osteoclasts (OC) in various stages of osteoarthritis (OA). Methods Twenty healthy adult male SD rats were made the model by modified Hulht procedure,the left knee served as the control group and the right knee as the OA model group. The total knee joint (n=5) was collected at postoperative 1, 2,4,8 weeks, fixed at 4 ℃ 4% poly formaldehyde (PFA) liquid, embedded by paraffin for conducting sections, and stained by TRAP,toluidine blue(TB) and safranine O (Saf O)fast staining. Then the cartilage morphology change was observed and OC positive cells number with TRAP staining were semi-quantitatively detected,the OA cartilage destruction progression was evaluated by Mankin's method and SPSS17.0 statistics software was used to conduct statistical analysis. Results OC in the two groups showed the transient change of rapidly increasing and then decreasing. The OC number at 1 week in the control group (left knee) was (65.20±4.12) cells/mm2 ,and was gradually reduced at 2,4 weeks,which were (47.20±4.31) cells/mm and (26.20±3. 87) cells/mm2 ,which at 8 weeks was almost invisible, the number of cells was (7.00 ± 2.28) cells/mm2 ;the OC number at 1 week in the OA model group (right knee) was (70.40 ± 5.46) cells/mm2 ,increased to (86.20± 5.42)cells /mm2 at 2 weeks, reduced to (38. 0 ± 3.16) cells/mm2 at 4 weeks , was almost invisible at 8 weeks, the number of cells was (6.21 ± 2.93 ) cells/mm2 . The OC number at 2,4 weeks in the model group was significantly higher than that in the control group, the difference had statistical significance (P<0.05). Conclusion Large numbers of osteoclasts are proliferated in the early and middle stages of rat knee osteoarthritis, which indicating that OC might be involved in the formation of osteoarthritis.