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@#Abstract: Objective To explore and analyze the diagnostic value of multicolor melting curve analysis (MMCA) for the resistance of five anti-tuberculosis drugs, so as to clarify the clinical value of MMCA in detecting drug resistance of Mycobacterium tuberculosis. Methods From April 2021 to May 2022, 200 patients with positive Mycobacterium tuberculosis admitted to the Fourth People's Hospital of Qinghai Province were selected as research objects, and sputum specimens were taken from the patients. Traditional Mycobacterium tuberculosis drug sensitivity test (modified Löwenstein-Jensen medium method) and MMCA analysis were respectively given to detect the resistance of five anti-tuberculosis drugs, including isoniazid, ethambutol, streptomycin, rifampicin and isoniazid, respectively. Those samples with inconsistent results between the two diagnosis methods were subjected to gene sequencing verification, and the diagnosis efficiency of MMCA for the five anti-tuberculosis drugs was compared. Results Using Mycobacterium tuberculosis drug sensitivity as the gold standard for drug resistance diagnosis, the sensitivity of MMCA for detecting drug resistance of rifampicin, ethambutol, streptomycin, isoniazid and levofloxacin were 95.83% (46/48), 93.75% (15/16), 100.00% (15/15), 100.00% (20/20) and 70.00% (7/10), respectively, with statistical differences between groups (P<0.05). There were no statistically significant differences in the specificity, positive predictive value, negative predictive value and accuracy of MMCA for the five anti-tuberculosis drugs (P>0.05). For the 8 samples with inconsistent results between MMCA and modified Löwenstein-Jensen medium method, gene sequencing was performed and compared with the results of gene sequencing. After comparison with gene sequencing results, it was found that the coincidence rate of MMCA and gene sequencing results was 75.00% (6/8). Conclusions In the detection of drug-resistant mutations in TB patients, multi-color probe fusion curve analysis has high diagnostic efficacy for first-line anti-tuberculosis drugs, but is not sensitive to second-line anti-tuberculosis drug levofloxacin. Therefore, for the detection of first-line anti-tuberculosis drugs, MMCA has a good clinical application prospect.
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Objective:To provide data reference for using Chinese rhesus macaques as research model by studying the immunophenotype and function of peripheral blood lymphocytes in Chinese rhesus macaques.Methods:By optimizing antibody clones and fluorescent colors, the lymphocyte subset assay and T cell function assay panels were determined. Then the panels were used to analyze the proportion of T, B, NK and other cell subsets in peripheral blood mononuclear cells (PBMCs) in 15 healthy Chinese rhesus monkeys, and the ability of T cells to secrete cytokines after non-specific stimulation.Results:Two multi-color flow cytometry analytic panels were established. Panel 1 could simultaneously detect a variety of lymphocyte subsets, including cytotoxic T lymphocytes, follicular helper T cells, regulatory T cells, B cells and NK cells. Panel 2 could detect the functions of multiple T cell subsets and the expression of immune checkpoint moleculars. The mean percentages of T, B, NK, Tfh, Treg, CD16 + NK and CD56 + NK cells in PBMCs of the Chinese rhesus macaques were (75.32±7.73)%, (13.22±7.50)%, (0.88±0.48)%, (0.73±0.27)%, (0.75±0.43)%, (47.87±22.35)% and (10.69±12.41)%. After non-specific stimulation, the proportion of CD4 + T cells secreting IL-2 and TNF-α was higher than that of CD8 + T cells, and the proportion of CD8 + T cells secreting CD107a and IFN-γ was higher than that of CD4 + T cells, while the proportion of CD4 + and CD8 + T cells secreting IL-17A was low. Conclusions:This study established a multi-color flow detection scheme that could simultaneously detect multiple cellular surface molecules and cytokines at the single cell level and could accurately and comprehensively analyze the immune cell subsets, functions and the immune checkpoint molecules in peripheral blood of Chinese rhesus macaques, providing a new experimental method and basic data for the development of vaccines and drugs against infectious diseases.
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Purpose: To compare multicolor imaging (MCI) with Optos color fundus photography (OCFP) for the evaluation of morphology and extent of preretinal membranes in diabetic tractional retinal detachments (TRD). Methods: In this retrospective study, 30 eyes with diabetic TRDs were imaged using the MCI feature of the Heidelberg Spectralis Spectral?domain optical coherence tomography (SD?OCT) and color photo using the Optos Daytona ultra?widefield fundus camera. Two investigators independently graded and determined the agreeability between the two modalities with respect to the extent of the TRD and preretinal membranes on the SD?OCT B?scan images. Results: The MCI provided better visualization of the attachments and traction points of the posterior hyaloid face and preretinal membranes and is comparable to the SD?OCT B?scan images. The inter?rater agreeability rates for OCFP had a Kappa (?) value of 0.37, while the MCI had a ? value of 0.46. When comparing between images of different wavelengths, grading using infrared reflectance (IR) had a poor agreement (?0.04 ± 0.04) while green reflectance (GR) (0.46 ± 0.32) and blue reflectance (BR) (0.53 ± 0.19) had a moderate agreement. The composite MCI and GR images also had comparatively higher intraclass coefficient when compared to the OCFP (0.25 [?0.09–0.55]) and IR (?0.03 [?0.39–0.34]) images. Conclusion: MCI is more sensitive for determining the extent of TRDs and for the detection of secondary membranes when compared to OCFP, thus, aiding in better surgical planning.
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In this study,a fluorescent(FL)aptasensor was developed for on-site detection of live Salmonella typhimurium(S.T.)and Vibrio parahaemolyticus(V.P.).Complementary DNA(cDNA)of aptamer(Apt)-functionalized multicolor polyhedral oligomeric silsesquioxane-perovskite quantum dots(cDNA-POSS-PQDs)were used as encoded probes and combined with dual-stirring-bar-assisted signal amplification for pathogen quantification.In this system,bar 1 was labeled with the S.T.and V.P.Apts,and then bar 2 was functionalized with cDNA-POSS-PQDs.When S.T.and V.P.were introduced,pathogen-Apt complexes would form and be released into the supernatant from bar 1.Under agitation,the two complexes reached bar 2 and subsequently reacted with cDNA-POSS-PQDs,which were immobilized on MXene.Then,the encoded probes would be detached from bar 2 to generate FL signals in the supernatant.Notably,the pathogens can resume their free state and initiate next cycle.They swim between the two bars,and the FL signals can be gradually enhanced to maximum after several cycles.The FL signals from released encoded probes can be used to detect the analytes.In particular,live pathogens can be distinguished from dead ones by using an assay.The detection limits and linear range for S.T.and V.P.were 30 and 10 CFU/mL and 102-106 CFU/mL,respectively.Therefore,this assay has broad application potential for simultaneous on-site detection of various live pathogenic bacteria in water.
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Objective:To investigate the significance of multicolor flow cytometry (MFC) monitoring of minimal residual disease (MRD) in the course of allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CD19-chimeric antigen receptor(CAR)-T cell immunotherapy for patients with refractory, relapsed B-cell acute lymphoblastic leukemia (r/r B-ALL).Methods:37 patients with r/r B-ALL admitted to Hebei Yanda Lu Daopei Hospital from January to July 2019, aged 15 (6, 19) years old, including 24 males and 13 females, were treated with CD19-CAR-T cell immunotherapy bridging allo-HSCT. MFC with cytoplasmic CD79a antibody to set up B-cell gates was used to monitor patients′ bone marrow (BM), cerebrospinal fluid (CSF), and tissue samples on day 0 (prior to the CAR-T cell immunotherapy), day 15, day 28 post CAR-T cell immunotherapy, and post transplantation.The MRD values of these samples were analyzed to evaluate the residual tumor cells and metastasis. The killing effect of the CAR-T cells was evaluated by the recovery of CD19+B cells before transplantation and the period between the timepoint when CD19+B cells was recovered and the timepoint when CAR-T cells were infused. Peripheral blood CAR-T cells were counted at different time points. Statistic analysis was performed by Kaplan-Meie assay and Log-rank test to analyze the difference of univariate cumulative survival.Results:(1)Among the 37 patients, 8 died and 29 survived. 5 patients relapsed after transplantation, of which 4 relapsed patients died and 1 survived. (2)MFC MRD negative remission rate of the death group was lower than that of the survival group at the following time points: post-CAR-T therapy and prior to transplantation (5/8 vs. 28/29, χ 2=7.540, P=0.006); day 15 of the CAR-T cell reinfusion (3/8 vs. 24/29, χ 2=6.512, P=0.011); day 28 of the reinfusion (3/8 vs. 276/29, χ 2=10.065, P=0.002). The probability of extramedullary MFC MRD positive tumor infiltration in the death group was higher than that in the survival group(7/8 vs. 14/29, χ 2=3.931, P=0.047). After CAR-T cell immunotherapy, the recovery period of CD19-positive cells in the death group, or the time for CAR-T cells to kill CD19-positive cells, was shorter than that in the survival group [42.00 days(30.00,49.00) vs. 55.00 days(41.50,73.50), Z=0.022, P=0.020]. Conclusion:The positive results of MRD by MFC at the following timepoints may predict unfavorable outcomes, such as post-CAR-T therapy and prior to transplantation, day 15 and 28 of the CAR-T cell immunotherapy, which may provide some guidance for clinical management.
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Objective:To observe the characteristics of macular telangiectasia (MacTel) in multi-color and multi-mode fundus images.Methods:An abservational case series study was conducted.Sixteen eyes of 12 patients diagnosed with MacTel by fluorescein fundus angiography (FFA) from January to November 2019 in Shandong Eye Hospital were analyzed.There were 8 cases (8 eyes) with MacTel type Ⅰ, among which 4 cases were male and 4 cases were female, with an average age of (62.3±12.5) years.The other 4 cases (8 eyes) had MacTel type Ⅱ, all of which were female, with an average age of (58.7±10.5) years.Best corrected visual acuity, slit lamp microscopy, color fundus photography, multicolor scanning laser imaging, FFA, optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) were carried out in all the patients.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Shandong Eye Institute (No.2019S003).Results:In color fundus images of MacTel type Ⅰ eyes, annular macular exudation with macular edema occurred in 6 eyes, macular edema without hard exudates in 1 eye, and hard macular exudates without macular edema in 1 eye.However, the transparency of retina in temporal fovea in MacTel type Ⅱ eyes decreased, showing a gray color.In multi-color fundus images of MacTel type Ⅰ eyes, punctate granular yellow macular exudation and yellow-green macular edema were observed, which were clearer than those in color fundus images.Punctate exudation was seen in both the blue and green reflectance images, which was clearest in green reflectance image, followed by blue reflectance image and then the infrared reflectance image.In OCT images of MacTel type Ⅰ eyes, cystoid edema of inner retina or uneven reflection signal of outer plexiform retina were observed.Loss of inner and outer retinal structures and cavities were observable in MacTel type Ⅱ eyes, and outer retinal atrophy appeared in 2 eyes.In OCTA images, the destruction of superficial and deep capillary plexus in macular area were observed in both MacTel typeⅠand type Ⅱ eyes, and the destruction of deep capillary plexus was more obvious.In addition, more obviously increased vascular space, decreased vessel density, and increased foveal avascular zone were found in MacTel type Ⅱ eyes.In early stage of FFA, delayed capillary filling near fovea was seen in MacTel typeⅠeyes, and dilated temporal vessels in fovea, some of which showed tumor-like dilation, and the limited tumor-like dilation was enhanced in the later stage.Different degrees of dilated parafoveal blood vessels in the early stage, and the capillary in the temporal side of the macula showing diffuse strong fluorescence in the late stage of FFA was observed in MacTel type Ⅱ eyes.Conclusions:Multi-color scanning laser imaging can be used to observe the morphological characteristics of MacTel, and the imaging features of different types of MacTel are significantly different.
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Objective:To investigate the diagnostic value of fluorescence quantitative method and G6PD/6PGD ratio method in glucose-6-phosphate dehydrogenase (G6PD) deficiency and the type of gene mutation.Methods:A total of 1 201 patients (711 males and 490 females) with suspected G6PD deficiency in Shanghai Children′s Hospital were collected from June 2018 to March 2021. Fluorescence quantification method, G6PD/6PGD ratio method and multicolor melting curve were used to detects enzyme activity, ratio and gene mutation type. Comparison of each index and evaluation of its diagnostic efficiency were performed.Results:Among 1 201 suspicious samples, 163 cases (135 males and 28 females) were finally diagnosed. 156 cases were diagnosed by fluorescence quantitative method with a detection rate of 95.71%, and 140 cases were diagnosed by G6PD/6PGD ratio method with a detection rate of 85.89%. enzymatic activity of G6PD and ratio of G6PD/6PGD in male were significantly lower than female, and the differences were statistically significant ( U=642.5, 734.5, P<0.001). 112 cases received G6PD gene mutation detection and 92 cases were diagnosed, 74 were hemizygous mutations, 1 were homozygous mutations, 15 were heterozygous mutations, and 2 were compound heterozygous mutations. Among 15 cases of heterozygous mutations, 11 cases were diagnosed by fluorescence quantitative method, the diagnosed rate was 73.33%, 4 cases were diagnosed by G6PD/6PGD ratio method, and the diagnosed rate was 26.67%. A total of 7 mutation sites were detected and the proportions were c.1388G>A (32.22%), c.1376G>T (30.00%), c.871G>A (13.33%), c.1024C>T (11.11%). c.95A>G (7.78%), c.487G>A (4.44%), c.392G>T (1.11%). The enzymatic activities of c.1376G>T and c.1024C>T, c.487G>A were statistically significant ( P<0.001,0.015); the G6PD/6PGD ratios of c.1024C>T and c.1388G>A, c.1376G>T were statistically significant ( P=0.017,0.002,0.011,0.013). Fluorescence quantitative method had sensitivity of 100%, specificity of 95.65%, and the area under the curve (AUC) is 0.972. The sensitivity of the G6PD/6PGD ratio method was 100%, the specificity was 94.57%, and the AUC was 0.979. The sensitivity of fluorescence quantitative method combined with G6PD/6PGD ratio was 96.7%, the specificity was 100%, and the AUC was 0.992. Conclusions:Compared with fluorescence quantification, the G6PD/6PGD ratio method might not be able to diagnose female heterozygotes effectively; The panel of G6PD fluorescence quantification and G6PD/6PGD ratio was helpful to reduce the missed diagnosis. Combined with gene mutation analysis, it could improve the diagnosis rate of G6PD deficiency in the children.
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Objective:To investigate the features of multicolor imaging in the macular region of central serous chorioretinopathy (CSC) patients.Methods:A cross-sectional study was conducted.Thirty-four acute CSC patients (34 eyes) treated in Renmin Hospital of Wuhan University from August 2017 to January 2018 were enrolled.Among the 34 subjects, there were 21 males (21 eyes) and 13 females (13 eyes). The subjects were 26 to 61 years old, with an average age of (37.41±9.35) years.The course of the disease was 5 to 45 days, with an average course of (12.00±2.29) days.All the subjects were examined by color fundus photography, fluorescein angiography (FFA), indocyanine green angiography (ICGA), multicolor imaging, spectral-domain optical coherence tomography (SD-OCT). The image features of each patient were compared and analyzed.The diagnostic accordance rate for leakage point and serous retinal neuroepithelial detachment of multicolor imaging and color fundus photography was calculated according to FFA/ICGA and OCT.This study protocol adhered to the Declaration of Helsinki and was approved by an Ethics Committee of Renmin Hospital of Wuhan University (No.WDRY2019-K037).Results:The serous retinal detachment region showed green light reflection area with clear boundary in 33 eyes (97.06%) in the standard as well as blue and green enhanced multicolor image, with not clear boundary in 1 eye (2.94%). The serous retinal detachment region showed weakly reflective area in 17 eyes (50%) in blue reflectance image, showed weak reflection with clear boundary in 32 eyes (94.11%) in green reflectance image, showed weakly reflection with clear boundary in 33 eyes (97.06%) in infrared reflectance image.The fluorescein leakage point in FFA image was found micro retinal pigment epithelium detachment (PED) in 19 eyes (55.88%), rough light band of retinal pigment epithelium (RPE) in 12 eyes (35.29%), and large PED in 3 eyes (8.82%) in SD-OCT image.The RPE leakage showed red mottled changes in the area of neuroepithelial detachment in 29 eyes (85.29%) in the standard as well as blue and green enhanced multicolor images, presented strong reflection spots in blue reflectance images in 2 eyes (5.88%), showed strong reflective spots in green reflectance in 5 eyes (14.70%), showed strong reflection spot in the weakly reflective area in 33 eyes (97.06%) in infrared reflectance images.Taking FFA/ICGA and OCT as the gold standard, the diagnostic accordance rate of standard multicolor, blue and green enhanced multicolor and infrared reflectance images for serous retinal neuroepithelial detachment and leakage points was higher than that of color fundus photography, and the differences were statistically significant (all at P<0.05). Conclusions:Standard multicolor, blue and green enhanced multicolor and infrared reflectance images can reflect the leakage point and retinal neuroepithelial detachment of acute CSC.Green reflectance image can show serous retinal neuroepithelial detachment of acute CSC.Multicolor imaging can be used as the auxiliary diagnosis method of acute CSC.
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Objective To observe the imaging features of cystoid macular edema (CME) in multicolor imaging (MC),and to evaluate the value of MC in the diagnosis of CME.Methods Descriptive case series study.From August 2017 to June 2018,42 eyes of 37 patients with CME diagnosed in the people's Hospital of Wuhan University were included in the study.Among them,there were 24 males and 13 females,with an average age of 48.51 ± 10.29 years.There were 14 eyes with diabetic retinopathy,14 eyes with central retinal vein occlusion,8 eyes with branch retinal vein occlusion,4 eyes with uveitis,and 2 eyes with Eales disease.The macular color fundus photography (CFP) was performed with Visucam 200 non-mydriatic fundus camera of Zeiss company in Germany.MC,frequnce domainoptical OCT (SD-OCT) and FFA were examined by Spectralis HRA2 + OCT of Heidelberg company in Germany.According to the MC standard method,five images,including 488 nm blue reflection (BR),515 nm green reflection (GR),820 nm infrared reflection (IR) imaging and standard MC and blue-green enhancement (BG),were obtained at the same time.Compared with SD-OCT,CFP and MC images were scored.Friedman M test and Wilcoxon signed rank test were used for statistical analysis.Results The standard MC and BG images showed blue-green uplift area or petal-shaped appearance,surrounded by green reflection areas with clear boundaries.BR image can be seen in the low reflexes area.On the GR image,there were patches or cystic low reflection areas,surrounded by a slightly high reflection.On the IR image,patches or cystoid high reflexes can be seen,surrounded by low reflection dark areas with clear boundaries.The average scores of CFP,standard MC,GB,IR,GR and BR were 1.20± 0.94,3.05± 0.99,2.90± 1.04,2.55± 1.27,2.00± 0.94,0.51 ± 0.85 respectively,and the differences were statistically significant (χ2=151.61,P=0.000).The score of CFP were significantly lower than that of standard MC (Z=-5.421),BG (Z=-5.354),IR (Z=-4.714),GR (Z=-4.438) and higher than that of BR (Z=-3.435).The differences were statistically significant (P=0.000,0.000,0.000,0.000,0.001).Conclusions The quality of MC imaging is better than that of CFP.Combined with SD-OCT,it can be used as an assistant method to diagnose CME.
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Interpretation of optic disc cupping and neuroretinal rim is challenging in myopic eyes because of large, shallow, and tilted disc, myopic crescent, and macular degeneration. Color and red-free optic disc photographs do not reveal the cup/disc ratio often in myopic eyes. We report characteristics of multicolor optic disc photography in three myopic eyes. All eyes underwent multicolor imaging Spectralis SDOCT system (Heidelberg Engineering, Germany). Owing to use of three laser lights in a confocal design, multicolor optic disc photography was able to delineate the neuroretinal rim and optic cup more clearly than color and red-free optic disc photographs.
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We describe the multicolor imaging findings in two cases of Torpedo maculopathy (TM). Multicolor imaging can be a useful tool in identifying the level of retinal and choroidal layer involvement in TM. This imaging modality further confirms the hypothesis that TM is a localized congenital abnormality of retinal pigment epithelium pigmentation which leads to outer retinal and inner choroidal degeneration.
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Objective: To explore the factors affecting cell loss in multicolor flow cytometry of mouse immune cells. Methods: C57BL/6J mice were used in the experiment. Splenocytes were isolated and resuspended twice with buffers of different types, compositions and pH; then the number of viable cells was counted to observe which factors affected the amount of cell loss during staining. Results: During flow cytometric labeling of mouse immune cells, buffer types significantly affected the amount of cell loss. Washing with PBS buffer containing potassium ions caused obvious cell loss, while slight shift in buffer pH and whether or not FBS/BSA was added had no significant effect on cell loss. Moreover, centrifugation speed also significantly affected cell loss. Conclusion: The use of potassium-containing PBS buffer during flow cytometric staining of mouse immune cells resulted in significant cell loss. The potassium-free PBS buffer and moderate centrifugation speed should be recommended.
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Choroidal nevi are benign fundus lesions that require regular follow with documentation. Conventional color fundus photography (CFP) has traditionally been used to images these lesions. Multicolor imaging (MCI) available on Spectralis spectral domain optical coherence tomography system is increasingly been tested vis-à-vis conventional CFP in various retinal diseases. We present data of the right eye of a 59-year-old gentleman with choroidal nevus who underwent conventional CFP as well as MCI. Nevus appeared orange red on MCI and its size appeared larger than the same measured on conventional CFP. We also report infrared reflectance and near infrared autofluorescence features of choroidal nevus.