Kidney transplantation is the definitive treatment for end stage renal disease (ESRD), offering improved quality of life and survival benefit over remaining on dialysis. There is, however, a prevailing significant mismatch between patients awaiting transplantation and available donor kidneys. Over time, initial stringent donor criteria have broadened and organs from extended criteria donors (ECDs) and older donors are now being accepted for transplantation. The spectrum of living donors has also undergone a change from close family members to an increasingly non-related, non-directed altruistic donors, newly classified as ‘unspecified’ donors. Unspecified elderly donors could be a potential untapped resource to expanding the kidney donor pool globally.We present a case of an 85 year and 8 months old individual, who donated to an unrelated non-directed matched recipient in the national deceased donor transplant waiting list with excellent donor and recipient outcomes at 7 years.To our knowledge she is one of the oldest reported unspecified living kidney donors in the world to date. This case illustrates that elderly donors in good health can come forward to donate, knowing that it is safe and valuable. Once the immediate perioperative challenges after kidney donation are managed, elderly donors rarely encounter long term sequelae. We therefore report this case to increase awareness and refocus attention of transplant teams on elderly donors as a potential untapped group to help address the organ shortage problem in renal transplantation. Kidney transplantation remains the preferred treatment for ESRD . HoweverElderly donors are generally classified as donors > 60 years and donation among carefully selected older individuals is associated with minimal perioperative risk and little or no added long-term renal dysfunction or ESRD . Only feAll these reported cases were uneventful with no perioperative complications, demonstrating feasibility, safety and good recipient outcomes with octogenarian donors. In this case report, we share our experience of an 85-year-old lady who donated her kidney altruistically as an unspecified donor to a recipient on the national kidney transplant waiting list with excellent outcome in both donor and recipient. To the best of our knowledge she is one of the oldest unspecified living kidney donors in the world to date.In 2013, a woman born in 1927 came forward to Lancashire Teaching Hospitals, one of our satellite referring centres expressing a desire to be an unspecified kidney donor. She was a retired teacher who in her own words, was healthy, had led a satisfactory life and was keen to contribute to society by this donation which would benefit someone on the national waiting list. The only significant past medical history was open fixation of right ankle fracture in 1990 and carpal tunnel decompression in 2009. Over several interviews with detailed discussions of risks of donor nephrectomy and long-term outcomes, she reiterated her full understanding of the risks and benefits and strong conviction to proceed. An independent psychological assessment confirmed full mental capacity and altruistic motive. The physical examination was normal. Her body mass index (BMI) was 31.2. An exhaustive donor work-up with additional cardiac tests in view of her age was completely satisfactory. An echocardiogram showed an ejection fraction of 74% and a myocardial perfusion scan was normal. A Tc99m-MAG3 isotopic scan confirmed excellent renal function with an isotopic GFR of 93 ml/min. The DMSA scan showed the left kidney contributing 46% and right 54% to overall renal function. The CT angiogram, for vascular anatomy demonstrated 2 renal arteries bilaterally with a normal urogram. Her case was then discussed by the multidisciplinary transplant team and a unanimous decision made not to disqualify her on basis of age as other parameters were entirely satisfactory. She was then registered on the United Kingdom Living Kidney Sharing Scheme (UKLKSS) as an unspecified donor and matched to a 53-year-old male recipient, with no potential living donors and listed on the deceased donor national waitlist, at a different centre. The UKLKSS is a highly successful scheme set up to benefit difficult to match patients on the deceased donor waiting list who don’t have living donors. Unspecified donors contributing to this scheme, provide opportunities for these recipients to have living donor (LD) transplants. The recipient had ESRD secondary to Polycystic Kidney Disease (PKD) and was 1–1-0 HLA mismatched with the donor. The donation operation was by a left-sided hand-assisted laparoscopic donor nephrectomy (LDN) lasting 1 h 58 min. There were no intraoperative problems. Post-operative care was with opioid based analgesia, early ambulation, incentive spirometry, laxatives and standard anti-thromboembolic prophylaxis with TED stockings and Dalteparin (5000iu) till discharge on day 4. The creatinine level at discharge was 93 μmol/L and eGFR 50 ml/min. Four weeks after discharge she had resumed driving and was back to near-normal routine and independence. On the most recent review her creatinine was documented to be 73umol/L with an eGFR > 60 ml/min. Currently, she is 94 years old and continues to lead an active, independent life and still continues to drive. She is aware of the successful recipient outcome anonymously, which has given her satisfaction with the donation and outcome and consents to sharing her experience in order to encourage other older donors to do same for the benefit to society.st artery and 50 min for 2nd artery; total ischemic time 5 h 26 min. The recipient achieved immediate graft function; and over last years has remained well with no rejection episodes, enjoying stable function, creatinine averaging 156 μmol/L and eGFR 40 ml/min  in 2017 . TransplOlder donors particularly, require thorough pre-operative evaluation and consideration of anaesthetic and surgical risks inherent with ageing. At the minimum, an echocardiogram, myocardial perfusion scan and pre-operative anaesthetic review should be done for all the donors. The use of the ESRD risk calculator tool  introducGrams, et al.concluded that many older persons had low estimates of long-term ESRD risk even in the presence of health characteristics, such as low eGFR or mild hypertension, that are often considered contraindications to donation . Data exMany transplant centres use a ‘normal-for-now’  standardnd post-operative day but it settled with no anti-hypertensives. A meta-analysis by Boudville, et al.reported a 5-mm Hg increase in blood pressure within 5 to 10 years after donation over that anticipated with normal aging [The risk of hypertension post kidney donation remains controversial. Our patient had high systolic blood pressure of 150 – 160 mmHg by the 2al aging .Another important parameter considered in donor selection is the BMI because higher BMIs are said to be more prone to complications , 13. OurHourmant et al.  reportedFrom the recipients’ perspective, living donation even from elderly donors provides shorter wait times,  less diaOur donor on a recent annual review continues to express satisfaction with her outcome, with good incisional scars and no herniae, confirming Klop et al.’s findings that elderly donors have better cosmetic outcome after LDN . There wFor transplant programs these good outcomes should alleviate concerns surrounding safety of utilization of kidney from older donors both from the donor and recipient perspectives.It is hence disappointing therefore that despite excellent outcome in recipients of kidneys from older donors,  utilizathttps://www.organdonation.nhs.uk).The reason for this primarily may be the lack of awareness that donation from the elderly is safe and valuable. Burnapp, et al. have recommended raising awareness and engaging select target audiences to encourage unspecified kidney donations in order to increase transplant opportunities for patients with ESRD . FurtherIn addition, an unspecified living donor can enable pre-emptive transplantation or provide elective nature for donor/ recipient surgeries.This case report of an 85-year-old living unspecified kidney donor, demonstrates excellent outcomes in both donor and recipient. It adds to a limited number of similar reports and seeks to refocus attention to older donors as a potential untapped group for increasing donor availability to address the increasing numbers of ESRD patients on transplant waiting lists. It also reinforces the need for professionals to disseminate the fact that older individuals in relatively good health can donate in a non-directed fashion and may trigger a chain donation thereby benefiting multiple recipients, difficult – to—match patients on the deceased donor waitlist who do not have LDs and will get LD kidney transplants and those with incompatible living donor pairs. Though the average cold ischemia time (CIT) is more prolonged with the kidney exchange schemes where kidney travel rather than donors, overall graft function remains at par with non – kidney exchange transplantation .Age alone taken as an individual factor is not a contraindication to kidney donation. While this is only a case report with its inherent limitations, it signposts transplant teams to a potential untapped source of healthy older individuals in society who may be prepared to donate in an altruistic non—directed fashion for the benefit of ever-increasing ESRD patients on transplant waiting lists the world over.


In in vitro assay, it was notable that PRRSV-infected porcine alveolar macrophages (PAMs) yielded inconsistent pGM-CSF protein- and mRNA-level, suggesting a post-transcriptional inhibition of pGM-CSF mRNA was employed by PRRSV. Meanwhile, concurrent analysis of pGM-CSF levels in serum samples from PRRSV-infected piglets suggested that effect of PRRSV infection demonstrated minimum effect on pGM-CSF levels regardless of PRRSV virulence phenotypes. Moreover, in vitro treatment of PAMs with pGM-CSF prior PRRSV inoculation did not inhibit PRRSV replication in PAMs although genes downstream of pGM-CSF in PAMs could be upregulated by pGM-CSF treatment. Meanwhile, knockdown of pGM-CSF using siRNA did not enhance PRRSV replication as well. Intriguingly, therapeutic antibody treatment of HP-PRRSV-infected piglets led to significantly increased serum pGM-CSF levels, thus aligning with low pneumonia incidence and low intracellular PRRSV-RNA levels in PAMs of therapeutic antibody treated piglets. Furthermore, transcriptome analysis of PAMs from infected piglets revealed increased serum pGM-CSF levels correlated with activation of downstream signal of pGM-CSF in PAMs as evidenced by a M1-like phenotypes of gene expression pattern, implying a potential host-protective role played by pGM-CSF for PRRSV infection in vivo. In conclusion, our results demonstrated developments of a highly sensitive and specific ELISA for pGM-CSF and revealed a potential protective role conferred by pGM-CSF during PRRSV infection.Granulocyte-macrophage colony stimulating factor (GM-CSF), participates in diverse biological processes associated with innate and adaptive immunity, has unknown effects during PRRSV infection. Here, a double-antibody sandwich ELISA for pGM-CSF was developed in-house for evaluation of pGM-CSF level during PRRSV infection both  Porartevirus  and Betaarterivirus suid 2 , are serotypically distinct and share only approximately 60% nucleotide sequence identity with each other (Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-strand RNA virus belonging to the genus rtevirus , 2, famiovirales . Since dovirales , 5. The ovirales . The twoch other . Neverthch other .via multiple strategies  was first identified as an inducer of differentiation and proliferation of granulocytes and macrophages derived from hematopoietic progenitor cells . Since t pGM-CSF . This re pGM-CSF , implyinnfection .Although little is known regarding pGM-CSF functions in infectious porcine disease, human GM-CSF (hGM-CSF) has been studied extensively and appears to possess both stimulatory and suppressive functions that can influence disease processes associated with autoimmunity, inflammation and cancer , 28. Indin vivo PRRSV infection results. Meanwhile, in vitro treatment of PAMs with pGM-CSF prior PRRSV inoculation or knockdown of pGM-CSF using siRNA did not inhibit PRRSV replication although genes downstream of pGM-CSF in PAMs could be upregulated by pGM-CSF treatment. However, application of a PRRSV-specific broad-spectrum neutralizing mAb to treat in vivo PRRSV infection of piglets led to significantly increased serum pGM-CSF levels. Thus, pGM-CSF may play a positive role in controlling PRRSV infection and/or in enhancing PRRSV clearance. Moreover, protective role of pGM-CSF in PAMs may be PAMs-dependent, as evidenced by activation of pGM-CSF-dependent genes in PAMs. Taken together, in the present study a pGM-CSF ELISA was developed, applied, and shown to be a suitable tool for evaluating pGM-CSF levels. Importantly, ELISA results revealed that pGM-CSF level was not influenced by PRRSV infection in vivo. Nevertheless, a high serum pGM-CSF level was positively correlated with PRRSV clearance and inhibition of viral replication, implying that pGM-CSF may play protective roles during PRRSV infection.In this study, a double-antibody sandwich ELISA incorporating with our homemade mouse monoclonal antibody (mAb) and rabbit polyclonal antibody against pGM-CSF was generated and demonstrated higher sensitivity than commercial ELISA kits. It is notable that PRRSV infection of its natural target cells, porcine alveolar macrophages (PAMs), induced a higher level of pGM-CSF mRNA expression as compared to that of non-infected PAMs. Nevertheless, pGM-CSF protein was not detected in either western blots or ELISA, suggesting that a PRRSV infection-induced post-transcriptional control mechanism blocked protein-translation of pGM-CSF mRNA in PAMs, these observations were also consisted with MARC-145 and BHK21 cells were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum (FBS) , penicillin (100 U/ml), and streptomycin (100 μg/ml). HEK-293T cells were maintained in DMEM as described above for MARC-145 and BHK-21 cell cultures. Porcine alveolar macrophages (PAMs) were prepared from bronchoalveolar lavage of 4-week-old PRRSV-negative pigs as previously described . PAMs wePRRSV strain VR2385 (GenBank: JX044140.1) was recovered from infectious clone pIR-VR2385-CA as previously described , 35. TheThe siRNAs used for knock-down pGM-CSF expression were artificially synthesized by GenePharma Co., Ltd . The sequence of siRNAs used in this study was listed as ® Paque Plus . PBMCs were seeded into wells of 12-well plates at a density of 2 × 106 cells/well, stimulated with LPS  at a concentration of 0.25 mg/mL, then cultured for 24 h. After cells were harvested, total RNA was isolated using RNAiso Plus  then RNA was reverse transcribed using the PrimeScript® RT reagent Kit (TaKaRa) according to the manufacturer’s instructions. The pGM-CSF sequence was amplified from cDNA of PBMCs using Q5® High-Fidelity DNA Polymerase  and ligated to pET-28a vector to encode a fusion protein comprised of GM-CSF with the 8×His-tag at its C-terminal. After verification of construct correctness using DNA sequencing, it was transformed into Escherichia coli strain BL21 (DE3) for recombinant protein expression. For transient expression in a mammalian system, the cDNA sequence of pGM-CSF was cloned into EcoRI and NotI sites of the VenusC1 vector then expressed as a Venus-fused protein. Transfection of plasmid into HEK-293T cells was conducted using FuGENE® HD Transfection Reagent  according to the manufacturer’s instructions.Porcine peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood (after addition of anticoagulation factor) using Ficoll−ΔΔCt method. For quantification of PRRSV-RNA copy numbers in serum samples or PAMs, Taqman probe synthesized by Tsingke Biotech  was used and qPCR was conducted using PerfectStarII Probe qPCR SuperMix . The pBAC-PRRSV-SD16 infectious clone was used for standard curve calculations. Sequences of primers and the Taqman probe used in this study were listed in For qPCR-based evaluation of relative expression of target genes, total RNA was extracted from PAMs using TRizol Reagent (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Reverse transcription and qPCR were conducted using a PrimeScript RT reagent Kit (TaKaRa) and 2×RealStar Power SYBR Mixture  as previously described . TranscrE. coli strain BL21 (DE3) followed by culturing of cells in LB medium at 37°C for 1.5 h until the OD value of the bacterial culture reached 0.6 to 0.8. Next, protein expression was induced by addition of isopropyl β-D-thiogalactoside  followed by culturing of bacteria at 37°C for an additional 6 h. After IPTG induction, pelleting of bacteria, and sonication of bacterial cells, inclusion bodies remained that contained recombinant pGM-CSF-8×His. After washing of inclusion bodies with phosphate-buffered saline (PBS), inclusion body proteins were reconstituted in 8 M urea  then pGM-CSF-8×His was purified using Ni+ affinity chromatography (Transgene). Dialysis of eluted recombinant pGM-CSF-8×His was conducted stepwise against a series of solutions with decreasing urea concentrations until the dialysis buffer was completely replaced with PBS. Dialyzed proteins in PBS were quantified using a BCA protein assay kit (Thermo Fisher Scientific).The pET-28a-pGM-CSF plasmid containing cDNA of pGM-CSF fused to DNA encoding a C-terminal 8×His tag was transformed into cells of via limited dilution and antibody isotyping was conducted using Mouse Monoclonal Antibody Isotyping Reagents (Sigma-Aldrich) according to the manufacturer’s instructions.Six-week-old Balb/C mice and healthy 3-month-old New Zealand White rabbits were obtained from Dashuo Biotech . Protocols used for animal handling and experimentation were reviewed and approved by the Animal Welfare Committee of Northwest A&F University (No. CVM-2019-M&R03). All animals were monitored on a daily basis for clinical signs. Every effort was made to minimize suffering of animals such that animals were euthanized to ensure a humane endpoint was reached according to our protocol. Experimental procedures for immunization of mice and fusion of splenocytes with S/p20 cells were conducted as previously described . Selectevia enzyme-linked immune immunosorbent assay (ELISA). CNBr-activated Sepharose 4B  was washed with a cold solution of 1 mM HCl then was conjugated with recombinant pGM-CSF protein following the manufacturer’s instructions using a ratio of 1 mL (Sepharose 4B) to 10 mg (pGM-CSF protein). Rabbit serum previously filtered through a 0.45-μm membrane  was added to pGM-CSF-conjugated Sepharose 4B then bound antibodies were eluted using 0.1 M glycine (pH 2.7). Eluted antibody was further dialyzed in PBS buffer overnight at 4°C then antibody concentration was measured at a wavelength of 280 nm.Rabbits were immunized with 100 μg of recombinant pGM-CSF-8×His (2 mg/mL) mixed with an equal volume of adjuvant for each immunization, with immunizations repeated a total of 5 times separated by two-week intervals. Freund’s Complete Adjuvant (Sigma-Aldrich) was used for the primary immunization, while Freund’s Incomplete Adjuvant (Sigma-Aldrich) was used for the remaining immunizations. Serum was collected after it met the minimum antiserum titer cutoff of 1:106 as determined Recombinant pGM-CSF or BHK21 cells transfected with VenusC1-pGM-CSF were suspended in 1×Laemmli Sample Buffer . Constituent proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) then transferred onto PVDF membranes (Millipore) for WB as previously described . Membran® 555 labeled goat anti-mouse IgG conjugate (Thermo Fisher Scientific). Coverslips were mounted onto slides using ProLong® Gold Antifade Reagent containing 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) and observed using a Leica DM1000 fluorescence microscope . All images were captured and processed using Leica Application Suite X .BHK21 cells transfected with VenusC1-pGM-CSF or empty vector were fixed with 4% paraformaldehyde (Sigma-Aldrich) and permeabilized with PBS containing 0.5% Triton X-100 (Sigma-Aldrich), then blocked with PBS containing 1% BSA (Sigma-Aldrich). Purified mAb against pGM-CSF (mAb-2A4H11) was used to probe transfected BHK21 cells. Specific interactions between antibody and corresponding target were detected using Alexa FluorThe 96-well Polystyrene Microplates  were coated with 1 μg/well of mAb-2A4H11 in 100 μL of ELISA coating buffer  at 4°C overnight. Unbound mAb was removed by washing wells with PBS-T buffer and wells were further blocked by addition of PBS-T buffer containing 5% skim milk for 3 hours at 37°C. Samples were diluted 20-fold in PBS-T dilution in a final volume of 100 μL before adding to mAb-coated microplates and followed by incubation for 1 hour at 37°C. After washing with PBS-T buffer for three times, 100 μL of PBS-diluted rabbit anti-pGM-CSF polyclonal antibody (1.6 μg/mL) was added to wells followed by incubation for 1 h at 37°C. Interaction between anti-pGM-CSF polyclonal antibody and target were detected by addition of HRP-conjugated goat anti-rabbit secondary antibodies (Transgene)  followed by incubation of plates for 1 h at 37°C. Results were visualized after addition of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate . A standard curve for the pGM-CSF sandwich ELISA was created using dilutions of recombinant pGM-CSF protein of known concentrations  that was plotted using GraphPad Prism version 5.0 . The specificity of the pGM-CSF sandwich ELISA was analyzed using 300 pg of recombinant pGM-CSF protein in a volume of 100 μL. The results were compared to results obtained using equivalent concentrations of recombinant porcine IFN-α , porcine IFN-β , porcine IFN-γ , and porcine IFN-λ3 (homemade). Absorbances of wells were measured using a VictorX5™ Multilabel Plate Reader  at a wavelength of 450 nm.Measurement of repeatability of the pGM-CSF sandwich ELISA. The repeatability of the double-antibody sandwich ELISA was assessed by including positive samples as an internal control within each plate. Within-plate precision was calculated from 20 replicates on one plate and inter-plate precision was calculated from a single sample tested using 10 different plates (runs). Repeatability was assessed from coefficient of variation (CV) values (CV = SD/Mean). %CV, means, and standard deviations (SDs) were calculated using a previously reported protocol . GeneralMeasurement of reactivity. HEK-293T cells were transfected with VenusC1-pGM-CSF or empty vector for 72 h then cells were washed three times with PBS. Next, cells were lysed using PBS containing 0.5% Triton X-100 (Sigma-Aldrich) and 1× protease inhibitor cocktail . Cell lysate was clarified by centrifugation at 15,000 ×g for 10 min at 4°C then supernatants were transferred to fresh tubes. Next, supernatants were added to wells of antibody-coated microplates followed by incubation of plates for 1 h at 37°C before homemade anti-pGM-CSF rabbit polyclonal antibody were added. After washing wells using TBS-T buffer for three times, anti-rabbit HRP-conjugate (Transgene) was added followed by visualization using TMB substrate (Tiangen Biotech). Absorbances of each wells were measured using a VictorX5™ Multilabel Plate Reader (Perkin Elmer) using a wavelength of 450 nm.in vivo samples, serum samples from piglets were diluted for 20-fold in PBS-T buffer before conducting the ELISA.Evaluation of test samples. Cell supernatants from cultures of PAMs infected with various PRRSV isolates were collected for further testing to detect and quantify pGM-CSF levels using the pGM-CSF double-antibody sandwich ELISA developed here. Next, to test whether the ELISA could be used to measure amounts of pGM-CSF in For evaluation of porcine IFN-γ and IL-13 from serum samples, commercial ELISA kit for IFN-γ (Thermo Fisher Scientific) and IL-13  were purchased and conducted following manufacturers’ instruction.in vivo challenge experiment, four-weeks-old piglets were obtained from a PRRSV-free pig farm near Yangling, Shannxi and further screened for CSFV, PRRSV, PCV2 and ASFV along with corresponding antibodies. Only piglets (n=15) negative for all examined pathogens and antibodies against PRRSV and ASFV were selected. Piglets were randomly divided into three groups (n=5) and housed in separate rooms. One group was inoculated with HP-PRRSV-XJA1 whereas other two groups were inoculated with two attenuated PRRSV vaccines: Ingelvac PRRS MLV (herein named MLV) and TJM-F92 representing vaccines against classic PRRSV and HP-PRRSV, respectively. On the other hand, the animal experiments involving challenge with highly pathogenic PRRSV-JXA1 and administration of a PRRSV-specific broad-spectrum neutralizing mAb as treatment for PRRSV infection of piglets were conducted in our previous report (For PRRSV s report . Briefly7 cells were lysed in 3 mL of TRizol Reagent (Thermo Fisher Scientific) for RNA purification, qPCR, and transcriptome analysis. RNA-seq was conducted by GENEWIZ Co., Ltd. . RNA-seq libraries were generated using the Illumina system (RS-122-2201) according to the manufacturer’s protocols. The reference pig genome assembly (Sus scrofa v11.1) was downloaded from the Ensemble website. Transcript abundances were converted to transcripts per million (TPM) units using Kallisto then the TPM value for a given gene was defined using the most abundant transcript associated with that particular gene. Transcript abundances of selected genes were used to generate heatmaps, which were drawn using the R package tool “pheatmap” from The R Project for Statistical Computing. RNA-seq datasets generated in this study can be found in the aforementioned NCBI Gene Expression Omnibus (GEO Accession No. GSE156504).Fresh PAMs harvested from piglets were centrifuged at 300 ×g for 10 min. Next, 3 × 10P-value of <0.01 was considered extremely significant.Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software). Indicator differences between treatment groups and controls were assessed using Student’s t-test. A two-tailed P-value of <0.05 was considered statistically significant and a To obtain antibodies for development of pGM-CSF ELISA with higher sensitivity, cDNA sequence of pGM-CSF was cloned from LPS-stimulated PBMCs . As demonstrated in in vivo , a NADC30-like China isolate HNhx strain, an attenuated HP-PRRSV vaccine strain TJM-F92, and classical strains VR2385 and VR2332. Based on our data, all PRRSV strains induced significant upregulation of pGM-CSF mRNA in PAMs, although several-fold differences were observed among different virus strains  as well. Furthermore, the supernatant of PRRSV-JXA1 infected PAMs with different inoculation time  was used to treat freshly isolated PAMs cells, qPCR analysis was conducted to understand the transcriptional change of M1-like cytokines  and M2-like cytokines , while these genes are typically upregulated in GM-CSF-treated monocytes/macrophages based on previous reports (p<0.05) whereas no significant alternation of mRNA level was observed for other genes in both categories  led to significant reduction of PRRSV infection-induced pathological lung changes  of pGM-CSF along with pGM-CSF itself were selected for transcriptome analysis for PAMs cells isolated from piglets  induced significant upregulation of pGM-CSF mRNA levels  can be found in the article/The animal study was reviewed and approved by Animal Welfare Committee of Northwest A&F University.CW designed this study. QJ, GQ, BL, YB, GW, RC, XZ, ZZ, and YY conduct the experiments. YY and CW analyze the data. CW secure the funds. YY and CW prepared the main body of this manuscript. CW revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a grant from the National Natural Science Foundation of China awarded to CW (No. 32172842) and a grant from the Natural and Science Development Program of Shaanxi Province awarded to CW (Grant No. 2020JM-158).Author RC was employed by the company Shaanxi Innolever Biotechnology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We developed a novel lentiviral vector, pseudotyped with the F and HN proteins from Sendai virus (rSIV.F/HN), that produces long-lasting, high-efficiency transduction of the respiratory epithelium. Here we addressed whether this platform technology can secrete sufficient levels of a therapeutic protein into the lungs to ameliorate a fatal pulmonary disease as an example of its translational capability. Pulmonary alveolar proteinosis (PAP) results from alveolar granulocyte-macrophage colony-stimulating factor (GM-CSF) insufficiency, resulting in abnormal surfactant homeostasis and consequent ventilatory problems. Lungs of GM-CSF knockout mice were transduced with a single dose of rSIV.F/HN-expressing murine GM-CSF ; mGM-CSF expression was dose related and persisted for at least 11 months. PAP disease biomarkers were rapidly and persistently corrected, but we noted a narrow toxicity/efficacy window. rSIV.F/HN may be a useful platform technology to deliver therapeutic proteins for lung diseases requiring long-lasting and stable expression of secreted proteins.  Efficient pulmonary gene transfer has been a limiting factor for development of gene therapy products targeting the lungs. This paper presents an efficient methodology for lung gene transfer and efficacy data for a life-limiting respiratory disease after a single dose of a novel lentiviral vector. This vector generates high transduction efficiency in a range of lung epithelial cells, including ciliated, goblet, and club cells as well as type 1 and 2 pneumocytes; a single dose produces expression for the lifetime of a mouse, and efficient generation of proteins is maintained after three or more transductions.,Efficient pulmonary gene transfer has been a limiting factor for development of gene therapy for chronic lung diseases such as cystic fibrosis (CF). To address this limitation, the UK Respiratory Gene Therapy Consortium  ranging from 0.1–100. mGM-CSF was quantified 4 days after treatment. Untreated cells and cells treated with rSIV.F/HN expressing a Gaussia luciferase (Glux) reporter gene (rSIV.F/HN-Glux) at an MOI of 100 were included as negative controls (n = 6/group). Dose-related mGM-CSF secretion was confirmed (Ex vivo transduction of fully differentiated human air-liquid interface (ALI) cultures at an MOI of ∼200  led to significant (p < 0.05) and persistent expression for at least 7 months after a single dose (To confirm that rSIV.F/HN transduction led to expression of murine GM-CSF (mGM-CSF) onfirmed A, reachigle dose B.Figure in vitro lentiviral vector transduction and compared with equal concentrations of a commercially available recombinant mGM-CSF protein (n = 6/group). Both proteins led to similar proliferation of FDC-P1 cells  cell proliferation assay. FDC-P1 myeloid cells were treated with known concentrations (0–10 ng/mL) of mGM-CSF produced after P1 cells C. These Dranoff et al.2). Respiratory mechanics, including elastance and pressure-volume (PV) curves, were also described for the first time. Differences in lung compliance, measured as the change in lung volume and pressure (PV curves), were not detected when comparing knockout and wild-type mice  scans were performed for knockout mice and age-matched littermates (n = 6/group). Although we observed a small but significant (p < 0.01) increase in lung density in GM-CSF knockout mice I, power ype mice J. Althouificance K and S1LGlux, n = 36) at the maximum feasible dose of 24e7 TU/mouse. The maximum feasible dose was defined as the maximum amount of vector (based on the vector concentration) that could be delivered in a 100-μL volume, the maximal allowable volume based on Home Office regulations.The lungs of PAP mice were treated with rSIV.F/HN-mGM-CSF via intranasal sniffing at doses ranging from 1e5 transduction units (TU)/mouse to the maximum feasible dose of 92e7 TU/mouse (n = 2–18/group) or with the irrelevant control vector  was detected in BALF and lung homogenates in mice treated with ≥ 1e7 TU/mouse A and 2B.x vector A and 2B.Glux) had any effect on the selected pulmonary biomarkers prior to embarking on a larger study. No significant changes were observed, suggesting that rSIV.F/HN-mGM-CSF was an appropriate vector to assess for therapeutic benefit  were reduced significantly (p ≤ 0.05–0.001) in rSIV.F/HN-mGM-CSF-treated PAP mice treated with 1e6 TU/mouse or greater, and SP-D levels in BALF were reduced significantly (p ≤ 0.01) with 1e5 TU/mouse or greater  and compared with rSIV.F/HN- greater D–2H.Glux (1e7 TU/mouse) and culled 1 week to 9 months after administration (n = 3–8/time point). mGM-CSF was detectable (p < 0.001) in lung tissue for the duration of the study (9 months) and in BALF for ∼2 months after transduction  as early as 1 week after transduction and remained significantly reduced (p ≤ 0.05) for the duration of the study (9 months after treatment) after a single administration of the vector C–3F.GM-CSF has several functions within the endogenous immune/inflammatory cascade, including differentiation of granulocytes  and monocytes/macrophages from precursors;In an initial dose-finding study, mice were treated with 1e7–92e7 TU/mouse. Glux-expressing control vector did not alter these baselines changes. Administration of rSIV.F/HN-mGM-CSF  led to dose-related histopathological changes in the lungs, liver, and kidneys , with histological assessment performed as described above. In the former group, we observed progressively more severe histopathological changes in the lungs 6–9 months after treatment, including tissue consolidation, inflammation, and increased numbers of pulmonary macrophages . As noted above, mice treated with these comparatively low doses showed significant improvements in PAP biomarkers even though levels of vector-induced mGM-CSF levels were below the lower limit of detection for the assay. Histopathological analysis at this time point showed no or moderate histopathology at 1e5 or 1e6 TU/mouse, respectively . We therHere we assess the feasibility of using a novel platform technology, rSIV.F/HN, to generate long-lasting protein secretion within murine lungs. As a therapeutic example, efficacy and toxicity were studied in a model of PAP, a disease with a considerable unmet need. mGM-CSF expression in murine lungs was dose related and persisted for the duration of the 11-month study after a single administration. Disease biomarkers were rapidly and persistently corrected. A narrow efficacy/toxicity window could be defined, with only low doses needing to be administered for therapeutic benefit, likely related to the long-lived mGM-CSF expression.,There is a considerable unmet need across a broad swathe of respiratory diseases of genetic and of multifactorial etiology; many of these are chronic and likely require sustained levels of therapeutic proteins. Recombinant proteins require frequent administration, are often associated with difficulties in achieving therapeutic levels at the required site of action, and are expensive. Thus, daily administration of inhaled GM-CSF resulted in modest improvements in pulmonary gas transfer and functional health status compared with placebo treatment in individuals with PAP;Reed et al.,We were readily able to achieve therapeutic GM-CSF doses over prolonged periods with our lentiviral vector. Above the relatively low titer of 1e7 TU/mouse, no further increases in pulmonary GM-CSF production or disease amelioration were seen. Although these data were encouraging at the 2-month time point, as the mice aged, we observed histopathology within and outside of the target organ. The histopathological changes observed were consistent with the known biological functions of mGM-CSF: differentiation of granulocytes and monocytes/macrophages from progenitor cellsin situ are required to stimulate macrophages to regulate surfactant levels. As the titer was reduced below 1e7, we could demonstrate a narrow efficacy/toxicity window and also noted a cumulative temporal effect of sustained GM-CSF production. These data suggest that only low titers of the rSIV.F/HN platform might be needed to generate therapeutic levels at the required site of action.Therefore, we next assessed the effect of lower viral titers. mGM-CSF levels were below the lower limit of detection (LLD) of the ELISA in BALF, lung homogenate, and serum in animals dosed with 1e6 TU/mouse, but a clear phenotypic effect could be seen. This is consistent with the fact that mGM-CSF levels in wild-type mice are also below the LLD for the ELISA, indicating that low levels of mGM-CSF produced ,This study was not focused on therapeutic translation, but because of the relatively narrow efficacy/toxicity window in mice, we took the opportunity to further refine the lentiviral vector cassette by removing its integration capability into the target cell genome , leading to more transient expression of GM-CSF or the option of using weaker or regulated promoters. However, a key element relates to whether efficacy and toxicity are altered in the presence of anti-GM-CSF autoantibodies, as is the case in man. Feretti et al.Here we have shown a proof of concept that a single administration of a lentiviral vector encoding a secreted protein can reverse the lung phenotype of a chronic respiratory disease. This may provide a useful platform technology for other chronic lung diseases requiring long-lasting exposure to a secreted therapeutic protein.Gaussia luciferase (Glux) cDNA under control of a hybrid elongation factor 1α promoter/Cytomegalovirus (CMV) enhancer were produced as described previously.,rSIV.F/HN carrying mGM-CSF (GenBank: AY950559) or Glux, or TSSM diluent buffer . mGM-CSF levels were quantified in medium using an mGM-CSF-specific ELISA  according to the manufacturer’s recommendations. The total protein concentration per well was quantified using cell lysates and Bio-Rad DC Protein Assay reagents . GM-CSF expression was expressed as picograms of mGM-CSF per milligram of total protein.Human adenocarcinoma alveolar basal epithelial cells  were cultured in complete medium  and transduced in Opti-MEM , with (1) rSIV.F/HN-mGM-CSF, (2) the control lentiviral vector rSIV.F/HN-Fully differentiated human air-liquid interface (ALI) cultures were purchased from Epithelix . ALIs were transfected with rSIV.F/HN-mGM-CSF  for 4 h in Opti-MEM or diluent control. Epithelial lining fluid was collected every 2–3 days by washing the apical surface of the ALIs with 100 μL PBS, and mGM-CSF levels were quantified by ELISA.in vitro by rSIV.F/HN-mGM-CSF (0.001–10 ng/mL), and values were compared with cells exposed to recombinant mGM-CSF . The “One Solution” cell proliferation assay (Promega) was used to estimate cell numbers according to the manufacturer’s recommendation.FDC-P1 cells  were exposed to increasing concentrations of mGM-CSF generated tm1Mlg/J, The Jackson Laboratory, Bar Harbor, Maine, USA) mice were used. Ages at the time of analysis ranged from 2–12 months, and treatment groups were generally aged matched. Mice were anesthetized with isoflurane  and transduced with rSIV.F/HN carrying mGM-CSF or the Glux reporter gene through nasal instillation of 100 μL total volume, which was then rapidly sniffed into the respiratory tract.All animal studies were approved by the Imperial College Animal Ethics Committee and carried out according to Home Office regulations. Wild-type  and GM-CSF knockout . Lung tissue was snap frozen in dry ice or fixed in 4% paraformaldehyde (PFA) for subsequent histopathological analysis. Other major organs were also collected and snap frozen for ELISAs and phosphatidylcholine (PC) and protein assays or fixed in 4% PFA for histopathological analysis. Bone marrow samples were isolated using a flushing technique, spun into cytospins , and the remaining cells were stored at −80°C in 10% dimethyl sulfoxide (DMSO) in fetal bovine serum (FBS).At the indicated time points, mice were culled through intraperitoneal injection of pentobarbital  and exsanguinated by femoral transection. Blood was collected from the heart, and whole-blood smears were prepared using standard procedures. The remaining blood was centrifuged at ∼7,200 × BALF turbidity was analyzed at 600-nm absorbance . Cells in BALF and from bone marrow were cytospun onto slides  and fixed with ice-cold methanol  for 5 min and left to air dry at room temperature. Slides were then stained with oil red O using standard procedures. Images (10/slide) were captured with an inverted light microscope . Using FIJI-ImageJ2 software, the size of wild-type and GM-CSF knockout mouse macrophages was measured .Snap-frozen lung tissue samples were thawed and homogenized using RNase/DNase-free Lysing Matrix D tubes  and cell lysis buffer 2 . Homogenized tissue was filtered through a QIAshredder disposable cell lysate homogenizer , and the supernatant was stored at −80°C. Total protein concentration of the lung homogenate was quantified using the Bio-Rad DC Protein Assay. The concentration of SP-D in BALF and lung homogenate was determined using the Quantikine ELISA Mouse SP-D Kit , following the manufacturer’s instructions. The concentration of mGM-CSF was determined using the Quantikine ELISA Mouse GM-CSF Kit, following the manufacturer’s instructions.This information is supplied in the H PAS 2) for automatic quantification of PAS-positive areas based on color intensity. Briefly, each montaged whole-lung coronal cross-section underwent color deconvolution to isolate only PAS-positive areas, which were enumerated and normalized against the total lung area to give a PAS percentage area readout. Surfactant deposition was defined as the percentage of PAS-positive area of the total lung area analyzed. In addition, other major organs were fixed in PFA, cut, and stained with H&E using standard procedures to quantify histopathological changes.PFA-fixed lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using standard procedures. A blinded, semi-quantitative analysis was undertaken. PAS staining was performed according to standard procedures to quantify surfactant deposition in the lungs. Images were captured and analyzed using ImageJ software, utilizing the color deconvolution macro . Given that there were no statistical differences between the control groups in the different experiments, data were combined as a historic control cohort. Except where specified, data are presented as median with interquartile range. Statistical analysis was performed using Mann-Whitney or Kruskal-Wallis test with Dunn’s correction for multiple comparisons, as appropriate. The null hypothesis was rejected at p < 0.05. Statistical analysis was performed using GraphPad Prism 8 software.The data originated from 7 individual experiments; each experiment included a concurrent irrelevant vector control group (rSIV.F/HN-


To investigate the value of cerebrospinal fluid chloride (CSF-Cl), cerebrospinal fluid glucose (CSF-GS), cerebrospinal fluid microalbumin (CSF-MALB), and cerebrospinal fluid adenosine deaminase (CSF-ADA) in the differential diagnosis of secondary hydrocephalus.  103 patients with secondary hydrocephalus treated in our hospital from January 2018 to April 2022 were selected. According to different types, it is divided into the hemorrhagic hydrocephalus group and tumor hydrocephalus group. By detecting the levels of inflammatory factors such as CSF-Cl, CSF-GS, CSF-MALB, and CSF-ADA in the two groups, we can analyze the value of these inflammatory factors in the differential diagnosis of secondary hydrocephalus. P < 0.05). There was no significant difference in the levels of CSF-Cl, CSF-GS, and CSF-ADA between the two groups (P > 0.05). The area under ROC curve (AUC) of CSF-Cl, CSF-GS, CSF-MALB, and CSF-ADA in the differential diagnosis of secondary hydrocephalus was 0.438, 0.553, 0.750, and 0.542, respectively, sensitivity was 15.1%, 45.3%, 79.2%, and 18.9%, respectively, and specificity was 96.0%, 80.0%, 80.0%, and 94.0%, respectively.  The level of CSF-MALB in the hemorrhagic hydrocephalus group was significantly higher than that in the tumor hydrocephalus group, and the difference between the two groups was statistically significant ( The inflammatory reaction of hemorrhagic hydrocephalus was significantly greater than that of tumor hydrocephalus. Moreover, CSF-MALB is closely related to the pathogenesis of hemorrhagic hydrocephalus. At the same time, CSF-MALB can be used as a good index for rapid differential diagnosis of secondary hydrocephalus. Secondary hydrocephalus is a common critical disease in neurosurgery. It often has the characteristics of acute onset, rapid progress, many complications, and high disability rate . If the Some studies have shown that there are differences in the expression of some inflammatory indexes in different neurosurgical diseases , 12. DetLaboratory tests are the gold standard for surgical diagnosis. We expect to identify different types of hydrocephalus through these laboratory inflammatory factors. However, there are few studies on this aspect at present. At present, there are few studies on the inflammatory indexes of cerebrospinal fluid chloride (CSF-Cl), cerebrospinal fluid glucose (CSF-GS), cerebrospinal fluid microalbumin (CSF-MALB), and cerebrospinal fluid adenosine deaminase (CSF-ADA) in the literature at home and abroad , 16. Then = 53) and tumor hydrocephalus group . There were 25 males and 28 females in the H-HCP group, aged 12–83 years, with an average age of 54.1 years. There were 17 males and 33 females in the T-HCP group, aged 2–73 years, with an average age of 36.9 years. There was no significant difference in gender, age, and course of disease between the two groups. Inclusion criteria were as follows: it meets the diagnostic criteria of hemorrhagic hydrocephalus and tumor hydrocephalus; CT or MRI showed enlargement of the ventricular system; the patients had no diseases such as cardiopulmonary disease and diabetes; and the patient's clinical data are complete. Exclusion criteria were as follows: the patient has heart lung disease or diabetes; the patient does not cooperate with the treatment or cannot perform lumbar puncture; and the patient's clinical data are incomplete.103 patients with secondary hydrocephalus treated in the Affiliated Hospital of Guangdong Medical University from January 2018 to April 2022 were selected. According to different etiological types, they were divided into the hemorrhagic hydrocephalus group , we analyzed the value of CSF-Cl, CSF-GS, CSF-MALB, and CSF-ADA in the differential diagnosis of secondary hydrocephalus. P < 0.05 was statistically significant.The study used SPSS statistical software for data analysis. The counting data were analyzed by the chi-square test and Fisher exact probability method. The measurement data are expressed as mean ± standard deviation, and the measurement data of two independent samples are tested by the P < 0.05). There was no significant difference in CSF-Cl, CSF-GS, and CSF-ADA between the two groups (P > 0.05)  .The area under ROC curve (AUC) of CSF-Cl, CSF-GS, CSF-MALB, and CSF-ADA in the differential diagnosis of secondary hydrocephalus was 0.438, 0.553, 0.750, and 0.542, respectively, sensitivity was 15.1%, 45.3%, 79.2%, and 18.9%, respectively, and specificity was 96.0%, 80.0%, 80.0%, and 94.0%, respectively .The treatment plan and prognosis of secondary hydrocephalus are related to different causes , 22. EarP > 0.05). At the same time, we found that AUC was 0.438 (ACU <0.7), sensitivity was only 15.1%, and specificity was 96.0%. This shows that the accuracy of CSF-Cl in the diagnosis of secondary hydrocephalus is not high, and its sensitivity is not strong, which is inconsistent with the standard that we need to screen different types of secondary hydrocephalus. CSF-Cl cannot quickly distinguish hemorrhagic hydrocephalus from neoplastic hydrocephalus.The normal value of CSF-Cl is the normal value of the chloride content in cerebrospinal fluid and the reference range of the normal value of chloride in cerebrospinal fluid , 28. CalThe normal value of CSF-GS is 2.5–4.4 mmol/L , 30. TheCSF-MALB is a sensitive index and specific inflammatory factor in the early stage of blood-brain barrier permeability change and participates in the process of immune regulation. When the blood-brain barrier is destroyed, cerebrospinal fluid microalbumin is very easy to enter cerebrospinal fluid from blood by relying on a large concentration difference. Therefore, it has high sensitivity and can be used as a specific evaluation index for the initial change of blood-brain barrier permeability. The results of this study showed that there were differences in CSF-MALB levels between the hemorrhagic hydrocephalus group and tumor hydrocephalus group. The level of CSF-MALB in the hemorrhagic hydrocephalus group was significantly higher than that in the tumor hydrocephalus group. The difference between the two groups was statistically significant. It shows that hemorrhagic hydrocephalus destroys the blood-brain barrier more than tumor hydrocephalus, and the inflammatory response of hemorrhagic hydrocephalus is greater than tumor hydrocephalus. Therefore, the level of CSF-MALB in cerebrospinal fluid will be greater. The AUC was 0.750 (AUC >0.7), sensitivity was 79.2%, and specificity was 80.0%. It shows that CSF-MALB has certain accuracy in diagnosing the types of secondary hydrocephalus, and its sensitivity and specificity are high. It can be used as a biochemical measurement index to distinguish the two types of secondary hydrocephalus: bloody hydrocephalus and tumor hydrocephalus.CSF-ADA is an important enzyme in the purine nucleoside metabolism. CSF-ADA is a sensitive index reflecting brain injury. It can be used as one of the routine examination items of brain function and can reflect the enzymatic changes of central nervous system function. In the process of secondary hydrocephalus, CSF-ADA is also one of the specific indicators of inflammation in secondary hydrocephalus. By comparing the HCP group and CSF-ADA group, we found that there was no significant difference between them. The AUC of CSF-ADA was 0.542 (AUC <0.7), sensitivity was 18.9%, and specificity was 94.0%. This shows that CSF-ADA is not accurate in distinguishing the types of secondary hydrocephalus, and its sensitivity is not high. It cannot be used as a biochemical measurement index to distinguish hemorrhagic hydrocephalus and neoplastic hydrocephalus.In conclusion, the inflammatory reaction of hemorrhagic hydrocephalus was significantly greater than that of tumor hydrocephalus. CSF-MALB is involved in the change of disease mechanism of secondary hydrocephalus. The detection of the CSF-MALB level can provide a basis for the early identification of the types of secondary hydrocephalus and the evaluation of the disease, which is helpful to guide the clinical medication and surgical treatment.


H3 K27M somatic variant in cerebrospinal fluid (CSF) by using digital polymerase chain reaction (dPCR). Optimization of the protocol was carried out stepwise, including preamplification of the H3 target region and adjustment of dPCR conditions. The optimized protocol allowed detection of the mutant allele starting from DNA quantities as low as 9 picograms. Analytical specificity was tested using a representative group of tumor tissue samples with known H3 K27M status, and no false-positive cases were detected. The protocol was applied to a series of CSF samples collected from patients with CNS tumors (n = 18) using two alternative dPCR platforms, QX200 Droplet Digital PCR system (Bio-Rad) and QIAcuity Digital PCR System (Qiagen). In three out of four CSF specimens collected from patients with H3 K27M-positive diffuse midline glioma, both platforms allowed detection of the mutant allele. The use of ventricular access for CSF collection appears preferential, as lumbar CSF samples may produce ambiguous results. All CSF samples collected from patients with H3 wild-type tumors were qualified as H3 K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach.Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the  High-grade gliomas are rare tumors which constitute 10%–15% of the central nervous system (CNS) neoplasms in pediatric patients . HoweverH3 K27M variant in diffuse midline gliomas (DMG). This variant is observed in up to 94% of diffuse brainstem gliomas, 65% of thalamic gliomas, and 60% of spinal cord gliomas [H3 K27M mutation is a very unfavorable prognostic factor. Accordingly, a distinct nosological entity ‘Diffuse midline glioma H3 K27M-mutant’ was added to the 2016 update of the WHO classification of CNS tumors [H3F3A mutational status. Importantly, the detection of H3 K27M mutation in an infiltrating midline glioma implicates assignment of the tumor as WHO grade IV by definition.Basic findings of the last decade have substantially advanced the understanding of molecular pathogenesis of glial tumors in pediatric patients. Wu et al. identified K27MH3 K27M substitution, in particular, is a prerequisite for the inclusion in currently running trials of anti-DRD2 agent ONC201 as a therapeutic option for H3 K27M-mutant DMG . In this regard, the possibility of using liquid biopsy—an approach based on the presence of cell-free DNA (cfDNA) of tumor cells in biological fluids—is of particular interest. Repeated sampling of body fluids  provides access to the dynamics of genetic aberrations arising at different stages of tumorigenesis and allows monitoring of the therapy effectiveness [Molecular profiling of malignant gliomas, enabled by modern advances in molecular oncology, allows ultimate specification of the diagnosis and selection of optimal therapies targeted at specific aberrations. Molecular inclusion criteria are increasingly used in clinical trials. The EGFR mutations in plasma specimens was approved by FDA as a companion diagnostic test in non-small cell lung cancer patients with poorly accessible tumors [EGFR mutations determined by using either liquid or conventional biopsy showed similar survival rates [PIK3CA variants in cfDNA isolated from plasma of the patients with ER-positive HER2-negative locally advanced or metastatic breast cancer [Plasma is certainly the most common source of cfDNA for analysis. Representation of somatic mutations in plasma has been demonstrated for various solid tumors . In 2016e tumors . Liquid al rates . In 2019t cancer . For patt cancer ,12.Examination of cerebrospinal fluid (CSF) samples is a promising diagnostic tool for neurooncology. However, the search for tumorigenic genetic aberrations in CSF is nontrivial due to the low concentrations of cfDNA ,14. The H3 K27M variant in cfDNA isolated from CSF of pediatric patients with CNS tumors.The aim of this study was to develop a reliable cross-platform dPCR protocol for detection of n = 25) was intended for the protocol development and its analytical characterization on formalin fixed/paraffin embedded (FFPE) tumor tissues. For a prospective cohort (n = 18), FFPE and CSF samples were matched for diagnostic characterization and clinical validation of the protocol. All patients of the prospective cohort, presenting with newly diagnosed or recurrent radiographically-confirmed CNS tumors, were enrolled in the Molecular Diagnostics in Pediatric Neurooncology and Liquid Biopsies Project at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. An exclusion criterion was complete surgical resection (R0) determined on the basis of postoperative MRI data, unless CSF sampling has occurred during or before surgery. The Informed Consent for participation in the study was obtained from parents/guardians of all patients. The study was approved by the Institutional Review Board at the Dmitry Rogachev Center.Overall design of the study is presented in n = 19), collected either intraoperatively, or through a ventricular access device, or by lumbar puncture, were clarified by centrifugation at 1,100 g for 10 min and stored in 1 ml aliquots at −80°C. The samples were thawed immediately before cfDNA isolation. Cell-free DNA was isolated with the use of QIAmp Circulating Nucleic Acid Kit . Concentrations of amplifiable fragments were measured by DNA Fragmentation Quantification Assay . The cfDNA profiles were analyzed with the use of Cell-free DNA ScreenTape Assay Reagent Kit (Agilent Technologies).Clinical and analytical data for CSF donors (prospective cohort) are summarized in n = 25, H3F3A K27M status determined by Sanger sequencing  for FFPE tumor tissues specimens of the retrospective cohort n = 25,  with thequencing . GenomicH3F3A target in cfDNA, the preamplification step was introduced to increase the sensitivity of dPCR assay. The reactions were set in 25 µl of 1× PCR buffer B  containing 50 nM of primers, 0.25 mM dNTPs, 2.5 mM MgCl2 and 0.1 U/µl Taq polymerase (Syntol LLC) with 50–100 copies of 150 bp DNA fragments per reaction. Preamplification was run in a T100 thermal cycler (Bio-Rad) at 98°C for 1 min, followed by eight cycles of 98°C for 10 s, 58°C for 30 s and 72°C for 30 s, followed by 72°C for 2 min. The product was diluted 1:1 with TE buffer and used in dPCR with LNA-modified allele-specific probes. The reactions were set in duplicates, each comprising 20 µl of 1× ddPCR Supermix for Probes (No dUTP)  with the primers and probes added to final concentrations of 1,800 nM and 500 nM, respectively. The process was run in a C1000 Touch Thermal Cycler (Bio-Rad) equipped with a QX200 Droplet Digital PCR (ddPCR) system (Bio-Rad) at 95°C for 10 min, followed by 40 cycles of 94°С for 30 s (ramp 2°C/s) and 57°C for 60 s (ramp 2 °C/s), followed by 98°C for 10 min. The data were processed and analyzed in QuantaSoft Version 1.6.6.0320 (Bio-Rad). Fluorescence thresholds  were set manually.Due to the expected low concentrations of the H3 K27M: 5′-GGTAAAGCACCCAGGAAG-3′; reverse H3 K27M: 5′-CAAGAGAGACTTTGTCCC-3′; wild-type probe: 5′-HEX-TC+GC+A+A+GA+GT+GC-RTQ1-3′; K27M probe: 5′-6-FAM-TC+GC+A+T+GA+GTGC-RTQ1-3′, with “locked” nucleic acid-modified (LNA) bases indicated by+. LNA-modified allele-specific probes are known to increase dPCR specificity. Distinctive structural feature of LNA residues is the methylene bridge between atomic positions O2′ and C4′ of furanose, which locks the sugar in 3′-endo conformation thus enhancing hybridization of the probe with the template. Introducing LNA-monomers into oligonucleotide sequences significantly improves their discriminating capacity in the recognition of single-nucleotide substitutions [Oligonucleotide sequences of primers and probes previously published in  were as itutions .The optimized protocol was tested for compatibility with the nanoplate-based digital PCR system (Qiagen) technologically different from the water-oil emulsion droplet-based PCR platform (Bio-Rad). The mixtures were set up in duplicates with QIAcuity Probe PCR Kit in Nanoplate 8.5K 96-well plates (Qiagen). The reactions, containing 3 µl 4× Probe PCR Master Mix, 1.2 µl primer–probe mix, 5 µl DNA template, and 2.8 µl RNase-free water in 12 µl total volume each, were run in a QIAcuity One instrument (Qiagen). The analysis was carried out with the use of QIAcuity Software Suite (Qiagen). Fluorescence thresholds for optimal resolution and cloud clustering, selected manually to fit all reactions, constituted 40 and 20 RFU for FAM and HEX, respectively. To enhance the accuracy of concentration measurements, the Volume Precision Factor (VPF), adjusting for tiny variations in nanoplate geometry, was applied as recommended by the manufacturer.10 copies/µl. Non-paired measurements  were excluded from the analysis.To quantitatively describe the dependence of dPCR outputs on reaction parameters (preamplification cycle number and template copy number), regression lines and determination coefficients were calculated in Microsoft Excel 2016. Bland–Altman test was used to assess quantitative agreement between the Droplet Digital™ PCR (Bio-Rad) and the QIAcuity™ Digital PCR System (Qiagen) based on the mean difference and standard deviation. The estimated concentrations of the target in starting samples were expressed in logH3F3A target preamplification protocol, as well as ddPCR settings and conditions, were optimized in order to increase the analytical sensitivity and specificity of the H3 K27M variant determination. The optimality was defined as a combination of high number of positive droplets with robust cloud separation and minimal “rain” effects (a fraction of droplets showing sub-threshold fluorescence).The H3F3A fragment. The analyses were set in duplicates for each sample. The data indicate that eight cycles of preamplification of the H3F3A gene fragment are necessary and sufficient for optimal cluster density and accurate interpretation of the results of ddPCR with 50–100 copies of the template at the start of preamplification. Representative examples of primary ddPCR data for different reaction conditions are shown in The optimization was carried out by varying preamplification cycle number , annealing temperatures and concentrations of the oligos, as well as the number of DNA copies at the start of the reaction. The exponential dependence of the ddPCR-detectable amount of DNA target on the preamplification cycle number  allows sH3 K27M substitution. Such results were considered as false positive (ambiguous sample). A sample containing at least 50 droplets with above-the-threshold fluorescence in the FAM channel and reproducible in two technical replicates was considered positive. In contrast, a sample containing more than 50 droplets with above-the-threshold fluorescence in the HEX channel and less than 50 droplets with above-the-threshold fluorescence in the FAM channel in the total number of duplicates and reproducible in two technical replicates was considered negative.The protocol was further optimized by adjusting annealing temperatures of ddPCR primers (within the range of 53–61°C) and final concentrations of the primers and probes in the reactions. Oligonucleotide concentrations were tested extensively within the range of 900–2,700 nM with a step of 300 nM for the primers and 250–750 nM with a step of 125 nM for the probes. Reducing the probe concentration to values below 500 nM resulted in the reporter fluorescence in HEX channel too low for reliable cloud separation. Elevated concentrations of oligonucleotides in the mixture increased the overall intensity of fluorescence but negatively affected specificity of the reaction. High concentration of the FAM-labeled fluorescence probe specific for the H3 K27M allele led to the non-specific annealing of the probe to the normal allele. This resulted in the ambiguous appearance of the small amount  of positive droplets with fluorescence above 2,000 RFU in the FAM channel in samples with absence of the The optimal fluorescence levels and maximal ddPCR efficiency were achieved at eight cycles of preamplification with 50–100 copies of DNA template and annealing temperatures of 58°C and 57°C for preamplification and ddPCR, respectively. The clearest cluster separation efficiency was achieved with 1,800 nM of the primers and 500 nM of the probes. The uniform threshold values of the fluorescence intensity, above which the droplets were regarded as positive, were set manually. The thresholds for FAM and HEX signals constituted 2,000 and 1,500 RFU, respectively. Typical results of ddPCR obtained before and after the optimization experiments are shown in H3 K27M variant, with concentrations descending from 600 to 1 pg/μl of DNA at the start of preamplification. The reactions were carried out in two technical replicates. The sensitivity threshold was determined by minimal dilution affording at least 50 droplets with above-the-threshold fluorescence in at least one of the channels in total counts for the duplicates (H3 K27M variant determined with the use of optimized ddPCR protocol was 9 pg of DNA (corresponding to 3 haploid genomes).Analytical sensitivity of the modified ddPCR protocol was tested using a series of 10 sequential two-fold dilutions of genomic DNA comprising the studied plicates . The limH3 K27M status determined by Sanger sequencing  or lumbar CSF (pt. 9). For pt. 11, both ventricular and lumbar CSF samples were available. Interpretation of the results for ventricular CSF was straightforward whereas analysis of the lumbar sample produced ambiguous results—solitary high-intensity signals in two channels  independently of dPCR platform. Comparative representation of these data is given in In sum, 14 ventricular (6 collected intraoperatively and eight from a ventricular access device), and 5 lumbar CSF samples were analyzed, corresponding to 18 patients with CNS tumors grade I–IV. In three out of four For pt. 10, only lumbar CSF with extremely low yields of cfDNA was available for the analysis, producing the same uninterpretable result despite preamplification.H3 K27M-negative tumors (n = 11) produced negative results in dPCR for H3 K27M substitution, which indicates high diagnostic specificity of the developed protocol.All CSF samples from patients with H3 K27M mutant allele into the analyzed pool of molecules increases. To describe the influence of the initial abundance of the mutant allele on the ddPCR outcome, the H3F3A target was amplified from 65, 50, 25, or 12 copies of the template (sample #9) at the start of preamplification. Comparative representation of the outcomes for different amounts of the template is given in The accuracy of quantitative analysis for samples containing small amounts of genetic material (10–100 copies of DNA template) is limited by stochastic sampling. For small DNA copy numbers, the variation in the number of mutant alleles taken from the extracted volume of cfDNA and introduced into the preamplification reaction is described by the classical Bernoulli test scheme, and for copy numbers of 100 and above, according to the Moivre-Laplace limit theorem, by the Gaussian  distribution . In ordit can be calculated as follows:n is DNA copy number after PCR, k—DNA copy number at the start of the reaction, v—reaction rate, t—time, and f(k) is a threshold function of k, f(k) = {0 at k < h; 1 at k≥h}, with h regarded as the threshold value.The number of copies of the DNA fragment-of-interest produced in the reaction at time k = 65 DNA copies at the start of the reaction corresponds to n = 1,574 positive droplets in the FAM channel, we get exp(tv) = 24.2. Accordingly, at k = 50, the expected number of amplified molecules will be n = 1,210, but only if k is not below the threshold value h; otherwise, n = 0. Thus, the empirical value of n = 3 is an artifact and should be replaced with a zero value. Similarly, at k = 25, the expected number of amplified copies will be n = 605, and at k = 12 it will be n = 290. In accordance with this model, the numbers of positive droplets  are valid only for the case of 65 copies of the template at the start of the reaction. However, this value could vary depending on VAF in the tumor and should not be considered as a threshold level, but only as the explanation for the appearance of single positive droplets. It’s important to note that in the described case #9 H3 K27M VAF was relatively low—14%.The known number of DNA copies at the start of the reaction enables estimation of the expected number of amplified DNA fragments. Considering that ETV6-NTRK3-positive (sample #1), posterior fossa ependymoma, group A (sample #7), diffuse pediatric-type high-grade glioma, H3-wildtype, and IDH-wildtype (sample #8) and pilocytic astrocytoma, KIAA1549 (exon 15)—BRAF (exon 9)—positive (sample #14). The presence of such droplets confirms their artefactual origin. The explanations of the appearance of the solitary droplets are either spontaneous cleavage of the fluorescence probe or its unspecific binding to the wild-type allele.The appearance of the low amount (<2) of the positive droplets has been seen after preamplification in several samples with confirmed H3 wild type status belonging to patients with the infant-type hemispheric glioma, H3 K27M altered tumors provided adequate positive results beginning from significantly lower amounts of the input DNA  compared to 65 copies/µl. Remarkably, the VAF in both of this cases were higher , comparing to the sample #9. Single mutant allele copies in the starting material could provide solitary positive droplets (samples#10 and 11.5), but extremely low overall cfDNA input prevents an unambiguous interpretation of the results.At the same time, CSF derived cfDNA samples from patients with The optimized protocol was tested using an alternative digital PCR technology platform (QIAcuity™) to evaluate its universality. Analytical consistency between the platforms was evaluated using Bland–Altman method. The analysis was performed on cfDNA with and without preamlification step. A total of 43 paired measurements were analyzed; four measurements, performed on a single dPCR platform, were excluded from the analysis given the lack of pairness. Due to high volume of CSF collected from patients 11 and 12, nucleic acids were isolated in quadruplicates and in duplicates, respectively ; see 10 copies/µl, with a standard deviation (SD) of 0.15 log10 copies/µl. For the vast majority of measurements , the results showed marked pairwise consistency of concentration values for mutant and wild-type alleles obtained with the two platforms, falling within the ±1.96 SD limit of agreement (−0.12…0.46 log10 copies/µl). The rest of the differences  were outside the limits of agreement. For three of these outliers (two wild-type and one comprising the mutation), concentration values obtained with QIAcuity exceeded the differences, ranging from 0.52 to 0.60 log10 copies/µl. For one of the outliers, the concentration of mutant allele obtained with QX200 was higher (−0.22 log10 copies/µl). It should be noted that all differences falling outside the limits of agreement corresponded to non-preamplified cfDNA. Concentration values obtained with the use of preamplification had lower discrepancy between the platforms (−0.01…0.37 vs. −0.22…0.6 log10 copies/µl).Bland-Altman analysis  showed tH3F3A K27M mutant and H3F3A wild-type target quantification obtained with the QIAcuity dPCR system were comparable with those obtained with the QX200 ddPCR platform, which indicates universality of the developed protocol.Overall, the results of Current standards for the diagnosis and management of CNS tumors include histological examination of tumor tissue followed by search for molecular markers . Cell-frex vivo CSF, concentration and quality of CSF-derived cfDNA should be expertly evaluated prior to genetic testing. It should be noted that representation of mutant alleles in cfDNA is normally reduced due to 1) intra-tumor heterogeneity and 2) the admixture of non-tumor cfDNA. According to Pan et al., concentrations of cfDNA obtained from CSF of patients with CNS tumors fall within a wide range of 5.5–1,845.0 pg/μl with a median of 25 pg/μl; notably, for high-grade tumors, diagnostic sensitivity to somatic variants in cfDNA is significantly higher [However, the use of liquid biopsies for CNS tumors is challenging. Firstly, there are no clear indications for molecular examination of CSF. The efficacy depends on combinations of many factors, including tumor characteristics , as well as the methods for CSF collection (ventricular access or lumbar puncture) and its timing . Secondly, in connection with the low stability of cells and cell-free nucleic acids in y higher . Wang ety higher . In our As reported by Zhao et al., patients with glial tumors invading ventricular cavities or bordering subarachnoid cisterns have significantly higher CSF levels of cfDNA than patients with similar tumors confined within the anatomical boundaries of nervous tissue . Connollcf. 7.31 pg/μl, respectively [.In the context of liquid biopsies, the topography of CSF sampling matters. Undoubtedly, the use of CSF samples obtained by relatively non-invasive methods from patients with suspected brain tumors would be a preferable option. Presence a ventriculoperitoneal shunt enables CSF collection with the minimal invasive burden. However, a closer look at published evidence on liquid biopsies from CNS tumors reveals the predominance of intraoperative sampling ,19-21. Mectively ), hence IDH1, TERT, or H3 genes in six of seven high-grade glioma cases [. At the same time, On et al. were able to unequivocally identify H3 K27M mutation in only 1 of 10 CSF samples obtained by lumbar puncture from newly diagnosed DMG patients; for three other cases in this cohort, the result was ambiguous, as only 1-2 mutant droplets were observed in ddPCR charts [H3 K27M-positive DMG patients, similar to those described by On et al. [Despite the significantly reduced concentrations of cfDNA in CSF derived by lumbar puncture, Fujioka et al. detected mutations in ma cases . At the R charts . In our n et al. —with lowIn addition to the difficulties in CSF sample collection and primary handling, their advanced molecular analysis is sophisticated. Modern strategies for analysis of somatic mutations in cfDNA are based on the use of targeted high-throughput sequencing -14,19-21H3 mutant and wild-type templates, obtained with alternative dPCR platforms by Bio-Rad and Qiagen, showed lower discrepancy upon the use of preamplification which, for its part, had no influence on the VAF estimates by dPCR.Preamplification step can be introduced in the protocol in order to increase representation of the target at the start of dPCR. This adjustment to the protocol is especially recommended for low quantities of starting material, as it ensures higher sensitivity of the analysis via target-specific enrichment. Increased numbers of target molecules at the start of the reaction allows it to outrun the background resulting from trace degradation of the probe . The conH3F3A K27M in three patients with DMG, with VAFs within the range of 7.0–17.3% at cfDNA concentrations within the range of 4.8–37.5 pg/μl (with a median of 7.25 pg/μl) [H3 K27M variant in liquid biopsies of high-grade glioma patients (n = 4) with significantly higher VAFs in cfDNA (18–65%) [H3 K27M in lumbar and ventricular samples of CSF collected at autopsy from a patient with diffuse intrinsic pontine glioma. Indeed, concentration of target H3 K27M variant in CSF collected from lateral ventricles was 2.5 times higher than in lumbar liquor  [Martínez-Ricarte et al. studied the mutational profile of CSF-derived cfDNA for a cohort of 20 patients with diffuse glioma. By using ddPCR, the authors identified 5 pg/μl) . In a st(18–65%) . It shouctively) ,16.H3 K27M somatic variant in CSF of pediatric patients with CNS tumors. The optimized universal dPCR protocol allows detection of solitary copies of the mutant allele with excellent analytical sensitivity. Thorough analytical characterization of cfDNA pools in terms of fragment lengths and concentrations is required to diminish the risk of false negatives. This requirement, along with insufficient quality and low representation of tumor DNA in CSF, limit standardization of the approach and its clinical prospects. Altogether, ventricular CSF sampling is more informative compared with lumbar sampling. Multicenter study designs are needed to evaluate clinical relevance of CSF liquid biopsies for rare CNS tumors.The study provides a reliable tissue-validated technical basis for detection of


Brain metastases (BM) include brain parenchymal (BPM) and leptomeningeal metastases (LM), which are associated with a poor prognosis and high mortality rate. Early and accurate diagnosis and timely, effective treatment are crucial for improving the overall survival of LM patients. Cerebrospinal fluid (CSF) biopsy technology has attracted widespread attention for its diagnostic value in diverse cancers, including LM. We summarized studies to compare the potential diagnostic value of CSF liquid biopsy techniques in BM patients with meta-analysis.The study protocol was prospectively registered in PROSPERO, registration number CRD42022373263. We obtained the literature on liquid biopsy for BM from 7 databases . Then, a systematic review of those studies was performed according to PRISMA criteria.Nine publications have been obtained, and we found CSF liquid biopsy techniques to be more suitable for diagnosing LM. We analyzed the sensitivity, specificity, and area under the curve (AUC) of CSF liquid biopsy. The overall sensitivity, specificity, and AUC of CSF liquid biopsy in the diagnosis of LM were 0.65 (95% CI: 0.48 - 0.79), 0.70 (95% CI: 0.50 - 0.86), and 0.69, respectively. Then, we compared the diagnostic advantages of CSF liquid biopsy techniques and CSF cytology in LM. The results show that CSF liquid biopsy is superior to CSF cytology in LM diagnosis.Our meta-analysis suggested that CSF liquid biopsy is more suitable for LM diagnosis and has higher accuracy than CSF cytology. Brain metastasis (BM) is an intracranial tumor that results from malignant tumors elsewhere in the body that metastasize to the brain. In the central nervous system (CNS) tumors, as a neurological complication of systemic malignancies, the incidence of BM accounts for about 20% of CNS malignancies . The rapCurrent advanced neuroimaging techniques could help to identify BM but are insufficient to make a definitive diagnosis . SurgicaBM include brain parenchymal metastases (BPM) and leptomeningeal metastases (LM). Liquid biopsy techniques are of great value in diagnosing BM. However, studies have shown that fluid biopsy of cerebrospinal fluid (CSF) appears more promising in LM due to anatomical differences between BPM and LM . TherefoWe scoured in PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI), and Wanfang Data knowledge service platform for published studies on liquid biopsy and brain metastases as of July 2022. We searched PubMed using the following strategy: (“brain metastasis”[Title/Abstract] OR “brain metastases”[Title/Abstract] OR “metastatic brain tumour”[Title/Abstract]) AND (“ctDNA”[Title/Abstract] OR “cfDNA”[Title/Abstract] OR “circulating tumor dna”[Title/Abstract] OR “circulating free dna”[Title/Abstract]). We searched Embase using the following strategy: brain metastasis OR brain metastases OR metastatic brain tumour AND ctDNA OR cfDNA OR circulating tumor DNA OR circulating free DNA. We searched Web of science using the following strategy: (TS=(brain metastasis) OR AB=(brain metastases OR metastatic brain tumour)) AND (TS=(ctDNA) OR AB=(cfDNA OR circulating tumor DNA OR circulating free DNA)).Inclusion criteria were as follows (1): studies related to BM and liquid biopsy techniques (2); Provides the diagnostic ability of liquid biopsy technology for BM; (3) All patients with BM have been diagnosed; (4) Data related to BM and liquid biopsy techniques, including true positive (TP), false positive (FP), false negative (FN), and true negative (TN), can be extracted or calculated from the article. The exclusion criteria are: (1) non-human experimental studies; (2) Studies unrelated to BM and liquid biopsy techniques; (3) duplicate articles or data, as well as studies with insufficient data; (4) Abstracts, letters, editors, expert opinions, reviews, case report types of articles.We independently assessed the quality of the included studies using the 9-star Newcastle-Ottawa Scale (NOS). We scored the included studies based on three aspects of the NOS assessment . We also assessed the quality of diagnostic studies by applying the diagnostic accuracy study quality assessment-2 (QUADAS-2) criteria. The QUADAS-2 included 14 items . Each doTwo reviewers independently extracted all data from the included studies using standardized forms and double-checked the data. We discussed all the disputes and had them resolved by a third reviewer.2I to assess statistical heterogeneity. We used 2I to assess statistical heterogeneity, with 2I > 50% indicating significant heterogeneity and 2I < 50% indicating that heterogeneity was not significant. If 2I > 50%, the random effects model is used for analysis and further performed sensitivity analysis. If 2I < 50%, the fixed-effect model is used for analysis without a sensitivity analysis. P values<0.05 was considered statistically significant.We used Review Manager 5.3  and Meta-disc to perform statistical analysis of all data. The sensitivity, specificity, positive and negative likelihood ratios (PLRs and NLRs), diagnostic advantage ratios (DORs), and 95% confidence intervals (CI) of the data were analyzed by applying Meta-disc software. We used We obtained 215 articles by searching databases such as PubMed, EMBASE, Web of Science, CNKI, and Wanfang Data knowledge service platform. By reviewing the title and abstract of the article, we excluded 3 duplicate articles and 179 articles that did not meet the inclusion criteria. We then read the full text of the remaining 33 articles, excluding 24 articles according to the exclusion criteria and incorporating 9 articles . This analysis was performed using a fixed-effects model and did not require sensitivity analysis because there was no significant heterogeneity between studies . These data are shown in CSF biopsy techniques offer superior advantages in diagnosing LM than BPM, and the difference is statistically significant  and 0.70 (95% CI: 0.50 - 0.86), respectively. The pooled PLR was 1.94 (95%CI:1.04 – 3.63), NLR was 0.54 (95%CI:0.32 - 0.92), and DOR was 3.67 (95%CI: 1.22 - 11.07) . Because of significant heterogeneity between studies , random effects models were used. These data are shown in CSF liquid biopsy is superior to CSF cytology in the diagnosis of LM, and the difference is statistically significant (OR2I = 54% > 50%), so we need to perform a sensitivity analysis. Sensitivity analysis found that Fitzpatrick’s research data was a dominating source of heterogeneity. After excluding Fitzpatrick’s studies, there was no statistical heterogeneity between the studies . This meta-analysis, again analyzed using fixed-effects models, showed that CSF liquid biopsy was still superior to CSF cytology in LM diagnosis  . We supposed that that CSF liquid biopsy is more accurate and valuable in diagnosing LM. Some previous studies also support our results. De Mattos-Arruda et al. found that CSF ctDNA analysis is more sensitive than CSF cytology in detecting LM , specificity of 0.70 (95% CI: 0.50 - 0.86), and AUC of 0.69 for the CSF liquid biopsy technique in the diagnosis of LM. The results suggest that CSF biopsy has a certain degree of sensitivity, specificity, and diagnostic accuracy in LM diagnosis. DOR is the value of the combination of sensitivity and specificity, and the higher the DOR value, the better the diagnostic ability. The DOR is 3.67 (95%CI: 1.22 - 11.07), indicating that the overall merger accuracy is not high. The pooled PLR was 1.94, indicating a 1.94-fold increase in the probability of LM occurring when CSF liquid biopsy was positive. In addition, the NLR was 0.54, which means that when the CSF liquid biopsy was negative, the probability of developing LM increased by 46%. PLR>10 and NLR< 0.1 represents a high diagnostic accuracy . These rcting LM . White ecting LM . Based octing LM . Targetecting LM . With thThere are some limitations to our study. First, despite our efforts to search for relevant studies, we may still have overlooked some studies that were not published online. Second, studies with positive results are more likely to be published, which may improve the accuracy of the overall diagnosis. Third, because of the CSF liquid biopsy technique is a broad concept, there are different methods of detecting CSF among the included studies, which may increase heterogeneity among the included studies and affect the reliability of these findings. In addition, the primary tumors and test indicators of LM patients included in this study may be different, which may also cause heterogeneity between studies and affect the reliability of the results. Finally, the small number of studies included in this study leads to the limited statistical power of these results.Our results suggested that CSF liquid biopsy are more suitable for diagnosing LM than that in BPM. In addition, we found that the overall diagnostic value of CSF liquid biopsy is low, and the diagnostic accuracy for LM is not as high as expected. However, by comparing the diagnostic advantages of the CSF liquid biopsy and CSF cytology in LM, our result showed that CSF liquid biopsy is superior to CSF cytology. Future studies are warranted to confirm our analysis.Conception and design: CL, LZ. Acquisition of data: HW, LW. Drafting the article: HW, LW. Critically revising the article: CL, LZ, and CF. All authors contributed to the article and approved the submitted version.