Periorbital purpura can be the only initial symptom of primary light chain amyloidosis:A case report

BACKGROUND Primary light chain amyloidosis is a rare and complex disease with complex clinical features and is highly susceptible to misdiagnosis and underdiagnosis in the early stages.CASE SUMMARY We report a case of a 47-year-old female patient whose only initial symptom was periorbital purpura,which was not taken seriously enough.As the disease progressed,pleural effusion gradually appeared,and after systematic diagnosis and treatment,she was diagnosed with“primary light chain amyloidosis”.She achieved rapid hematological remission after treatment with a daratumumab+bortezomib+cyclophosphamide+dexamethasone regimen.CONCLUSION Periorbital purpura can be the only initial symptom of primary light chain amyloidosis;we should pay attention to the cases where the initial clinical symptoms are only periorbital purpura.

Preliminary research on myosin light chain kinase in rabbit liver

AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction(RT-PCR); the MLCK was obtained from rabbit liver, and its activity was analyzed by gamma-(32)P incorporation technique to detect the phosphorylation of myosin light chain. RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg x L(-1), the activity was at the highest level. CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.

IMMUNOLOGICAL DIAGNOSIS OF LIGHT CHAIN DISEASE ANALYSIS OF 11 CASES FOUND IN FUJIAN PROVINCE

The present paper reports 11 cases of light chain disease (LCD) sequently found in several citles over Fujian province, Immunological classification of this group of LCD ww as follows: six of me cases belonged to type λ, four of them were type κ, and another one was a double LCD. We found that LCD was common in Fujlan only next to multiple myeloma (MM) of IgG class and accounted for 20% of the total 55 MM cases found in recent yean.It to well known that In matt patients of LCD M protein or Bence Jones proteinemia (BJPemia) to not detectable by conventional electro-phoresis. Our studies show that by making serum protein along with urinary BJP electrophoresis on the same one gel plate the sltuation can be greatly Improved It not only favour* the recognition of smail and faint band or bands of free light chain in serum, but also provides a repid and sensitive way, i. e. , immunofixation, to directly detect urinary light chain on the gel plate Immediately after electrophresis has been run.

Persistent diarrhea with petechial rash-unusual pattern of light chain amyloidosis deposition on skin and gastrointestinal biopsies:A case report

BACKGROUND Amyloidosis is a rare disease characterized by extracellular deposition of misfolded protein aggregated into insoluble fibrils.Gastrointestinal involvement in systemic amyloidosis is common,but is often subclinical or presents as vague and nonspecific symptoms.It is rare for gastrointestinal symptoms to be the main presenting symptom in patients with systemic amyloidosis,causing it to be undiagnosed until late-stage disease.CASE SUMMARY A 53 year-old man with diarrhea,hematochezia,and weight loss presented to a community hospital.Colonoscopy with biopsy at that time was suspicious for Crohn disease.Due to worsening symptoms including nausea,vomiting,and a new petechial rash,an abdominal fat pad biopsy was done.The biopsy showed papillary and adnexal dermal amyloid deposition,in a pattern usually seen with cutaneous amyloidosis.However,Cytokeratin 5/6 was negative,excluding cutaneous amyloidosis.The patterns of nodular amyloidosis,subcutaneous amyloid deposits and perivascular amyloid were not seen.Periodic Acid-Schiff stain was negative for lipoid proteinosis,Congo red was positive for apple green birefringence on polarization and amyloid typing confirmed amyloid light chain amyloidosis.Repeat endoscopic biopsies of the gastrointestinal tract showed amyloid deposition from the esophagus to the rectum,in a pattern usually seen in serum amyloid A in the setting of chronic inflammatory diseases,including severe inflammatory bowel disease.Bone marrow biopsy showed kappa-restricted plasma cell neoplasm.CONCLUSION Described is an unusual presentation of primary systemic amyloidosis,highlighting the risk of misdiagnosis with subsequent significant organ dysfunction and high mortality.

Pomolidomide for relapsed/refractory light chain amyloidosis after resistance to both bortezomib and daratumumab:A case report

BACKGROUND Immunoglobulin light chain(AL)amyloidosis is a rare disease characterized by deposition of ALs essentially in any organ or tissue,with cardiac involvement being very frequent(61%).Early diagnosis is of high importance because early initiation of treatment in AL amyloidosis may improve outcomes.Despite the administration of immunotherapeutic agents,in particular bortezomib and daratumumab,which have improved the outcomes of AL amyloidosis,antiplasma cell therapy remains suboptimal for some patients.CASE SUMMARY We report the case of a 55-year-old man presenting with heart failure who was diagnosed with cardiac AL amyloidosis by an endomyocardial biopsy.He experienced a short-term hematological remission with no organ response after being administered a bortezomib-daratumumab containing regimen.The treatment was switched to pomolidomide due to pulmonary involvement and progressive pleural effusion,in which flow cytometry analysis showed abnormal plasma cells.After two cycles of this regimen,the pleural effusion was controlled effectively with no recurrence.CONCLUSION This case emphasizes the crucial role of endomyocardial biopsy in early diagnosis of cardiac amyloidosis and suggests that pomolidomide may be an effective treatment for patients with AL amyloidosis that is relapsed/refractory to both bortezomib and daratumumab.

Secondary light chain amyloidosis with Waldenstr?m’s macroglobulinemia and intermodal marginal zone lymphoma:A case report

BACKGROUND The co-existence of Waldenstr?m’s macroglobulinemia(WM) with internodal marginal zone lymphoma(INMZL) is rare and often associated with poor prognosis.CASE SUMMARY We present a Chinese female patient who developed secondary light chain amyloidosis due to WM and INMZL and provides opinions on its systemic treatment.A 65-year-old woman was diagnosed with WM 6 years ago and received Bruton tyrosine kinase inhibitor monotherapy for two years.Her INMZL was confirmed due to left cervical lymphadenopathy.The patient presented with oedema in both lower limbs one year ago,and was diagnosed with secondary light chain amyloidosis.Treatment with the BC regimen(rituximab 375 mg/m~2 monthly for 6-8 courses,and bendamustine 90 mg/m~2 per day × 2,monthly for six courses) was initiated,but not tolerated due to toxic side effects.Bortezomibbased therapy was given for two months,including bortezomib,dexamethasone,and zanubrutinb.Oedema in both lower limbs was relieved and treatment efficacy was evaluated as partial remission.CONCLUSION A detailed clinical evaluation and active identification of the aetiology are recommended to avoid missed diagnosis and misdiagnosis.

Role and mechanism of phosphate myosin light chain in chronic allograft nephropathy of rats

Objective To investigate role and mechanism of phosphate myosin light chain ( pMLC) in rat kidney of chronic allograft nephropathy ( CAN) model. Methods Left donor kidneys from Fisher ( F344) rats were ortho-topically transplanted into Lewis recipients,Meanwhile, F344 rats and LEW rats with resection of right

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Flowcytometric detection of immunoglobulinlight chain in hematolymphoid immunophenotyping

During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of variable,diverse,and joining gene segments.A single mature B-cell expresses an IgH chain and either a kappa or lambda light chain,which is known as allelic or isotypic exclusion.In normal or reactive conditions,lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression.

Clathrin light chains negatively regulate plant immunity by hijacking the autophagy pathway

The crosstalk between clathrin-mediated endocytosis(CME)and the autophagy pathway has been reported in mammals;however,the interconnection of CME with autophagy has not been established in plants.Here,we report that the Arabidopsis CLATHRIN LIGHT CHAIN(CLC)subunit 2 and 3 double mutant,clc2-1 clc3-1,phenocopies Arabidopsis AUTOPHAGY-RELATED GENE(ATG)mutants in both autoimmunity and nutrient sensitivity.Accordingly,the autophagy pathway is significantly compromised in the clc2-1 clc3-1 mutant.Interestingly,multiple assays demonstrate that CLC2 directly interacts with ATG8h/ATG8i in a domain-specific manner.As expected,both GFP-ATG8h/GFP-ATG8i and CLC2-GFP are subjected to autophagic degradation,and degradation of GFP-ATG8h is significantly reduced in the clc2-1 clc3-1 mutant.Notably,simultaneous knockout of ATG8h and ATG8i by CRISPR-Cas9 results in enhanced resistance against Golovinomyces cichoracearum,supporting the functional relevance of the CLC2-ATG8h/8i interactions.In conclusion,our results reveal a link between the function of CLCs and the autophagy pathway in Arabidopsis.

Hepatomegaly and jaundice as the presenting symptoms of systemic light-chain amyloidosis: A case report

BACKGROUND Light chain(AL)amyloidosis is a plasma cell dyscrasia characterized by the pathologic production and extracellular tissue deposition of fibrillar proteins derived from immunoglobulin AL fragments secreted by a clone of plasma cells,which leads to progressive dysfunction of the affected organs.The two most commonly affected organs are the heart and kidneys,and liver is rarely the dominant affected organ with only 3.9%of cases,making them prone to misdia-gnosis and missed diagnosis.CASE SUMMARY A 65-year-old woman was admitted with a 3-mo history of progressive jaundice and marked hepatomegaly.Initially,based on enhanced computed tomography scan and angiography,Budd-Chiari syndrome was considered and balloon dilatation of significant hepatic vein stenoses was performed.However,addi-tional diagnostic procedures,including liver biopsy and bone marrow-exami-nation,revealed immunoglobulin kapa AL amyloidosis with extensive liver involvement and hepatic vascular compression.The disease course was progre-ssive and fatal,and the patient eventually died 5 mo after initial presentation of symptoms.CONCLUSION AL amyloidosis with isolated liver involvement is very rare,and can be easily misdiagnosed as a vascular disease.

Immunophenotypic analysis of abnormal plasma cell clones in bone marrow of primary systemic light chain amyloidosis patients

Background Primary systemic light chain amyloidosis （AL） is a rare plasma cell disease,our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients,and explore whether the detection of abnormal plasma cell clones in bone marrow by flow cytometry （FCM） could be used as an important indicator of AL diagnosis.Methods Fresh bone marrow samples were collected from 51 AL,21 multiple myeloma （MM）,and 5 Waldenstr（o）m＇s macroglobulinemia （WM） patients.The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45,CD38,CD138,CD117,CD56,and CD19.Results In AL,light chain restriction could be identified in 31 cases （60.9%）,in which the λ light chain restriction was found in 24 cases （77.4%）.In MM,κ light chain restriction was found in 13 cases （61.9%）,and λ light chain restriction in eight cases.CD45 on abnormal plasma cells was negative to weakly positive in both AL and MM,but was positive to strongly positive in WM.In the bone marrow plasma cells of the 51 AL,78.4% were CD56＋,68.6% were CD117＋,and 88.2% were CD19-.While in the 21 MM cases,66.7% were CD56＋,38.1% were CD117＋,and 90.4% were CD19-.The plasmacytoid lymphocytes in the five WM patients were CD19＋ and CD56-,CD117-.Conclusion Detection of abnormal plasma cell clones in bone marrow by FCM is valuable for the diagnosis of AL.

Clathrin light chains regulate hypocotyl elongation by affecting the polarization of the auxin transporter PIN3 in Arabidopsis

PIN-FORMED(PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers.Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.

The sphingosine-1-phosphate/RhoA/Rho associated kinases/myosin light chain pathway in detrusor of female rats is down-regulated in response to ovariectomy

Background:Dysuria is one of the main symptoms of genitourinary syndrome of menopause,which causes serious disruption to the normal life of peri-menopausal women.Studies have shown that it is related to decrease of detrusor contractile function,but the exact mechanism is still poorly understood.Previous results have suggested that the sphingosine-1-phosphate(S1P)pathway can regulate detrusor contraction,and this pathway is affected by estrogen in various tissues.However,how estrogen affects this pathway in the detrusor has not been investigated.In this study,we detected changes of the S1P/RhoA/Rho associated kinases(ROCK)/myosin light chain(MLC)pathway in the detrusor of ovariectomized rats in order to explore the underlying mechanism of dysuria during peri-menopause.Methods::Thirty-six female Sprague-Dawley rats were randomly divided into SHAM(sham operation),OVX(ovariectomy),and E groups(ovariectomy+estrogen),with 12 rats in each group.We obtained bladder detrusor tissues from each group and examined the mRNA and protein levels of the major components of the S1P/RhoA/ROCK/MLC pathway using quantitative real-time polymerase chain reaction and Western blotting,respectively.We also quantified the content of S1P in the detrusor using an enzyme linked immunosorbent assay.Finally,we compared results between the groups with one-way analysis of variance.Results::The components of the S1P pathway and the RhoA/ROCK/MLC pathway of the OVX group were significantly decreased,as compared with SHAM group.The percent decreases of the components in the S1P pathway were as follows:sphingosine kinase 1(mRNA:39%,protein:45%)(both P<0.05),S1P(21.73±1.09 nmol/g vs.18.86±0.69 nmol/g)(P<0.05),and S1P receptor 2/3(S1PR2/3)(mRNA:25%,27%,respectively)(P<0.05).However,the protein expression levels of S1PR2/3 and the protein and mRNA levels of SphK2 and S1PR1 did not show significant differences between groups(P>0.05).The percent decreases of the components in the RhoA/ROCK/MLC pathway were as follows:ROCK2(protein:41%,mRNA:36%)(both P<0.05),p-MYPT1(protein:54%)(P<0.05),and p-MLC20(protein:47%)(P<0.05),but there were no significant differences in the mRNA and protein levels of RhoA,ROCK1,MYPT1,and MLC20(all P>0.05).In addition,all of the above-mentioned decreases could be reversed after estrogen supplementation(E group vs.SHAM group)(all P>0.05).Conclusion::In this study,we confirmed that ovariectomy is closely associated with the down-regulation of the S1P/RhoA/ROCK/MLC pathway in the rat detrusor,which may be one mechanism of dysuria caused by decreased contractile function of the female detrusor during peri-menopause.

Neurofilament light chain in demyelinating conditions of the central nervous system: a promising biomarker

Neurofilaments are the major structural proteins of the neuronal cytoskeleton and are classified according to molecular weight into heavy,intermediate,and light chains.They are released into the interstitial fluid and cerebrospinal fluid(CSF)as a consequence of axonal damage.In particular,the light chain(NfL)represents the most abundant and soluble subunit and has been demonstrated to be increased in the CSF of patients with inflammatory,degenerative,vascular,or traumatic injuries in correlation with clinical and radiological activity.Similar results have been obtained measuring serum NfL with high-sensitivity single-molecule array,which enables reliable and repeatable measurement of the low NfL concentrations in serum.In particular,CSF and serum NfL values are strongly correlated in patients with multiple sclerosis(MS)and have been demonstrated to be increased in patients with MS and clinically isolated syndromes(CIS)in accordance with clinical and radiological activity.NfL levels increase in patients with a recent relapse and seem to predict cognitive impairment,long-term outcome,and conversion of CIS to MS.The few available data on patients with other demyelinating diseases suggest that NfL levels are also increased in neuromyelitis optica spectrum disorders and related conditions in correlation with attack severity,suggesting that axonal damage may occur in these disorders.We herein report and discuss published data on the role of NfL as a possible predictor of disease activity,clinical outcome and treatment response in patients with demyelinating conditions of the central nervous system.

CHANGES OF MYOSIN LIGHT CHAIN PATTERNS IN MUSCLE FIBERS AFTER CROSS-REINNERVATION

In the course of analysis of single fibers randomly excided from the fast and slow muscles of the rat for myosin light chain patterns, we have found that the light chains of all fibers taken from extensor digitorum longus (EDL) show the same as but different from those of fibers taken from soleus (Sol) in pattern. This characteristic differentiation of

5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

Long myosin light chain kinase (L-MLCK) contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, re- spectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determina- tion of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle forma- tion caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.

Submucosal hematoma is a highly suggestive finding for amyloid light-chain amyloidosis:Two case reports

The clinical and endoscopic features of amyloid lightchain(AL) amyloidosis are diverse and mimic various other diseases.Endoscopically,few reports on submucosal hematomas of the gastrointestinal(GI) tract are available in the literature.Here,we report two cases of AL amyloidosis presenting as submucosal hematomas in the absence of clinical disease elsewhere in the body.The 2 cases were referred to our hospital because of hematochezia.The endoscopic findings in both cases were similar in submucosal hematoma formation.However,the clinical courses differed.In the first case,there was no evidence of systemic amyloidosis and the disease was conservatively managed.In the second case,the disease progressed to systemic amyloidosis and the patient died within a short time.We conclude that the endoscopic detection of a submucosal hematoma in the setting of GI bleeding should raise suspicion of AL amyloidosis.Referral to a hematologist should be done immediately for treatment while the involvement is limited to the GI tract.

Blood diagnostic and prognostic biomarkers in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease for which the current treatment approaches remain severely limited.The principal pathological alterations of the disease include the selective degeneration of motor neurons in the brain,brainstem,and spinal cord,as well as abnormal protein deposition in the cytoplasm of neurons and glial cells.The biological markers under extensive scrutiny are predominantly located in the cerebrospinal fluid,blood,and even urine.Among these biomarke rs,neurofilament proteins and glial fibrillary acidic protein most accurately reflect the pathologic changes in the central nervous system,while creatinine and creatine kinase mainly indicate pathological alterations in the peripheral nerves and muscles.Neurofilament light chain levels serve as an indicator of neuronal axonal injury that remain stable throughout disease progression and are a promising diagnostic and prognostic biomarker with high specificity and sensitivity.However,there are challenges in using neurofilament light chain to diffe rentiate amyotrophic lateral sclerosis from other central nervous system diseases with axonal injury.Glial fibrillary acidic protein predominantly reflects the degree of neuronal demyelination and is linked to non-motor symptoms of amyotrophic lateral sclerosis such as cognitive impairment,oxygen saturation,and the glomerular filtration rate.TAR DNA-binding protein 43,a pathological protein associated with amyotrophic lateral sclerosis,is emerging as a promising biomarker,particularly with advancements in exosome-related research.Evidence is currently lacking for the value of creatinine and creatine kinase as diagnostic markers;however,they show potential in predicting disease prognosis.Despite the vigorous progress made in the identification of amyotrophic lateral sclerosis biomarkers in recent years,the quest for definitive diagnostic and prognostic biomarke rs remains a formidable challenge.This review summarizes the latest research achievements concerning blood biomarkers in amyotrophic lateral sclerosis that can provide a more direct basis for the differential diagnosis and prognostic assessment of the disease beyond a reliance on clinical manifestations and electromyography findings.

Storage time affects the level and diagnostic efficacy of plasma biomarkers for neurodegenerative diseases

Several promising plasma biomarker proteins,such as amyloid-β(Aβ),tau,neurofilament light chain,and glial fibrillary acidic protein,are widely used for the diagnosis of neurodegenerative diseases.However,little is known about the long-term stability of these biomarker proteins in plasma samples stored at-80°C.We aimed to explore how storage time would affect the diagnostic accuracy of these biomarkers using a large cohort.Plasma samples from 229 cognitively unimpaired individuals,encompassing healthy controls and those experiencing subjective cognitive decline,as well as 99 patients with cognitive impairment,comprising those with mild cognitive impairment and dementia,were acquired from the Sino Longitudinal Study on Cognitive Decline project.These samples were stored at-80°C for up to 6 years before being used in this study.Our results showed that plasma levels of Aβ42,Aβ40,neurofilament light chain,and glial fibrillary acidic protein were not significantly correlated with sample storage time.However,the level of total tau showed a negative correlation with sample storage time.Notably,in individuals without cognitive impairment,plasma levels of total protein and tau phosphorylated protein threonine 181(p-tau181)also showed a negative correlation with sample storage time.This was not observed in individuals with cognitive impairment.Consequently,we speculate that the diagnostic accuracy of plasma p-tau181 and the p-tau181 to total tau ratio may be influenced by sample storage time.Therefore,caution is advised when using these plasma biomarkers for the identification of neurodegenerative diseases,such as Alzheimer's disease.Furthermore,in cohort studies,it is important to consider the impact of storage time on the overall results.