Calcium Pyrophosphate Dihydrate Research Articles

Lumbar Spinal Involvement in Calcium Pyrophosphate Dihydrate Disease: A Systematic Literature Review.

BackgroundCalcium-pyrophosphate-dihydrate-disease (CPPD) is a crystal-induced arthropathy. The lumbar-spinal involvement is rare and often under-diagnosed. This study aimed to report the case of a lumbar spine CPPD involvement and to perform a systematic review of clinical, imaging features of lumbar involvement in CPPD patients, and treatments that have been implemented.MethodsThis systematic review was conducted in accordance with the Preferred-Reporting-Items-for-Systematic-Reviews and Meta-Analyses (PRISMA) guidelines.ResultsOne hundred and sixty-seven articles met the search criteria using electronic databases searches. We retained 28 articles (20 case reports, 2 case series, 1 family survey, 4 retrospective studies, and 1 prospective study) involving a total of 62 patients. The age ranged between 39 and 89 years old. Among patients with lumbar spine CPPD, 32 were women. The duration of symptoms varied between one day and 8 years. The affection has been discovered during back pain in most cases. In 5 studies, the diagnosis was made on histological specimens of patients operated on for another pathology. X-ray showed calcifications in 2 cases. CT-scan detected calcium deposit in 7 cases. MRI showed lesions going from the increased signal of the disk, to calcified or not-cystic lesion of the facet joints, an intramedullary mass mimicking a schwannoma. Histological examination established the diagnosis of CPPD in 21 patients in all studies. Medical treatment included NSAIDs, Colchicine, Interleukin-1-receptor-antagonist, and antibiotics. Surgery was performed on 13 patients and allowed to establish the histological diagnosis.ConclusionIn the case of inflammatory back pain in elderly subjects, without an infectious gateway, diagnosis of CPPD should be considered, especially for patients with a history of spinal surgery or degenerative radiography changes. CT scan is more sensitive than conventional radiographs. The discovertebral biopsy is the Gold-Standard and should be performed whenever the diagnosis was uncertain. Treatment includes the medical and surgical components.

Drugs Recommended in Adult Rheumatic Diseases, But Considered for Off-Label Use in Argentina

BackgroundOff-label (OL) drug use is the prescription of a drug for indications other than those authorised in its technical datasheet. The objective of this study was to identify drugs recommended in rheumatology but considered for off-label use in Argentina. MethodsA list of medications for certain selected rheumatic conditions was compiled. A drug was considered recommended if it was endorsed by a) at least one Argentine or Pan-American treatment guideline or consensus, or b) two international treatment guidelines, or c) one international treatment guideline and one selected textbook. Approval of these drugs for any condition in Argentina until December 31st, 2018 was explored, and medicines were divided into those with on-label indications and those considered for OL use. ResultsOne hundred and thirty-six medications were analysed in 13 clinical conditions. Sixty-seven OL recommendations (49%) were found, and several drugs had more than one. All the conditions included the recommendation of at least 1 OL drug except osteoporosis and rheumatoid arthritis. The frequency of OL recommendations for the following conditions was 100%: calcium pyrophosphate dihydrate crystal deposition disease, polymyalgia rheumatica, Sjögren syndrome, and systemic sclerosis. The drugs with the highest number of OL recommendations were methotrexate (in 7 conditions), and glucocorticoids and mycophenolate (in 4). There were 2 OL recommendations for rituximab and 1 for abatacept. ConclusionsAlmost all the rheumatic disorders analysed involved the recommendation of at least 1 OL medication, and in 4 conditions all the recommendations were OL. Most OL drugs recommended in rheumatology are neither biological nor small-molecule therapies.

Drugs Recommended in Adult Rheumatic Diseases, But Considered for Off-Label Use in Argentina.

Off-label (OL) drug use is the prescription of a drug for indications other than those authorised in its technical datasheet. The objective of this study was to identify drugs recommended in rheumatology but considered for off-label use in Argentina. A list of medications for certain selected rheumatic conditions was compiled. A drug was considered recommended if it was endorsed by a) at least one Argentine or Pan-American treatment guideline or consensus, or b) two international treatment guidelines, or c) one international treatment guideline and one selected textbook. Approval of these drugs for any condition in Argentina until December 31st, 2018 was explored, and medicines were divided into those with on-label indications and those considered for OL use. One hundred and thirty-six medications were analysed in 13 clinical conditions. Sixty-seven OL recommendations (49%) were found, and several drugs had more than one. All the conditions included the recommendation of at least 1 OL drug except osteoporosis and rheumatoid arthritis. The frequency of OL recommendations for the following conditions was 100%: calcium pyrophosphate dihydrate crystal deposition disease, polymyalgia rheumatica, Sjögren syndrome, and systemic sclerosis. The drugs with the highest number of OL recommendations were methotrexate (in 7 conditions), and glucocorticoids and mycophenolate (in 4). There were 2 OL recommendations for rituximab and 1 for abatacept. Almost all the rheumatic disorders analysed involved the recommendation of at least 1 OL medication, and in 4 conditions all the recommendations were OL. Most OL drugs recommended in rheumatology are neither biological nor small-molecule therapies.

Case Report: Posterolateral Epidural Supra-C2-Root Approach (PESCA) for Biopsy of a Retro-Odontoid Lesions in Same Sitting After Occipitocervical Fixation and Decompression in a Case of Crowned Dens Syndrome With Brainstem Compression and Displacement

Background‘Crowned dens syndrome' (CDS) is a special form of calcium pyrophosphate dihydrate deposition disease which is characterized radiologically by a halo-like or crown-like distribution in the periodontoid region and clinically by cervical pain. Herein, we will describe our experience of posterolateral epidural supra-C2-root approach (PESCA) for biopsy of retro-odontoid lesions in one surgical session after occipitocervical fixation and decompression in a patient with CDS and massive brainstem compression.Case PresentationA 70-year-old woman presented to our department with a 4-week history of progressive walking impairment, neck pain, neck rigidity, fever, dizziness, slight palsy of the left hand, and multiple fall episodes. Magnetic resonance imaging (MRI) of the craniovertebral junction (CVJ) and cervical spine revealed a lesion of the odontoid process and the retro-odontoid region with mainly solid components, as well as small cystic components, and brainstem compression and displacement. In first step, fusion surgery of the CVJ C0–C4 was performed with occiptocervical decompression. After fusion and decompression the lower lateral part of the C1 arc and the lateral superior part of the left side of the C2 arc were removed. The entry point was located directly above the superior part of the C2 root. A biopsy of the lateral portions of the lesions was obtained by bioptic forceps under microscope guidance. Pathologic examination of the mass revealed deposition of birefringent crystals compatible with calcium pyrophosphate. In addition to the clinical symptoms (especially neck pain), the diagnosis of CDS was made. Non-steroidal inflammatory drugs (NSAIDs) and colchicine (and later magnesium) were started. At follow-up examination 6 months after surgery, an MRI scan of the cervical spine revealed regression of the pannus and the cyst with replacement of the brainstem, clinical improvement of walking, and increased strength of the left hand.ConclusionsThis study demonstrates that PESCA can be used to obtain tissue for pathological analysis in one surgical sitting after fusion and decompression and that fusion, decompression, and PESCA (in the same session) together with subsequent conservative management could be a good alternative for the treatment of CDS.

CHARACTERIZATION OF THE CALCIFICATIONS IN THE POSTERIOR HORN OF HUMAN MENISCUS VIA RAMAN MICROSPECTROSCOPY

Purpose: Calcifications in joint soft tissues, such as meniscus and articular cartilage, are closely associated with osteoarthritis (OA) progression and could act as a potential target for disease-modifying agents in OA therapy. Basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals are the two most common forms of calcium crystals found in these tissues. Moreover, within the CPPD crystals, two different polymorphs - monoclinic and triclinic - have been identified in arthritic patients.

The utility of routine cultures, cell count, and crystal evaluation of aspirate from aseptic olecranon bursitis.

BackgroundAspiration of the olecranon bursa is a treatment option for acute olecranon bursitis (OB). Typically, the aspirate is sent for microbiologic analysis, cell count, and crystal analysis. This study investigates the utility of fluid aspirate analysis from patients with clinically diagnosed aseptic OB.MethodsIn this prospective study (IRB #i20-00986), patients presenting with acute aseptic OB were treated with aspiration as standard of care. Patients consented to participate in this study via phone. Patients with suspected infectious bursitis, open draining wound, and chronic OB were excluded. The aspirate was sent out for routine microbiologic analysis (aerobic and anaerobic cultures and Gram staining) and fluid analyses, including cell count with differential and crystal analysis. Nucleated and differential cell count was reported as absolute numbers per cubic millimeter and percentage, respectively. Compression wrap was applied after OB aspiration, and patients were asked to ice and take anti-inflammatory medications. Clinical follow-up was done after 6 weeks and at 3 months for resolution vs. recurrence of symptoms, and the mean time to resolution was reported.ResultsA total of 26 patients (28 cases) with aseptic OB were enrolled in this study. Two patients had bilateral OB. The mean time to aspiration after the onset of symptoms was 26.4 days. One patient had recurrence of swelling after the first aspiration and underwent repeat bursa aspiration. No organisms were isolated or reported on Gram staining on any of the aspirate samples. Two aspirates were reported positive for calcium pyrophosphate dihydrate crystals. No patient had monosodium urate crystals. All patients had resolution of swelling and symptoms without the development of postaspiration infection.ConclusionsThis study demonstrates limited clinical utility of routine microbiologic analysis (cell count, microbiologic, and crystal evaluation) of fluid aspirate from clinically diagnosed aseptic OB. Although 7% of fluid aspirates were positive for calcium pyrophosphate dihydrate crystals, it did not change the overall treatment.

Natural antibodies and CRP drive anaphylatoxin production by urate crystals

In gout, crystallization of uric acid in the form of monosodium urate (MSU) leads to a painful inflammatory response. MSU crystals induce inflammation by activating the complement system and various immune cell types, and by inducing necrotic cell death. We previously found that the soluble pattern recognition molecule C-reactive protein (CRP) recognizes MSU crystals, while enhancing complement activation. In the absence of CRP, MSU crystals still induced complement activation, suggesting additional CRP-independent mechanisms of complement activation. In the present study, we searched for additional MSU crystal-binding complement activators. We found that all healthy individuals, even unborn children, have MSU crystal-specific immunoglobulin M (IgM) in their blood. This indicates that innate IgM, also known as natural IgM, recognizes these crystals. In serum lacking IgM and CRP, MSU crystals showed negligible complement activation as assessed by the production of the anaphylatoxins C4a, C3a, and C5a (listed in order of production via the classical complement pathway). We show that IgM and CRP both activate the classical complement pathway on MSU crystals. CRP was more efficient at fixating active C1 on the crystals and inducing release of the most inflammatory anaphylatoxin C5a, indicating non-redundant functions of CRP. Notably, while CRP recognizes MSU crystals but not the related calcium pyrophosphate dihydrate (CPPD) crystals, natural IgM bound to both, suggesting common and distinct mechanisms of recognition of individual crystal types by complement activators.

Pseudogout, an unconventional imitator of the lumbosacral spine with associated chronic epidural hematoma: illustrative cases

BACKGROUNDPseudogout, or calcium pyrophosphate dihydrate (CPPD) disease, is an inflammatory joint disease that most commonly involves the joints of the knees, ankles, and wrists. Pseudogout has also been known to involve the spine, especially the atlanto-occipital joint of the cervical spine, but there is limited documentation of its involvement in the lumbar spine. Though the atypical presentation of spinal pseudogout with findings consistent with discitis and epidural abscess has been documented, its presentation with associated chronic spinal epidural hematoma is a rare entity.OBSERVATIONSThe authors present two separate cases of pseudogout involvement of the lumbar spine, one case presenting with a clinical and radiographic picture consistent with discitis and epidural abscess and the other with radiographic and operative findings consistent with a chronic epidural hematoma.LESSONSThis case series demonstrates rare and atypical presentations of pseudogout within the lumbosacral spine.

Fluorescence identification of arthropathic calcium pyrophosphate single crystals using alizarin red S and a xanthene dipicolylamine ZnII complex.

Calcium pyrophosphate deposition disease, previously known as pseudogout, is a type of chronic and painful joint arthropathy. Accurate identification of calcium pyrophosphate dihydrate (CPPD) single crystals is crucial for determining the best course of treatment. In this study, a two-step method involving alizarin red S (ARS) and a xanthene dipicolylamine ZnII (XDZ) complex was employed for the identification of CPPD single crystals in both triclinic and monoclinic forms using a fluorescence microscope and a microplate reader. The accurate identification method proposed in this study has the potential to advance the diagnosis and treatment of patients suffering from painful gouty arthritis.

Symptomatic retro-odontoid pseudotumor causing calcium pyrophosphate dihydrate deposition combined with multilevel cervical spondylotic myelopathy.

IntroductionSymptomatic retro-odontoid pseudotumor (ROP) caused by cervical compression and myelopathy is rare. Pathological diagnosis is recommended for differential diagnosis including the following: inflammatory disease, primary bone tumor, metastatic disease and calcium pyrophosphate dihydrate deposition (CPPD) also known as “crowned dens syndrome”. The authors report a rare case of ROP caused by CPPD deposition combined with multilevel cervical spondylotic myelopathy (MCSM) which was treated by tumor resectioning using a transoral approach combined with posterior decompression and fusion.Case presentationA 66-year-old male presented with progressive neck pain and spastic gait with no history of trauma. Radiographic imaging revealed degenerative change involving the atlanto-axial and atlanto-occipital joints with calcified enhancing soft tissue around the odontoid process causing cord compression and cervical instability at the C1-C2 level combined with MCSM and spinal cord compression at C3 to C7. Microscopic assisted transoral tumor resection combined with posterior decompression and fusion was performed at the occiput to T2. The pathology report describes a rhomboid-shaped crystal caused by calcium pyrophosphate dihydrate deposition (CPPD) disease. At the 6-month follow-up following the operation, the patient's neck pain and spastic gait were improved compared to the preoperative examination.DiscussionCervical compression and myelopathy from ROP causing CPPD combined with MCSM is rare. Pathology diagnosis and surgical management are highly recommended.ConclusionIn this case, a combined surgical approach: tumor resection using a transoral approach and a posterior approach for decompression and fusion at occiput to T2 was an effective option for this condition.

Multilevel calcium pyrophosphate dihydrate deposition in cervical ligamentum flavum: clinical characteristics and imaging features

BackgroundInvolvement in cervical ligamentum flavum is a rare manifestation of the calcium pyrophosphate dihydrate deposition disease. Only few cases of this condition have been reported. We revealed eighteen cases of CPPD in cervical ligamentum flavum that diagnosed at a single medical center. In our case series, clinical characteristics and magnetic resonance imaging findings of patients are described.MethodsWe retrospectively reviewed the medical charts and imaging studies of the eighteen patients with pseudogout attack of the cervical ligamentum flavum. In addition, we discussed the differences between this disease and ossification of ligamentum flavum in image manifestations.ResultsThere were fourteen men and four women aged between 59 and 87 years. Diabetes mellitus and hypertension were the most common comorbidities. Myelopathy and neck pain were presented in most patients. C4–5 and C5–6 were attacked most frequently, and multiple- rather than single-level involvement could be observed in our series. “Acute on chronic phenomenon” was a specific magnetic resonance image finding in patients whose symptom durations were between 2 to 5 months. Compared to ossification of ligamentum flavum, calcium pyrophosphate dihydrate crystal deposition had different image signs, including morphology, side of the involved ligament, no continuity with the lamina, acute on chronic phenomenon, and presence of retro-odontoid mass.ConclusionsNodular calcifications in cervical ligamentum flavum raise highly suspicion for calcium pyrophosphate dihydrate deposition and must be diagnosed by histological examination and polarized light microscopy. This disease is different from ossification of ligamentum flavum, and it could be recognized by specific image features.

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals—The Etiological Factor of Chondrocalcinosis

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg2+-dependent and also works against soluble species of pyrophosphate.

Accuracy of synovial fluid analysis compared to histology for the identification of calcium pyrophosphate crystals: an ancillary study of the OMERACT US Working Group - CPPD subgroup.

The aim of this study was to evaluate the accuracy of synovial fluid analysis in the identification of calcium pyrophosphate dihydrate crystals compared to microscopic analysis of joint tissues as the reference standard. This is an ancillary study of an international, multicentre cross-sectional study performed by the calcium pyrophosphate deposition disease (CPPD) subgroup of the OMERACT Ultrasound working group. Consecutive patients with knee osteoarthritis (OA) waiting for total knee replacement surgery were enrolled in the study from 2 participating centres in Mexico and Romania. During the surgical procedures, synovial fluid, menisci and hyaline cartilage were collected and analysed within 48 hours from surgery under transmitted light microscopy and compensated polarised light microscopy for the presence/absence of calcium pyrophosphate crystals. All slides were analysed by expert examiners on site, blinded to other findings. A dichotomic score (absence/ presence) was used for scoring both synovial fluid and tissues. Microscopic analysis of knee tissues was considered the gold standard. Sensitivity, specificity, accuracy, positive and negative predictive values of synovial fluid analysis in the identification of calcium pyrophosphate crystals were calculated. 15 patients (53% female, mean age 68 yo ± 8.4) with OA of grade 3 or 4 according to Kellgren-Lawrence scoring were enrolled. 12 patients (80%) were positive for calcium pyrophosphate crystals at the synovial fluid analysis and 14 (93%) at the tissue microscopic analysis. The overall diagnostic accuracy of synovial fluid analysis compared with histology for CPPD was 87%, with a sensitivity of 86% and a specificity of 100%, the positive predictive value was 100% and the negative predictive value was 33%. In conclusion synovial fluid analysis proved to be an accurate test for the identification of calcium pyrophosphate dihydrate crystals in patients with advanced OA.

A Case of Pseudogout Following Zoledronic Acid Administration

Calcium pyrophosphate dihydrate (CPPD) deposition in the joints, often referred to as pseudogout, can lead to debilitating arthritis. Rare cases of pseudogout associated with bisphosphonate therapy have been reported in the literature, although the mechanism for this remains unclear. We report a case of a 53-year-old female who developed an acute pseudogout attack following administration of zoledronic acid for treatment of osteoporosis.

AB0629 ACCURACY OF SYNOVIAL FLUID ANALYSIS FOR THE IDENTIFICATION OF CALCIUM PYROPHOSPHATE CRYSTALS: AN ANCILLARY STUDY OF OMERACT CRITERION VALIDITY STUDY FOR ULTRASOUND IN CPPD

Background:Synovial fluid analysis (SFA) via compensated polarized light microscopy is still considered the gold standard for the identification and diagnosis of Calcium Pyrophosphate Deposition disease (CPPD)-related arthropathies[1], but very few studies have been published about its diagnostic accuracy.Objectives:The aim of this study was to evaluate the accuracy of SFA in the identification of calcium pyrophosphate dihydrate (CPP) crystals compared to microscopic analysis of joint tissues as the reference standard.Methods:This is an ancillary study of an international, multicentre cross-sectional study performed by the CPPD subgroup of the OMERACT Ultrasound working group[2]. Consecutive patients with knee osteoarthritis (OA) waiting for total knee replacement surgery were enrolled in the study from 2 participating centres, Mexico and Romania. During surgical procedures synovial fluid (SF), menisci and hyaline cartilage were collected and analysed within 48 hours after surgery under transmitted light microscopy and compensated polarised light microscopy for the presence/absence of CPP crystals. All slides were analysed by expert examiners on site, blinded to other findings. A dichotomic score (absence/presence) was used for scoring both SF and tissues. Microscopic analysis of knee tissues was considered the gold standard. Sensitivity, specificity, accuracy, positive and negative predictive values (PPV and NPV) of SFA in the identification of CPP crystals were calculated.Results:15 patients (53% female, mean age 68yo ± 8.4) with OA of grade 3 or 4 according to Kellgren-Lawrence scoring were enrolled. 12 patients (80%) were positive for CPP crystals at SFA and 14 (93%) at tissues microscopic analysis. Among 12 SFA positive patients, all were positive for CPP crystals in either medial or lateral meniscus, and 11 were positive in both; 10 patients were positive at the hyaline cartilage, and all 10 were also positive for at least one meniscus. Regarding the 3 SFA negative patients, only one had no crystals in the examined tissues, while the other 2 patients had CPP crystals in both menisci and hyaline cartilage. The overall diagnostic accuracy of SFA compared to histology analysis for CPPD was 87%, with a sensitivity of 86% and a specificity of 100%, the PPV was 100% and the NPV was 33% (Table 1).Table 1.sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy of synovial fluid analysis compared to the reference standard. CI: Confidential Interval. SF: synovial fluid, in parentheses: numerators and denominators for all percentages provided.SensitivitySpecificityPPVNPVAccuracySF analysis86% (12/14)100% (1/1)100% (12/12)33% (1/3)87% (13/15)(0.65-0.99) CI 95%(0.0-0.25) CI 95%(0.65-0.99) CI 95%(0.0-0.25) CI 95%Conclusion:SFA demonstrated to be an accurate test for the identification of CPP crystals in patients with advanced OA. However, is not always feasible and carries some risks for the patient. Considering the availability of validated imaging techniques for the detection of CPPD, such as US, SFA could be used in those patients where imaging and clinical data are not definitely confirmatory of the disease.

Chondrocytes From Osteoarthritic and Chondrocalcinosis Cartilage Represent Different Phenotypes.

Basic calcium phosphate (BCP)-based calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with calcium pyrophosphate dihydrate (CPPD) deposition disease in the joint inducing OA-like symptoms. There is only little knowledge about the effect of CPPD crystals on chondrocytes and the signaling pathways involved in their generation. The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes. Cartilage samples of patients with CC, patients with severe OA, and healthy donors were included in this study. The presence of CC was evaluated using standard X-ray pictures, as well as von Kossa staining of cartilage sections. OA severity was evaluated using the Chambers Score on cartilage sections, as well as the radiological Kellgren–Lawrence Score. Patients with radiologically detectable CC presented calcification mainly on the cartilage surface, whereas OA patients showed calcification mainly in the pericellular matrix of hypertrophic chondrocytes. OA cartilage exhibited increased levels of collagen X and matrix metalloproteinase 13 (MMP13) compared with CC and healthy cartilage. This observation was confirmed by qRT-PCR using cartilage samples. No relevant influence of CPPD crystals on hypertrophic marker genes was observed in vitro, whereas BCP crystals significantly induced hypertrophic differentiation of chondrocytes. Interestingly, we observed an increased expression of p16 and p21 in cartilage samples of CC patients compared with OA patients and healthy controls, indicating cellular senescence. To investigate whether CPPD crystals were sufficient to induce senescence, we incubated chondrocytes with BCP and CPPD crystals and quantified senescence using β-gal staining. No significant difference was observed for the staining, but an increase of p16 expression was observed after 10 days of culture. Primary chondrocytes from CC patients produced CPPD crystals in culture. This phenotype was stabilized by mitomycin C-induced senescence. Healthy and OA chondrocytes did not exhibit this phenotype. BCP and CPPD crystals seem to be associated with two different chondrocyte phenotypes. Whereas BCP deposition is associated with chondrocyte hypertrophy, CPPD deposition is associated with cellular senescence.

Weight-bearing CT for Knee Osteoarthritis Assessment: A Story Unfolds

Weight-bearing CT for Knee Osteoarthritis Assessment: A Story Unfolds

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout.

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (Kd) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin-horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0-300 μg/mL.

Optical Biomarkers for the Diagnosis of Osteoarthritis through Raman Spectroscopy: Radiological and Biochemical Validation Using Ex Vivo Human Cartilage Samples.

Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation. Raman spectroscopy (RS) has been recently proposed as a label-free tool to detect molecular changes in musculoskeletal tissues. We used cartilage samples derived from human femoral heads to perform an ex vivo study of different Raman signals and ratios, related to major and minor molecular components of articular cartilage, hereby proposed as candidate optical biomarkers for OA. Validation was performed against the radiological Kellgren–Lawrence (K-L) grading system, as a gold standard, and cross-validated against sulfated glycosaminoglycans (sGAGs) and total collagens (Hyp) biochemical contents. Our results showed a significant decrease in sGAGs (SGAGs, A1063 cm−1/A1004 cm−1) and proteoglycans (PGs, A1375 cm−1/A1004 cm−1) and a significant increase in collagen disorganization (ColD/F, A1245 cm−1/A1270 cm−1), with OA severity. These were correlated with sGAGs or Hyp contents, respectively. Moreover, the SGAGs/HA ratio (A1063 cm−1/A960 cm−1), representing a functional matrix, rich in proteoglycans, to a mineralized matrix-hydroxyapatite (HA), was significantly lower in OA cartilage (K-L I vs. III–IV, p < 0.05), whilst the mineralized to collagenous matrix ratio (HA/Col, A960 cm−1/A920 cm−1) increased, being correlated with K-L. OA samples showed signs of tissue mineralization, supported by the presence of calcium crystals-related signals, such as phosphate, carbonate, and calcium pyrophosphate dihydrate (MGP, A960 cm−1/A1004 cm−1, MGC, A1070 cm−1/A1004 cm−1 and A1050 cm−1/A1004 cm−1). Finally, we observed an increase in lipids ratio (IL, A1450 cm−1/A1670 cm−1) with OA severity. As a conclusion, we have described the molecular fingerprint of hip cartilage, validating a panel of optical biomarkers and the potential of RS as a complementary diagnostic tool for OA.

Calcified chondroid mesenchymal neoplasms with FN1-receptor tyrosine kinase gene fusions including FGFR2, FGFR1, MERTK, NTRK1, and TEK: a molecular and clinicopathologic analysis.

Translocations involving FN1 have been described in a variety of neoplasms that share the presence of a cartilage matrix and may also contain a variable extent of calcification. Fusions of FN1 to FGFR1 or FGFR2 have been reported in nine soft tissue chondromas, mostly demonstrated indirectly by FISH analysis. Delineation of FN1 fusions with various partner genes will facilitate our understanding of the pathogenesis and diagnostic classification of these neoplasms. In this study, we present molecular, clinical, and pathologic features of 12 cartilaginous soft tissue neoplasms showing a predilection for the TMJ region and the distal extremities. We analyzed for gene fusions with precise breakpoints using targeted RNA-seq with a 115-gene panel. We detected gene fusions in ten cases, including three novel fusions, FN1-MERTK, FN1-NTRK1, and FN1-TEK, each in one case, recurrent FN1-FGFR2 fusion in five cases, FN1-FGFR1 in one case, and FGFR1-PLAG1 in one case. The breakpoints in the 5' partner gene FN1 ranged from exons 11-48, retaining the domains of a signal peptide, FN1, FN2, and/or FN3, while the 3' partner genes retained the transmembrane domain, tyrosine kinase (TK) domains, and/or Ig domain. The tumors are generally characterized by nodular/lobular growth of polygonal to stellate cells within a chondroid matrix, often accompanied by various patterns of calcification, resembling those described for the chondroblastoma-like variant of soft tissue chondroma. Additional histologic findings include extensive calcium pyrophosphate dihydrate deposition in two cases and features resembling tenosynovial giant cell tumor (TGCT). Overall, while the tumors from our series show significant morphologic overlap with chondroblastoma-like soft tissue chondroma, we describe findings that expand the morphologic spectrum of these neoplasms and therefore refer to them as "calcified chondroid mesenchymal neoplasms." These neoplasms represent a spectrum of chondroid/cartilage matrix-forming tumors harboring FN1-receptor TK fusions that include those classified as soft tissue chondroma as well as chondroid TGCT.