Quantitative lateral flow immunoassay for rapid detection of procollagen type I N-terminal propeptide in the monitoring of osteoporosis treatment.

Appropriate follow-up after treatment initiation in patients with osteoporosis is challenging. Serum biomarkers may offer more efficient monitoring of bone mineral density (BMD) than the currently used dual X-ray absorptiometry; however, significant changes in BMD often occur over at least 12 months. During teriparatide treatment for osteoporosis, monitoring with markers such as procollagen type I propeptide (PINP), which is derived from osteoblasts, can provide clinically useful information for disease management. However, rapid and cost-effective methods for detecting serum PINP are lacking, necessitating a point-of-care test (POCT) for enhanced follow-up efficiency in osteoporosis management. For the quantitative detection of PINP, we developed a high-sensitivity lateral flow immunoassay with a stacking pad (sLFIA). We established a calibration equation based on the test line/control line ratio obtained from our PINP sLFIA results of various nonspiked serum samples to calculate the PINP concentrations in 40 serum samples and compared the result with those obtained using a fully automated electrochemiluminescence immunoassay. PINP concentrations between these two methods exhibited excellent correlation (R = 0.991). In addition, we assessed the serum PINP concentrations of patients with osteoporosis treated with teriparatide. At the 3-month follow-up, their PINP levels were nearly twice as high as those at baseline, thus implying that our method can be used for osteoporosis treatment monitoring. Our findings thus indicate that the PINP sLFIA can serve as a POCT for monitoring medication response and managing osteoporosis.

Quantitative lateral flow immunoassay for rapid detection and monitoring of cerebrospinal fluid leakage following incidental durotomy.

Cerebrospinal fluid (CSF) leakage due to incidental durotomy is an inherent complication of spine surgery. With appropriate treatment, complications of CSF leakage, such as headache and even meningitis, can be reduced. CSF leakage could be detected on the basis of correlated clinical symptoms; diagnosis should be based on these symptoms and appropriate imaging studies. However, the diagnosis of CSF leakage remains a challenge, especially if incidental durotomy is unrecognized during surgery; even if incidental durotomy is detected and repaired intraoperatively, the severity of the leakage and quality of the primary dural repair are difficult to evaluate postoperatively. Rapid, inexpensive, and safe methods of detecting CSF-containing samples are currently lacking; hence, the development of a point-of-care test (POCT) method to improve diagnostic efficiency is necessary. We developed a high-sensitivity lateral flow immunoassay with a stacking pad (sLFIA) for quantitative detection of ß-trace protein (BTP), a specific CSF marker. The BTP concentration in 39 clinical samples was calculated using a calibration equation for test-line intensity and evaluated by a standard laboratory method. To avoid the hook effect, we diluted each sample prior to testing. The correlation coefficient between the enzyme-linked immunosorbent assay and our BTP sLFIA method was 0.991 A 75-fold sample dilution was applied owing to the hook effect point, identified as 175 ng mL-1. We established an optimal sample-specific cutoff point at a value of 4.0 µg mL-1 for CSF leakage in subfascial drainage samples following spinal posterior decompression. The sensitivity and specificity of the BTP sLFIA method were 90% and 97%, respectively, according to a receiver operating characteristic curve analysis. In addition, clinical samples from patients who underwent primary dural repair intraoperatively were tested, and CSF leakage was successfully diagnosed using our method. Finally, the quantitation of BTP in samples collected daily provided an accurate assessment of the severity of the residual leakage. Our results demonstrate that the BTP sLFIA method possesses the potential to serve as a POCT method for screening and monitoring postoperative CSF leakage.

Development of a multiplex and sensitive lateral flow immunoassay for the diagnosis of periprosthetic joint infection.

The diagnosis of periprosthetic joint infection (PJI) remains a challenge. However, recent studies showed that synovial fluid biomarkers have demonstrated greater diagnostic accuracy than the currently used PJI diagnostic tests. In many diagnostic tests, combining several biomarkers into panels is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases, and reducing cost. In this study, we prove that combining alpha-defensin and C-reactive protein (CRP) as biomarkers possesses the potential to provide accurate PJI diagnosis. To further verify the result, we developed a multi-target lateral flow immunoassay strip (msLFIA) with staking pad design to obtain on-site rapid response for clinical diagnosis of PJI. A total of 10 synovial fluid samples were tested using the msLFIA, and the results showed that the combined measurements of synovial fluid alpha-defensin and CRP levels were consistent with those obtained from a commercial enzyme-linked immunosorbent assay kit. In addition, we developed a multi-target lateral flow immunoassay strip (msLFIA) with staking pad design to obtain on-site rapid response for clinical diagnosis of PJI, which the multi-target design is used to increase specificity and the stacking pad design is to enhance detection sensitivity. As a result, the turnaround time of the highly sensitive test can be limited from several hours to 20 min. We expect that the developed msLFIA possesses the potential for routine monitoring of PJI as a convenient, low-cost, rapid and easy to use detection device for PJI.

Development a stacking pad design for enhancing the sensitivity of lateral flow immunoassay.

Lateral flow immunoassays (LFIAs) have wide application in point-of-care testing, particularly in resource-poor settings. To achieve signal amplification in a gold nanoparticle-based lateral flow assay without an additional procedure or the need for complex fabrication, a new and simple method was developed for using a "stacking pad" configuration that adds an additional membrane between the conjugation pad and test pad to the conventional AuNP-based LFIA format. This design helps to accumulate the antibody and antigen on the stacking pad, hence extending the antigen/antibody binding interactions to enhance the test's detection sensitivity. With the enhanced lateral flow assay, as low as 1 ng/mL of Protein A and 15.5 ng/mL of C-reactive protein can be visualized with the naked eye. We also successfully applied the stacking pad system in the analysis of C-reactive protein in human serum and synovial fluid samples. These results suggest that this stacking pad LFIA can provide sensitive and on-site prognosis for detection in synovial fluid and serum samples in resource-limited settings.

The unresolved problem of beta-2 microglobulin amyloid deposits in the intervertebral discs of long-term dialysis patients.

BACKGROUND: Dialysis-related destructive spondyloarthropathy caused by beta-2 microglobulin (ß2M) amyloid deposits in intervertebral discs is a major burden for patients undergoing long-term dialysis. This study aimed to quantify the presence of ß2M amyloid deposits in the intervertebral disc tissue of such patients and analyze whether there was a significant correlation between ß2M accumulation and the duration of dialysis. METHODS: Two groups of patients who had undergone surgery for degenerative spinal pathologies were selected: the dialysis group (n = 29) with long-term dialysis and the control group (n = 10) with no renal impairment. Tissue sections were prepared from specimens of intervertebral disc tissue obtained during spinal surgery and analyzed via histological staining, including immunohistochemistry (IHC) and Congo red. RESULTS: There was a statistically significant multifold increase of ß2M expression in the disc tissue of long-term dialysis patients when compared to non-dialysis patients, as shown by both IHC (0.019 ± 0.023 µm2 vs. 0.00020 ± 0.00033 µm2, respectively; p = 0.012) and Congo red staining (0.027 ± 0.041 µm2 vs. 9.240 × 10-5 ± 5.261 × 10-5 µm2, respectively; p = 0.047). We also note a moderate strength positive correlation between the duration of dialysis and positive IHC (r = 0.39; p = 0.015) and Congo-red staining (r = 0.42; p = 0.007). CONCLUSIONS: The problem of ß2M amyloidosis in long-term dialysis patients remains unresolved even with predominant use of high-flux dialysis membranes. This highlights the insufficiency of current dialysis modalities to effectively filter ß2M.

Effects of Strontium Ranelate on Spinal Interbody Fusion Surgery in an Osteoporotic Rat Model.

Osteoporosis is a bone disease that afflicts millions of people around the world, and a variety of spinal integrity issues, such as degenerative spinal stenosis and spondylolisthesis, are frequently concomitant with osteoporosis and are sometimes treated with spinal interbody fusion surgery. Previous studies have demonstrated the efficacy of strontium ranelate (SrR) treatment of osteoporosis in improving bone strength, promoting bone remodeling, and reducing the risk of fractures, but its effects on interbody fusion surgery have not been adequately investigated. SrR-treated rats subjected to interbody fusion surgery exhibited significantly higher lumbar vertebral bone mineral density after 12 weeks of treatment than rats subjected to the same surgery but not treated with SrR. Furthermore, histological and radiographic assessments showed that a greater amount of newly formed bone tissue was present and that better fusion union occurred in the SrR-treated rats than in the untreated rats. Taken together, these results show significant differences in bone mineral density, PINP level, histological score, SrR content and mechanical testing, which demonstrate a relatively moderate effect of SrR treatment on bone strength and remodeling in the specific context of recovery after an interbody fusion surgery, and suggest the potential of SrR treatment as an effective adjunct to spinal interbody fusion surgery for human patients.

Increased sulfatase 1 gene expression in degenerative intervertebral disc cells.

Sulfatase 1 (SULF1) plays a key role in cell signaling involving in cell growth, differentiation, proliferation, and migration. Abnormal SULF1 expression has been implicated in the development of various cancers and diseases of the skeletal and nervous systems. The present study aims to examine the difference in SULF1 expression between degenerative and non-degenerative intervertebral discs (IVDs) to provide an enhanced understanding of disc degeneration. Degenerative and non-degenerative disc tissues were surgically harvested from patients and experimental rats. Disc degeneration-specific genes were identified by microarray analysis. The gene expression of SULF1 was measured by sulfatase assay, reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blotting. Also, the presence of SULF1 in human and rat discs was confirmed by immunohistochemistry. More specifically in human cells, an increase of SULF1 gene expression was observed in degenerative cells at both mRNA and protein levels, as well as in time- and dose-dependent manner in response to TNF-α treatment. Increased staining of SULF1 was detected in degenerative discs compared to non-degenerative discs for humans and rats. These findings show an upregulation of SULF1 in degenerative discs for the first time, and suggest that there is a link between SULF1 and disc degeneration.

Symptomatic epidural hematoma after lumbar decompression surgery.

BACKGROUND CONTEXT: Postoperative symptomatic epidural hematoma (SEH) is a serious complication of lumbar spine surgery. Despite its rarity, this uncommon complication may result in devastating neurological sequelae, including lower limb weakness. PURPOSE: A retrospective study was made to identify possible risk factors of postoperative spinal epidural hematoma by reviewing the clinical cases of this rare complication and analyzing the postoperative evaluations of patients. METHODS: From 2002 to 2010, out of 15,562 who underwent lumbar decompression procedure with/without instrumentation, 25 patients required reoperation for epidural hematoma after the initial spinal surgery. For the control group, another 75 patients were randomly selected from the pool of patients who received lumbar decompression surgery during the same period of time. The medical records of preoperative, intraoperative and postoperative factors were collected to determine possible risk factors by comparing between the cases and controls, and the postoperative evaluations of muscle power, intractable pain, saddle anesthesia, time to detection and time to evacuation were analyzed to find if there is any significant relation within the case group. Mann-Whitney U test, two-sample t test, χ (2) test and Fisher's exact test were used for statistical analysis. RESULTS: The incidence of postoperative symptomatic epidural hematoma is 0.16%. After the initial procedure, 20 (80%) patients developed progressive decrease in muscle power (MP ≤ 3), 14 (56%) patients had intractable pain (VAS ≥ 7), and 19 (76%) patients had saddle anesthesia. Preoperative diastolic blood pressure, intraoperative use of gelfoam for dura coverage and postoperative drain output were statistically significant risk factors (p < 0.01). Within the SEH case group, postoperative symptom of decreased muscle power had significant relation with blood loss, laminectomy level and fusion level (p = 0.016, 0.021, 0.010). If the symptom of decreased muscle power or perianal anesthesia was not improved after hematoma evacuation, there was a tendency for permanent leg weakness after 1-year follow-up (p = 0.001, 0.003). CONCLUSIONS: The findings suggest that preoperative diastolic blood pressure, intraoperative use of gelfoam for dura coverage and postoperative drain output are risk factors for symptomatic epidural hematoma after lumbar decompression surgery. Major blood loss and multilevel surgical procedure could result in poor recovery of muscle power. After spine decompression surgery, early detection and evacuation of hematoma are the key to avoid neurologic deterioration and have better clinical outcomes.

Advanced glycation end products in degenerative nucleus pulposus with diabetes.

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100 µg/ml) for 3 days, and mRNA expressions of MMP-2 and RAGE were measured by real-time RT-PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP-2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP-2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration.