Measurement of D-dimer in cerebrospinal fluid using a luminescent oxygen channeling immunoassay.

Background: Measurement of D-dimer in cerebrospinal fluid (CSF) allows insight into coagulation system activation in the central nervous system and can be utilized to monitor intracranial hemorrhage as well as acute phase processes beyond hemostasis in inflammatory and neoplastic diseases. So far, the measurability of D-dimer in low and very low concentrations in CSF was limited in conventional immunoassays. Novel high-sensitivity chemiluminescent immunoassays such as the luminescent oxygen channeling immunoassay (LOCI®) are getting increasingly available but have not been validated in CSF. The aim of this study was to investigate the accuracy and linearity of the LOCI® in assessing D-dimer in CSF. Methods: INNOVANCE LOCI hs D-dimer reagent cartridge was used for the measurement of D-dimer in CSF of patients with different neurological diseases. For the evaluation of linearity, dilution series were performed in a pooled CSF sample with the determination of intra-assay precision (CV, coefficient of variation) in 3 individual samples with 20 replicates. Furthermore, D-dimer concentrations measured by LOCI® were compared with the respective results of a routinely available clinical latex-enhanced immunoassay (HemosiIL D-Dimer HS 500). Results: Linear regression analysis of the LOCI® method revealed a r 2 of 1.00 (p < 0.001) with a regression coefficient B of 1.012 ± 0.003 (CI: 1.005-1.019, p < 0.001) and an intercept of -1.475 ± 1.309 (CI: -4.493 to 1.543); the median intra-assay CV was 0.69% (range: 0.68-0.75). In total, 185 CSF samples were measured by LOCI® technology, showing a mean concentration of 204.84 ± 2,214.93 ng/ml. D-dimer concentration between LOCI and latex-enhanced immunoassay differed by a factor of 10.6 ± 13.6 on average with a maximum deviation by a factor of 61.3; the maximum deviation was found at low concentrations. Conclusion: D-dimer in CSF of patients with neurological disease can be reliably measured by the LOCI® method with high linearity and accuracy at low concentrations.

Analysis of CSF D-Dimer to Identify Intrathecal Fibrin-Driven Autoimmunity in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Proteins of the coagulation system contribute to autoimmune inflammation in patients with multiple sclerosis (MS). On blood-brain barrier (BBB) disruption, fibrinogen enters the CNS and is rapidly converted to fibrin, unfolding pleiotropic autoimmune mechanisms. Fibrin accumulation leads to subsequent proteolytic degradation that results in D-dimer generation. The primary objective of this study was to determine intrathecal levels of D-dimer in CSF as a measure of intrathecal coagulation cascade activation and to evaluate its diagnostic utility in patients with MS in contrast to healthy subjects. Key secondary objectives included analysis of CSF D-dimer in differential diagnoses of MS and its relation to routine clinical markers of disease activity. METHODS: Patients admitted for the assessment of suspected MS were prospectively recruited from October 2017 to December 2020. Blood plasma and citrated CSF samples were analyzed using a highly sensitive luminescent oxygen channeling immunoassay. Intrathecal generation of D-dimer was analyzed by adjusting for CSF/serum albumin (Qalb) and CSF/plasma D-dimer quotients (QD-dimer), and corresponding CSF fibrinogen levels were determined. Final diagnoses after full evaluation and clinical data were recorded. RESULTS: Of 187 patients, 113 patients received a diagnosis of MS or clinically/radiologically isolated syndrome. We found increased intrathecal CSF D-dimer generation levels (QD-dimer/Qalb-index) for patients with relapsing-remitting MS (RRMS; n = 71, median 4.7, interquartile range [IQR] 2.5-8.0) when compared with those for disease controls (n = 22, median 2.6, IQR 2.1-4.8, p = 0.031). Absolute CSF D-dimer values correlated with CSF fibrinogen levels (r = 0.463; p < 0 .001) and CSF leukocytes (r = 0.273; p = 0.003) and were elevated in MS patients with contrast enhancement (CE) compared with MS patients without CE on MRI (n = 48, median 6 ng/mL, and IQR 3-15.25 vs n = 41, median 4 ng/mL, and IQR 2-7; p = 0.026). Exploratory subgroup analyses indicated a correlation of intrathecal inflammatory activity and CSF D-dimer levels. DISCUSSION: D-dimer in CSF can be reliably determined and correlates with markers of CNS inflammation and CSF fibrinogen levels. Adjusted for BBB dysfunction, CSF D-dimer may allow the identification of intrathecal coagulation cascade activation in patients with MS. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that CSF D-dimer levels are elevated in patients with RRMS.

Measurement of prothrombin fragment 1+2 in cerebrospinal fluid to identify thrombin generation in inflammatory central nervous system diseases.

BACKGROUND: The interaction of central nervous system inflammation and coagulation system activation in multiple sclerosis (MS) receives increasing attention for its diagnostic and therapeutic potential. During blood-brain barrier (BBB) disruption, fibrinogen migrates into the CNS and contributes to inflammation. In the coagulation cascade, fibrinogen is converted into fibrin by thrombin, which itself is cleaved from prothrombin by activated factor XII. We hypothesized that the conversion of prothrombin to thrombin can be quantified by prothrombin fragment 1+2 (PF1.2) in cerebrospinal fluid (CSF). Primary endpoint was the correlation between PF1.2, D-dimer and fibrinogen in CSF of patients with neuroinflammatory diseases. Secondary endpoints were PF1.2 levels depending on presence of contrast enhancement (CE) on MRI, and correlation between PF1.2 with serum-CSF albumin quotient (Qalb). Additionally, an exploratory analysis of CSF PF1.2 levels to distinguish between MS-patients and controls without neurological disease was performed. METHODS: Patients admitted for a suspected inflammatory CNS disease were prospectively recruited from October 2017 to December 2020. Citrated CSF samples were obtained and analyzed for PF1.2, fibrinogen and D-dimer using a highly sensitive luminescent oxygen channeling immunoassay. Patient clinical data and final diagnoses were retrospectively collected and analyzed. RESULTS: 187 patients were included, of whom 116 received diagnoses of relapsing-remitting (RRMS), primary-progressive MS, clinically or radiologically isolated syndrome, or anti-aquaporin-4-/anti-myelin-oligodendrocyte-glycoprotein-antibody-related diseases. CSF analysis of those 116 patients revealed a correlation between PF1.2 and CSF fibrinogen (ρ=.315; p<.001) as well as between PF1.2 and CSF D-dimer (ρ=.531; p<.001). Among all 187 patients, CSF PF1.2 was increased in patients with CE on MRI (n=71; 147.38 pmol/l; IQR 83.68-215.36) compared to patients without CE (n=86; 100.03 pmol/l; IQR 33.87-162.80; p=.008). CSF PF1.2 correlated significantly with Qalb (ρ=.445; p<.001). No differences of CSF PF1.2 levels were observed between RRMS (131.48 pmol/l, IQR 42.75-204.10) and disease controls (102.28 pmol/l; IQR 55.60-159.94; p=.606). CONCLUSION: In patients with autoimmune inflammatory CNS diseases PF1.2 correlated strongly with fibrinogen and D-dimer in CSF, indicating coagulation system activation. The findings suggest that thrombin generation might require acute BBB dysfunction to exert autoimmune effects in the CNS.

Response to aspirin and clopidogrel monitored with different platelet function methods.

Laboratory non-response to aspirin or clopidogrel is defined as an inability to cause in vitro detectable platelet function inhibition. It would be beneficial to monitor response to aspirin or clopidogrel with widely available and routinely used platelet function methods, like the platelet function analyzer (PFA-100) or the fully automated coagulation analyzer BCT. The aim of this study was to assess the potential of the coagulation analyzer BCT and the platelet function analyzer PFA-100 in monitoring the response of aspirin and clopidogrel. A group of 125 consecutive patients with arterial occlusive disease treated either with aspirin 100 mg/day (82 patients) or clopidogrel 75 mg/day (43 patients) as only antiplatelet drug were investigated. For the first time platelet-enriched plasma (PRP), not adjusted to a fixed predetermined concentration of platelets, was used for aggregation studies and the effect of clopidogrel alone without combination of aspirin treatment on platelet function was investigated. Response to aspirin was observed in 85% (70/82) of patients using PFA-100, while performing the arachidonic acid-induced aggregation on the BCT showed an inhibitory effect to aspirin in 91% (75/82) of patients. Non-response to aspirin was assessed with both platelet function methods in 7% (6/82) of patients. Clopidogrel response was observed in 58% (25/43) of patients when performing ADP-induced aggregation on the BCT. On the PFA-100 the antiplatelet effect of clopidogrel could not be detected. In conclusion, measurement of platelet aggregation on the BCT using native platelet-enriched plasma allows the quantification of individual inhibitory effects to aspirin as well as to clopidogrel, while the PFA-100 seems only suitable to investigate the degree of platelet inhibition induced by aspirin but not by clopidogrel.