Plasma Eluate Research Articles

Dried Blood Spot Analyses for the Diagnosis of Anti-cytokine Autoantibodies

BackgroundHigh-titer neutralizing anti-cytokine autoantibodies have been shown to be involved in several acquired diseases, including pulmonary alveolar proteinosis, cryptococcal meningitis, and disseminated/extrapulmonary Nocardia infections (anti-GM-CSF autoantibodies), disseminated mycobacterial disease (anti-IFN-γ autoantibodies), and some cases of severe COVID-19 infection (anti-type 1 interferons). Currently, patient blood samples are shipped via courier and require temperaturecontrolled conditions for transfer. This method is expensive and requires patients to have access to medical personnel to draw the blood. However, the well-established technique of collecting blood on a paper card as a dried blood spot (DBS) for diagnosis offers a point of care alternative which can be performed with a simple finger prick. This method is less invasive, cheaper, and allows for easy transport of patient samples. Methods30 uL of whole blood from patients was blotted on filter paper and stored at 4C until use. The filter paper was hole punched and each punched spot was eluted with 150 uL of a 0.05% Tween PBS solution at room temperature overnight. The eluate was screened for anti-cytokine autoantibodies using a particle-based approach. Patient plasma was also screened in conjunction for comparison. ResultsWe confirmed the presence of autoantibodies in the DBS eluate from 4 previously diagnosed patients with anti-GM-CSF autoantibodies and 2 patients with anti-IFN-γ autoantibodies. Functional studies showed the DBS eluate from a patient with anti-GM-CSF autoantibodies was able to block GM-CSF-induced STAT-5 phosphorylation in normal PBMC. As a proof of concept and to increase the number of patients evaluated, we also confirmed the presence of anti-cytokine autoantibodies using dried plasma eluate from 9 patients with known anti-GM-CSF autoantibodies and 9 patients with anti-IFN-γ autoantibodies. Levels detected in DBS analyses were comparable to the levels found in plasma from the same patients not subjected to blotting and elution. Temperature studies showed that the autoantibodies were detected at similar levels when stored at 4C, 25C, and 40C for a week. ConclusionsThe diagnosis of pathogenic anti-cytokine autoantibodies should be considered in the context of unusual or adult-onset infections, and screening for this diagnosis can be performed with dried blood spot testing.

Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA.

Simple SummaryCancer treatment strategies and their follow-up monitoring are changing to personalized therapies, based on molecular genetic information from the individual person. Liquid biopsy, where this molecular information is derived from body fluids such as blood, has the potential to provide a systemic fingerprint of cancer dynamics, and, compared to tissue biopsy, is much less invasive for the patient. We used the previously published mediator probe PCR technology for liquid biopsy detection of several mutations in one reaction, so-called digital multiplex PCR. Quantification of point mutations in plasma eluates from follow-up patients using 4-plex digital assays showed a comparable performance to reference 2-plex assays. As a key feature, the presented multiplex assays require no laborious optimization as they use the same concentrations and cycling conditions for all targets. This allows for flexible design and interchangeable target panels, thus the assay is easily adaptable for individual patient monitoring and reduces sample consumption.There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (KRAS and BRAF) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

Evolution of cross-neutralizing antibodies and mapping epitope specificity in plasma of chronic HIV-1-infected antiretroviral therapy-naïve children from India.

Delineating the factors leading to the development of broadly neutralizing antibodies (bnAbs) during natural HIV-1 infection and dissecting their epitope specificities generates useful information for vaccine design. This is the first longitudinal study to assess the plasma-neutralizing antibody response and neutralizing determinants in HIV-1-infected children from India. We enrolled 26 and followed up 20 antiretroviral therapy (ART)-naïve, asymptomatic, chronic HIV-1-infected children. Five (19.2 %) baseline and 10 (50 %) follow-up plasma samples neutralized ≥50 % of subtypes A, B and C tier 2 viruses at an ID50 titre ≥150. A modest improvement in neutralization breadth and potency was observed with time. At baseline, subtype C-specific neutralization predominated (P=0.026); interestingly, follow-up samples exhibited cross-neutralizing activity. Epitope mapping revealed V3C reactive antibodies with significantly increased Max50 binding titres in follow-up samples from five infected children; patient #4's plasma antibodies exhibited V3-directed neutralization. A salient observation was the presence of CD4 binding site (CD4bs)-specific NAbs in patient #18 that improved with time (1.76-fold). The RSC3 wild-type (RSC3WT) protein-depleted plasma eluate of patient #18 demonstrated a more than 50% ID50 decrease in neutralization capacity against five HIV-1 pseudoviruses. Further, the presence of CD4bs-neutralizing determinants in patient #18's plasma was confirmed by the neutralizing activity demonstrated by the CD4bs-directed IgG fraction purified from this plasma, and competition with sCD4 against JRFLgp120, identifying this paediatric donor as a potential candidate for the isolation of CD4bs-directed bnAbs. Overall, we observed a relative increase in plasma-neutralizing activity with time in HIV-1-infected children, which suggests that the bnAbs evolve.

Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni.

Interactions between early developing Schistosoma mansoni larval stages and the hemolymph of its snail intermediate host represent the first molecular encounter with the snail’s immune system. To gain a more comprehensive understanding of this early parasite-host interaction, biotinylated sporocyst tegumental membrane (Mem) proteins and larval transformation proteins (LTP) were affixed to streptavidin-agarose beads and used as affinity matrices to enrich for larval-reactive plasma proteins from susceptible (NMRI) and resistant (BS-90) strains of the snail Biomphalaria glabrata. Nano-LC/MS-MS proteomic analyses of isolated plasma proteins revealed a diverse array of 94 immune-and nonimmune-related plasma proteins. Included among the immune-related subset were pattern recognition receptors (lectins, LPS-binding protein, thioester-containing proteins-TEPs), stress proteins (HSP60 and 70), adhesion proteins (dermatopontins), metalloproteases (A Disintegrin And Metalloproteinase (ADAM), ADAM-related Zn proteinases), cytotoxins (biomphalysin) and a Ca2+-binding protein (neo-calmodulin). Variable immunoglobulin and lectin domain (VIgL) gene family members, including fibrinogen-related proteins (FREPs), galectin-related proteins (GREPs) and C-type lectin-related proteins (CREPs), were the most prevalent of larval-reactive immune lectins present in plasma. FREPs were highly represented, although only a subset of FREP subfamilies (FREP 2, 3 and 12) were identified, suggesting potential selectivity in the repertoire of plasma lectins recognizing larval glycoconjugates. Other larval-binding FREP-like and CREP-like proteins possessing a C-terminal fibrinogen-related domain (FReD) or C-type lectin binding domain, respectively, and an Ig-fold domain also were identified as predicted proteins from the B. glabrata genome, although incomplete sequence data precluded their placement into specific FREP/CREP subfamilies. Similarly, a group of FReD-containing proteins (angiopoeitin-4, ficolin-2) that lacked N-terminal Ig-fold(s) were identified as a distinct group of FREP-like proteins, separate from the VIgL lectin family. Finally, differential appearance of GREPs in BS-90 plasma eluates, and others proteins exclusively found in eluates of the NMRI strain, suggested snail strain differences in the expression of select larval-reactive immune proteins. This hypothesis was supported by the finding that differential gene expression of the GREP in BS-90 and ADAM in NMRI snail strains generally correlated with their patterns of protein expression. In summary, this study is the first to provide a global comparative proteomic analysis of constitutively expressed plasma proteins from susceptible and resistant B. glabrata strains capable of binding early-expressed larval S. mansoni proteins. Identified proteins, especially those exhibiting differential expression, may play a role in determining immune compatibility in this snail host-parasite system. A complete listing of raw peptide data are available via ProteomeXchange using identifier PXD004942.

Discrimination of normal and esophageal cancer plasma proteomes by MALDI-TOF mass spectrometry.

Most patients presenting with symptoms of esophageal cancer (EC) have advanced disease. Even with resection, the cure rate is extremely low due to local recurrence and metastatic disease. Early detection and effective therapeutic intervention are essential to improve survival. This study tested the hypothesis that the presence of EC modulates concentrations of specific plasma proteins and peptides, potentially allowing discrimination between EC and controls based on mass spectrometric analysis of the respective plasma proteomes. Blood samples from 79 esophageal cancer patients and 40 age-matched normal subjects were processed to plasma, and protein/peptide sub-fractions were isolated using HIC8 or WAX-derivatized superparamagnetic beads. Triplicate matrix-assisted laser desorption time-of-flight mass spectra were acquired for specific plasma fractions from each subject. HIC8 and WAX-derivatized plasma eluates yielded 79 and 77 candidate features, respectively, and a Random Forest algorithm identified a subset of features whose peak intensities allowed discrimination between cancer patients and controls. Areas under the curve in receiver operating characteristic curves for HIC8 spectra were 0.88 and 0.83 for WAX spectra. The combined feature set discriminated EC from control plasma with 79 % sensitivity and 79 % specificity, with positive and negative test likelihood ratios of >14 and 0.17, respectively. These data lay the foundation for the development of a clinically useful test for esophageal cancer based on statistical analysis of proteomic spectra of patient plasma samples. This approach will be validated by analysis of larger patient cohorts, development of cancer-specific classifiers, and assessment of racial origin imbalances.

Investigating the Use of Protein Saver Cards for Storage and Subsequent Detection of Bovine Anti-Brucella abortus Smooth Lipopolysaccharide Antibodies and Gamma Interferon

Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.

Effect of warfarin treatment on thrombin activatable fibrinolysis inhibitor (TAFI) activation and TAFI‐mediated inhibition of fibrinolysis

Severe clotting deficiencies are associated with enhanced in vitro fibrinolysis due to insufficient thrombin activatable fibrinolysis inhibitor (TAFI) activation. Because oral anticoagulant therapy (OAT) with warfarin causes a partial deficiency of vitamin K-dependent factors, its effect on clot lysability remains unclear. To evaluate plasma and blood fibrinolytic capacity in patients under stable OAT (n = 221) as compared with controls (n = 132). Fibrinolysis resistance of plasma (turbidimetry) and blood (thromboelastography) clots was calculated as the lysis time of tissue factor-induced clots exposed to 30 and 100 ng mL(-1) t-PA, respectively. Plasma PAI-1 was similar in the two groups, whereas TAFI was slightly lower in patients. OAT plasma clots lysed faster than controls (P = 0.001). The addition of the TAFIa inhibitor PTCI reduced lysis time by 14% in OAT and 34% in controls, and the difference between the groups disappeared. Similar data were obtained with blood clots. Thrombin and TAFIa generation in OAT plasma amounted to roughly 50% of controls, supporting a reduced thrombin-dependent TAFI activation. Clot resistance of OAT plasma was normalized by Ba-citrate plasma eluate or prothrombin but not by BaSO(4) serum eluate, rFVIIa or FX. Surprisingly, circulating levels of TAFIa and its inactive derivative TAFIai were higher in warfarin patients (P < 0.0001) and correlated with plasmin-antiplasmin (P = 0.0001) but not with prothrombin F(1) (+) (2) . OAT enhances both plasma and blood fibrinolysis by reducing thrombin-dependent TAFI activation, a phenomenon largely determined by low prothrombin levels. At variance with in vitro data, 'basal' in vivo TAFIa/ai levels seem related to plasmin rather than thrombin generation.

Purification of anticardiolipin and lupus anticoagulant activities by using cardiolipin immobilized on agarose beads

We describe a novel method for the purification of aPL, in which pure CL is immobilized on octyl-sepharose beads by hydrophobic interaction. No lipid contamination was present in eluates, and the system could be reutilized three times without loosing extracting capacity. Four patients with antiphospholipid syndrome were studied. A marked decrease in aCL and LA activities was found in all patient plasmas after the passage through a CL-octyl-sepharose column. Both activities were recovered in eluates which contained β 2-GP-I and IgGs. β 2-GP-I was also present in normal plasma eluates, which showed no aCL and slight LA activity. This method represents an improvement in the purification of aPL, and could be useful in explaining the mechanism of action of antibodies that are obtained using pure phospholipid as extracting matrix.

Enhanced endothelial tissue factor but normal thrombomodulin in endotoxin-treated rabbits

Exposure of cultured endothelial cells to bacterial endotoxin induces an enhancement of cell procoagulant activity (PCA) and a simultaneous reduction of thrombomodulin activity (TM). We evaluated the effect of endotoxin on the expression of both endothelial PCA and TM in vivo, in rabbits. Animals were given a single i.v. injection of endotoxin ( E. coli 0111:B4 LPS, W, 10–200 μg/kg); the thoracic aorta was harvested after 2 or 4 hours and placed in an ad hoc device to expose the endothelial surface only. Endotoxin treatment resulted in a dose-dependent increase of endothelial PCA (p < 0.001, at 100 μg/kg or more), which was totally dependent on factor VII and thus identified as tissue factor. In contrast, endothelial TM activity, as measured by the rate of thrombin-induced protein C activation, was similar in control and endotoxemic rabbits, even when the animals were given two injections (50 μg/kg, 24 h apart), or a continuous infusion (40 μg/kg/h during 4 hours) of endotoxin. To explore the effect of endotoxin on TM activity at the microcirculation level, we measured the extent of protein C activation in vivo, induced by a continuous infusion of low doses of thrombin (1 NIH U/kg/min for 60 min). Again, endotoxin administration was not associated with significant changes in TM-dependent protein C activation, as assessed by the anticoagulant activity present in a barium citrate plasma eluate obtained at the end of thrombin infusion. Although reduction of TM during persistent endotoxemia cannot be definitively excluded, our data support a major role of endothelial PCA in LPS-induced coagulative changes.

飲酒（焼ちゅう）により血中に産生されるウロキナーゼ様線溶酵素

After normal human subjects (78 in number, aged 18-34 years) had taken the local Japanese spirit “Shochu, ” rice wine “Sake” and beer, almost no change in enzyme parameters of plasma fibrinolysis was observed. However, plasma eluate from [Nα-(e-aminocaproyl)-DL-homoarginine hexylester]-Sepharose affinity chromatography revealed extremely strong pyro-Glu-Gly-Arg-pNA (S-2444) amidolytic and fibrinolytic activities in comparison with those of the control. The highest activities were observed in the Shochu group (1, 133nmol pNA/dl, 470IU/dl plasma), followed by the Sake (811nmol pNA/dl, 415IU/dl plasma) and beer (707nmol pNA/dl, 313IU/dl plasma) groups, corresponding to an approximately 2.1, 1.7 and 1.5 times increase in plasma fibrinolysis compared to the control group (490nmol pNA/dl, 242IU/dl plasma).Fibrinolytic enzyme isolated from the Shochu group was partially inactivted by rabbit anti HMW-UK serum and could be adsorbed on an anti HMW-UK IgG-Sepharose immunoadsorbent column. The final preparation eluted from the immunoadsorbent column with 6M urea buffer revealed molecular weights of approximately 30, 000 (main band), 50, 000 and 30, 000 (minor bands) by zymography, and an active site of approximately 8% (assuming the molecular weight of 30, 000) by p-nitrophenyl-p′-guanidinobenzoate titration. The enzyme not only hydrolyzed the UK-specific substrate S-2444 but also strongly activated human Glu-plasminogen to plasmin. This indicated that some of the enzyme appearing in the plasma after drinking was “UK-like plasminogen activator”.

Mechanisms of proteinuria in human glomerulonephritis.

We evaluated glomerular barrier function in 28 patients with glomerulonephritis. Neutral dextrans of graded size were used to characterize the size-selective properties of the barrier. Charge selectivity was characterized by electrofocusing excreted urinary proteins. A fractional IgG clearance (relative to freely permeable inulin), smaller or greater than 100 x 10(-5) was used to distinguish patients with minor (group I, n = 13) and major (group II, n = 15) urinary IgG leakage, respectively. Fractional clearances of smaller dextrans (radii 20-50 A) were similar, but those of larger dextrans (radii 52-60 A) were elevated in group II relative to group I patients. A model of solute transport through a bimodal pore size distribution revealed the values for pore radius in the lower mode to approximate 51-55 A in both group I and group II patients. Pore radius in the upper mode, by contrast, was much larger in group II than in group I patients, approximating 87-97 vs. 72-77 A, respectively. Electrofocusing of urinary protein from group I patients revealed mostly albumin (isoelectric point 5.2). In group II patients, however, immunoglobulin excretion was copious. Moreover, the distribution of anionic, neutral, and cationic species (isoelectric points 5.5-8.5) in urinary and plasma eluates of IgG2 and IgG4 was similar. We conclude that when glomerulonephritis is associated with selective albuminuria, as in group I,, there is an isolated reduction of electrostatic retardation of relatively small anionic proteins. Major urinary IgG leakage (group II), however, appears to result from the development in the glomerular membrane of a subpopulation of enlarged pores that are highly permeable towards proteins of large size and varying charge.

Prothrombin Houston: a dysprothrombin identifiable by crossed immunoelectrofocusing and abnormal Echis carinatus venom activation

A 72-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.

Prothrombin Houston: a dysprothrombin identifiable by crossed immunoelectrofocusing and abnormal Echis carinatus venom activation

A 70-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed Immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.

A simplified radio-enzymatic assay for total catecholamines in plasma

A two step radioenzymatic procedure for determining plasma catecholamines (noradrenaline plus adrenaline) has been developed. Catecholamines are isolated from 2 ml of plasma (pH 8.6) by adsorption on alumina and subsequent acid elution. Aliquots of the acid eluate are assayed for total catecholamines using 3 H-Sadenosylmethionine and catechol O-methyltransferase (COMT). 3 H-normetanephrine formation is linearly related to noradrenaline concentration. Inhibition of COMT by plasma eluates necessitates internal standardization for each eluate. At plasma concentrations of 440 pg/ml the coefficient of variation is 4.3%. The method has sufficient sensitivity and precision to measure total catecholamines in small amounts of plasma and tissues.

Studies on thrombin formation.

SummaryPlasma accelerator globulin, antihemophilic factor as well as serum eluate were found to produce increased thrombin elaboration in a reaction mixture consisting of bovine plasma eluate, platelets and calcium ions. In the presence of both plasma accelerator globulin and antihemophilic factor, conversion of prothrombin to thrombin became accelerated. Addition of serum eluate to the plasma eluate enhanced the accelerating influence of either plasma accelerator globulin, of antihemophilic factor or of both in the utilization of prothrombin. Upon preincubation of those factors which are supposed to participate in the conversion of prothrombin, before addition of a plasma eluate to the incubation mixture, it was found that more prothrombin was converted in a shorter period of time than in the one-stage thrombin formation procedure. The greater velocitv of thrombin formation in the two-stage procedure indicated that precursory interactions of certain factors had taken place during the initial incubation. ...