Pooled Patient Serum Research Articles

Exosomal miR-208b related with oxaliplatin resistance promotes Treg expansion in colorectal cancer

Oxaliplatin resistance is a challenge in the treatment of colorectal cancer (CRC) patients. Regulatory T cells (Tregs) are well known for their immunosuppressive roles, and targeting Tregs is an effective way to improve chemosensitivity. Exosome-delivered microRNA (miRNA) might be used as a potential biomarker for predicting chemosensitivity. However, the relationship between Tregs and exosomal miRNAs remains largely unknown. TaqMan low-density array was performed to screen the differentially expressed serum miRNAs from pooled serum of patients who had FOLFOX treatment. Differential expression was validated using qRT-PCR in individual samples. Exosomes were isolated by sequential differential centrifugation, and they were verified by transmission electron microscopy. The RNA and protein levels were determined by quantitative real-time PCR and western blotting. A mouse xenograft model was adopted to evaluate the correlation between exosome-derived miR-208b and Tregs in vivo. We demonstrated that circulating miR-208b is a non-invasive marker for predicting FOLFOX sensitivity in CRC. miR-208b in colon cancer was secreted by tumor cells in the pattern of exosomes, and oxaliplatin-resistant cells showed the most obvious phenomenon of miR-208b increase. Colon cancer cell-secreted miR-208b was sufficiently delivered into recipient T cells to promote Treg expansion by targeting programmed cell death factor 4 (PDCD4). Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-208b resulted in tumor growth and oxaliplatin resistance. Our results demonstrate that tumor-secreted miR-208b promotes Treg expansion by targeting PDCD4, and it may be related to a decrease of oxaliplatin-based chemosensitivity in CRC. These findings highlight a potential role of exosomal miR-208b as a predictive biomarker for oxaliplatin-based therapy response, and they provide a novel target for immunotherapy.

Comprehensive assessment of biotin interference in immunoassays

BackgroundBiotinylated antibodies and analogues are currently used in many immunoassays while biotin is widely used as a dietary supplement. Thus, biotin interference is an emerging issue for clinical laboratories. MethodsVarious concentrations of biotin solutions were prepared using pooled patient serum samples. All analytes were measured by sandwich or competitive immunoassay on the Roche Cobas 8000 e602 platform. ResultsSome of the sandwich immunoassay results were falsely decreased to different extents by different biotin levels, while some of the competitive immunoassay results were falsely increased. The most notable false reductions were in high-sensitivity troponin T, thyroid-stimulating hormone, and follicle-stimulating hormone results, while the most notable false increases were in triiodothyronine and vitamin D results. Other immunoassay results were also affected to some extent by biotin interference. ConclusionsBiotin can interfere in immunoassays and result in aberrant test results. Clinicians should use caution in interpreting abnormal results in patients who ingest biotin.

P116 Complement component 1q assay lacks sensitivity and trends with de novo donor-specific antibody titers

Aim Despite major advancements in immunosuppression, long-term allograft survival rates from deceased and living donors remain poor following post-kidney transplantation. The presence of de novo donor-specific antibodies (dnDSA) and complement component 1q (C1q) following transplantation contributes to repetitive microvascular injury that correlates with worsening graft function and graft failure. Immune monitoring post-transplantation of dnDSA and C1q is commonly employed to assess patient graft failure risk and can guide clinical decision making regarding the patient’s baseline immunosuppression. In this investigation, we sought to determine if the C1q assay provided added clinically relevant data as C1q positivity was often associated with dnDSA titer and the use of pooled patient serum for quality control varied greatly among different runs of the same lot number. Methods Kidney transplant patients were monitored for the presence of human leukocyte antigen (HLA) class I and class II antibodies using the Labscreen Single Antigen Antibody Detection Tests and the C1qScreen (One Lambda, Inc., Canoga Park, CA). C1q risk scores were delineated as N = C1q negative, 2 = C1q weak positive, 3 = C1q moderate positive, 4 = C1q strong positive, 5–6 = very strong positive based on C1q mean fluorescent intensity (MFI) cutoff ranges. Results DSA titration (1:16, MFI = 3136) trended as N = C1q negative. DSA titration (1:256, MFI = 13136) trended as 5–6 = C1q very strong positive. Poor delineation of C1q risk at intermediate titrations may be due to confounding quality control variability in the daily pooled plasma serum measurements, which can alter baseline risk scoring of C1q MFI values. Conclusions C1q and dnDSA are often tracked to make risk assessments regarding kidney allograft health post-transplantation; however, C1q MFI measurements may not be additionally informative for clinicians as it often tracked with dnDSA antibody titer and the C1q assay lacks sensitivity at lower concentrations and potentially makes C1q risk scores ambiguous or misleading.

Therapeutic Drug Monitoring of Lithium: A Study of the Accuracy and Analytical Variation Between Laboratories in Denmark.

Serum lithium is monitored to ensure levels within the narrow therapeutic window. This study examines the interlaboratory variation and inaccuracy of lithium monitoring in Denmark. In 16 samples consisting of (1) control materials (n = 4), (2) pooled patient serum (n = 5), and (3) serum from individual patients (n = 7), lithium was measured in 19 laboratories using 20 different instruments. The lithium concentrations were targeted by a reference laboratory. Ion-selective electrode (n = 5), reflective spectrophotometric (RSM, n = 5), and spectrophotometric (n = 10) methods were used. Acceptable accuracy-interpreted as total differences from target values (bias) less than or equal to ±12%-was generally found in patient samples above 0.7 mmol/L. Below 0.7 mmol/L, 8 instruments had 2 or more patient samples exceeding a difference to the targeted reference value of >12%. Seven of these instruments had a systematic positive or negative bias and more so at lower lithium concentrations. Three poorly calibrated instruments were found in the ion-selective electrode group, 3 in the spectrophotometric group, and 2 in the RSM group. The instruments using reflectance spectrophotometry (RSM) were on average 21% positively biased when measuring control materials. However, this effect was not observed in patient samples. Large interlaboratory variation was found below 0.7 mmol/L because of 7 instruments with a poor accuracy and 1 with poor precision. Methods should be recalibrated or substituted. Controls below 0.7 mmol/L are recommended.

Use of In Vivo Gene Expression of Isolated Bone Marrow Mesenchymal Stromal Cells to Study the Pathophysiology of Osteoporosis in Patients With Severe Thalassemia

We compared osteoblast differentiation gene expressions in the isolated CD105 mesenchymal stromal cells from bone marrow in 10 patients with severe thalassemia and 12 normal donor controls. The fold expressions of osteoblast differentiation genes of CD105 cells from patients with thalassemia were lower than those of normal donors but increased after being cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Moreover, the fold expressions of these genes of CD105 cells from normal donors when cultured with 10% pooled serum of patients with thalassemia were lower than when cultured with 10% pooled serum of normal donors. We have also presented the evidence of reversible suppressed expression of these genes in CD105cells from patients with thalassemia when cultured in pooled serum of normal donors. Moreover, healthy donor CD105 cells exhibited lower expression of these genes when cultured in pooled serum of patients with thalassemia compared with pooled serum of normal donors indicating the existence of circulating factors in thalassemic serum impairing the differentiation of mesenchymal stromal cells to osteoblasts.

Application of five frozen human-pooled serum samples assigned by the International Federation of Clinical Chemistry and Laboratory Medicine reference procedure in a traceability investigation of γ-glutamyltransferase catalytic concentration measurements in China

The in vitro directive of the European Union requires traceability to the international recommended reference procedures. The application of the reference procedures is necessary in order to evaluate the accuracy of gamma-glutamyltransferase (GGT) assays of routine measurement systems in China. Five frozen patient-pooled serum samples were assigned values by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure in order to evaluate the traceability of the results of GGT catalytic activity from six homogeneous systems. One of the serum samples was used to calibrate seven non-homogeneous systems. All of the homogeneous systems, except the Dade system (Dade Bering Inc, IL, USA), achieved traceability within the measurement range. The Roche and Hitachi systems were better than the other systems. After calibration, the variance of the non-homogeneous systems decreased dramatically from between 14.50% and 25.23% to between 1.25% and 3.09% and the bias decreased from between -11.4% and -4.1% to between 0.5% and 3.5%. Manufacturers in China should ensure that their calibration systems correspond to the IFCC reference procedures. Fresh frozen pooled patient serum assigned by reference laboratories can be used to calibrate non-homogeneous systems in order to achieve traceability.

Two Cases with Unusual Vancomycin Measurements

Patient A was a 68-year-old woman with a history of lymphoplasmacytic lymphoma who presented after several weeks of mucosal bleeding. On admission, her cancer involved 95% of bone marrow cells. She was pancytopenic and febrile (38.3 °C). Leukocyte count was 0.39 × 103/μL (neutrophils, 0.03 × 103/μL), hematocrit 25%, platelet count 21 × 103/μL, relative serum viscosity 2.3 (reference interval, 1.4–1.8), blood urea nitrogen 6.4 mmol/L (18 mg/dL), creatinine 62 μmol/L (0.7 mg/dL), and estimated glomerular filtration rate >60 mL/min/1.73 m2. She had an IgMκ monoclonal component of 42.8 g/L. This patient was started on vancomycin 1 g intravenously (IV) every 12 h, ceftazidime 2 g IV every 8 h, and a course of chemotherapy. On day 3 after the beginning of antibiotic treatment, a trough specimen was collected for measurement of vancomycin. The concentration, measured with a Beckman Coulter Synchron competitive turbidimetric immunoassay, was <0.1 mg/L. The result, which was incompatible with ongoing vancomycin therapy, signaled a problem to the technologist. No analytical issues were evident upon review of calibration, controls, and results for other chemistry tests performed on the same specimen. In an attempt to resolve an apparent falsely low result, a 1:1 mix of the specimen was made with the Beckman liquid comprehensive control serum (level 3, 30.4 mg/L) and demonstrated near complete recovery (result of mix after adjusting for dilution, 28.6 mg/L). However, a 1:1 mix with pooled patient serum containing vancomycin (11 mg/L) led to only 15% recovery (result of mix after adjustment for dilution, 1.7 mg/L). The specimen was subsequently sent to another laboratory, where a vancomycin concentration of 9.8 mg/L was measured by use of a competitive enzyme-linked immunoassay (Emit, Olympus). Patient B was a 64 year-old woman with a history of non-Hodgkin lymphoma admitted …

The impact of hemolysis on Ortho-Clinical Diagnostic's ECi and Roche's elecsys immunoassay systems

Background: Hemolysis is regularly encountered in clinical specimens and often interferes with a variety of laboratory test methods. Although not widely recognized, immunoassays based on nonisotopic detection systems can also be affected by hemolysis. For this reason, we investigated the effect of differing amounts of hemolysis across a range of values for several immunoassays on the Ortho-Clinical Diagnostics ECi and Roche Elecsys platforms. Methods: Hemolysate was prepared from whole blood and spiked at varying concentrations into pooled patient serum samples for different analytes. Results: Out of the 21 analytes tested, six (28.6%) exhibited significant increases or decreases in measured concentrations with increasing amounts of hemolysis. Conclusions: Although immunoassays are generally thought to be impervious to hemolysis interference, hemolysis can interfere in immunoassay testing platforms. For these reasons, we recommend that laboratories conduct hemolysis interference studies for all laboratory test protocols.

Expression and evaluation of IgE-binding capacity of recombinant Pacific mackerel parvalbumin

ABSTRACT Background Parvalbumin is the major and cross-reactive allergen in fish. Sufficient amounts of IgE-reactive recombinant fish parvalbumin are needed for diagnosis and immunotherapy of fish allergy. Methods A DNA fragment corresponding to parvalbumin of the Pacific mackerel Scomber japonicus was synthesized and cloned into the expression vector pGEX-6p-3 to produce glutathione S-transferase (GST)-fusion parvalbumin in Escherichia coli. The GST-free recombinant parvalbumin was purified using the RediPack GST Purification Module (Amersham Pharmacia Biotech, Buckinghamshire, UK). Parvalbumins of seven species of fish (Japanese eel, horse mackerel, red sea bream, Pacific mackerel, skipjack, bigeye tuna and Japanese flounder) were purified by gel filtration and reverse-phase HPLC. The IgE-binding capacity was examined by ELISA and antigenic cross-reactivity by inhibition ELISA. Results The GST-free recombinant Pacific mackerel parvalbumin was obtained in an electrophoretically pure state. Data from ELISA and inhibition ELISA revealed that the recombinant parvalbumin contains most of the IgE-binding epitopes of the natural counterpart. In addition, the recombinant parvalbumin inhibited the IgE reactivities of the pooled patient serum to parvalbumins purified from six species of fish in almost the same magnitude as the natural Pacific mackerel parvalbumin. Conclusions Because the recombinant Pacific mackerel parvalbumin bearing the IgE-binding capacity of the natural counterpart is cross-reactive with various fish parvalbumins, it can be a useful tool for the diagnosis and immunotherapy of fish allergy.

Detection of Extrahepatic Hepatitis C Virus Replication by a Novel, Highly Sensitive, Single-Tube Nested Polymerase Chain Reaction

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction.

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Association of end-product adducts with increased IgE binding of roasted peanuts.

Recently, we have shown that roasted peanuts have a higher level of IgE binding (i.e., potentially more allergenic) than raw peanuts. We hypothesized that this increase in IgE binding of roasted peanuts is due to an increased levels of protein-bound end products or adducts such as advanced glycation end products (AGE), N-(carboxymethyl)lysine (CML), malondialdehyde (MDA), and 4-hydroxynonenal (HNE). To support our hypothesis, we produced polyclonal antibodies (IgG) to each of these adducts, determined their levels in raw and roasted peanuts, and examined their ability to bind to IgE from a pooled serum of patients with clinically important peanut allergy. Results showed that AGE, CML, MDA, and HNE adducts were all present in raw and roasted peanuts. Roasted peanuts exhibited a higher level of AGE and MDA adducts than raw peanuts. IgE was partially inhibited in a competitive ELISA by antibodies to AGE but not by antibodies to CML, MDA, or HNE. This indicates that IgE has an affinity for peanut AGE adducts. Roasted peanuts exhibited a higher level of IgE binding, which was correlated with a higher level of AGE adducts. We concluded that there is an association between AGE adducts and increased IgE binding (i.e., allergenicity) of roasted peanuts.

Comparison of methods to assess airborne rat and mouse allergen levels. II. Factors influencing antigen detection.

Potential factors influencing antigen detection in immunoassays for measuring rat or mouse aeroallergen (i.e., assay setup, antigen specificities, standard extracts used, and antigen decay) were investigated in a three-country study (the UK, The Netherlands, Sweden). An inhibition enzyme immunoassay (EIA) setup gave nominal rat urinary allergen (RUA) sample values seven times higher than a sandwich EIA setup utilizing identical antibodies and standards. In immunoblotting experiments, pooled patient serum and polyclonal rabbit antibodies partly detected different rat antigens; monoclonal antibody specificity could not be determined. Immunoblot detection of mouse urinary antigens (MUA) by the polyclonal rabbit antibodies from all laboratories was similar. In both the RUA and the MUA assays, urinary antigen standards were detected with similar potency, except purified Rat n 1, which was an inefficient inhibitor in the RUA RAST inhibition. In the sandwich EIA RUA assays, a rat room-dust extract was detected with 700800-fold less sensitivity than rat urine, whereas in the RAST RUA assay, dust inhibited equally with rat urine. Simulated decay did not decrease the potency of urinary antigen in any assay. Thus, assay setup and choice of detection antibodies strongly influence the nominal allergen levels. We recommend the use of standardized and characterized antibodies and standard extracts in sandwich EIAs to measure airborne rodent urinary allergens.

Epitope specificity and immunoaffinity purification of the major peanut allergen, Arah I

The antigenic and allergenic structure of Ara h I, a major allergen of peanuts, was investigated with the use of seven monoclonal antibodies obtained from BALB/c mice immunized with purified Ara h I. Previous work with monoclonal antibodies produced to allergens has primarily been done with inhalant allergens. Only recently have the major allergens of various foods been determined so that investigations with monoclonal antibodies into the allergenic epitopes could begin. When used as a solid phase in an ELISA, these monoclonal antibodies captured peanut antigen, which bound human IgE from patients with positive results to challenges to peanuts. The Ara h I monoclonal antibodies were found to be specific for peanut antigens when binding for other legumes was examined. In ELISA inhibition studies with the monoclonal antibodies, we identified four different antigenic sites on Ara h I. In related studies with pooled human IgE serum from patients with positive results to challenges to peanuts, we identified three similar IgE-binding epitopes. As a means of purifying the Ara h I allergen, we prepared an immunoaffinity column with monoclonal antibody 8D9. We eluted from this column the allergen Ara h I, which had a mean molecular weight of 63.5 kd and which bound human IgE from individual and pooled serum of patients with peanut sensitivity.

Allergen carriage by atmospheric aerosol: I. Ragweed pollen determinants in smaller micronic fractions

Outdoor air-bearing natural ragweed pollen burdens were drawn through 5 μm membrane filters during 24 hr periods; aerosols penetrating these barriers were collected on individual, in-line, 0.8 μm filters. The 0.8 μm filters were free of intact pollen grains and grain fragments when scanned in toto by transmitted light. Aqueous extracts of 0.8 μm filters were found to elicit wheal-and-flare skin test reactions in a ragweed pollen-sensitive subject but not in nonatopic controls. The same extracts produced significant inhibition of an IgG-ELISA system by use of ragweed pollen protein conjugated to polystyrene plate wells and pooled serum of patients on ragweed immunotherapy. No inhibition resulted from preincubation of serum with an unrelated allergen (hickory pollen extract). These data indicate the presence of airborne ragweed pollen allergen in aerosol fractions below 5 μm during seasonal periods of ragweed pollination, confirming the report of Busse et al. 9 Potential sources of such aerosols are prominent in nature, and their occurrence may help explain existing dose-response disparities in pollinosis.