Monoclonal Reagents Research Articles

The identification and enumeration of dendritic cell populations from individual mouse spleen and Peyer's patches using flow cytometric analysis

Dendritic cell (DC) research currently involves pooling of tissues from multiple animals followed by enrichment techniques to obtain sufficient numbers of DCs for analysis. Enrichment techniques take advantage of DC adherence, buoyant density properties, and/or positive or negative selection of cell populations using monoclonal antibodies. However, enrichment techniques may significantly change the maturation and/or activation status of DCs or selectively eliminate one or more subpopulations of DCs. To overcome these drawbacks, we designed a multicolor flow cytometric technique for simultaneous analysis of DC populations from tissues of individual mice. The spleens and Peyer's patches were mechanically and enzymatically digested, then incubated with a panel of six monoclonal antibody-fluorochrome direct conjugate reagents. A BD(R) Biosciences LSR II flow cytometer and FCS Express(R) software were used to identify three subtypes of mature DCs (myeloid, lymphoid, and plasmacytoid), precursor DCs, polymorphonuclear neutrophils, B lymphocytes, and Gr-1(+)/CD8alpha(+) memory T lymphocytes in the spleen. Likewise, we also identified these DC subpopulations and B lymphocytes in the Peyer's patches. The three key parameters in analysis of the DC populations were bi-exponential plotting in data analysis, collection of a minimum of 50,000 total events, and accurate color compensation. This procedure to analyze DCs from individual mice can lead to further understanding of the role of DCs in many other model systems as well as better understanding of how dietary or physiological factors may affect in vivo DC homeostasis.

Histopathological changes of the hippocampus neurons in brain injury.

The glial fibrillary acidic protein (GFAP) is known as a peculiar marker of mature astrocytes of the central nervous system (CNS). However, we found distinct immunopositivity to a monoclonal anti-GFAP reagent in the hippocampus neurons in head injury fatalities. The present study investigated the neuronal and neuroglial GFAP-immunopositivity in the hippocampus in a series of head injury cases, which included acute and subacute/delayed deaths (n=17 and n=73, respectively), and acute cardiac death (n=13), delayed death due to multiple organ failure from non-head injury (n=6), and pneumonia (n=9) cases were examined as controls. GFAP-immunopositivity in the neurons was frequently observed in CA4, CA3 and CA2 regions in cases of subacute/delayed head injury death that showed marked brain swelling accompanied by secondary brain stem hemorrhages, showing an inverse relationship to that in astrocytes. These findings suggest possible induction of GFAP or a related protein in hippocampus neurons depending on the severity of brain swelling following head injury.

One‐tube HLA‐B27/B2708 typing by flow cytometry using two “Anti‐HLA‐B27” monoclonal antibody reagents

Flow cytometry-based methods are widely used to detect the ankylosing spondylitis-associated HLA-B27/B2708 antigens. However, the generally used "HLA-B27" monoclonal antibodies (moabs) cross-react with many HLA specificities, including the common HLA-B7 antigen. Thus, using two "B27" moabs is highly recommended. The assay used two "HLA-B27" reagents, FITC and PE conjugated, respectively and a PE-Cy5 anti-CD3 antibody. Assay verification used 51 reference subjects possessing B*2705, B*2702, and B*2708 and a range of cross-reactive HLA antigens. A total of 1,006 consecutive patients' samples, referred for "HLA-B27 typing", were assayed alongside our standard flow cytometry method. A further 12 low frequency HLA-B*27 specificities were tested. Samples reacting with one "B27" moab only were B*27 allele typed by PCR using sequence-specific primers. All patient B27/B2708 positives (28.3%) were identified by our one-tube method which detected B*2705, B*2702, B*2708, and 8/12 other B*27 specificities. It was unaffected by HLA-B7 and other cross-reactive antigens but required a minor adjustment, a reduction in the volume of one of the "B27" moabs used, to avoid detecting a minority of HLA-B57 subjects. Our one-tube B27/B2708 assay is simple, robust, uses two "B27" moabs for typing precision and security, does not suffer from interference by HLA-B7 or other cross-reactive antigens and has the obvious advantage of using a single tube per typing.

Monoclonal Antibody Produced Against Calf Thymus Histone

The localization and modification of histone play a prime role in the regulation of unelucidated biochemical pathways, such as apoptosis and cell proliferatory-inhibitory activities. Thus studying histone reveals cell proliferation and its biochemical processes. Therefore anti-histone antibody production is important in order to isolate, purify, identify, and separate histone from cultured cells. In order to produce anti-histone antibodies by hybridoma, 8-week-old female BALB/c mice were immunized with commercially procured whole calf thymus histone. Subsequently the spleen cells of the immunized BALB/c and SP2/O-AG-14 myeloma cells were fused using polyethylene glycol, which resulted in 16 clones. Out of these 16 clones, only one, 1D7, was found to be specific to our antigen (calf thymus histone) by ELISA. The supernatant of the positive clone 1D7 was used in immunoblotting to characterize the specificity of the produced monoclonal antibody. Immunoblotting confirmed that the monoclonal antibody produced from 1D7 clone is specific to histone H1 epitope present in the calf thymus. Our results suggest that to generate more potent monoclonal antibody reagent specific for many different epitopes present on histone, a larger number of fusions are required. Further, if such potent monoclonal antibody reagent is produced, it will be an indispensable tool in elucidating the role of H1 in apoptosis and cell proliferatory-inhibitory activities. This reagent can be used for diagnosis of antihistone antibody in SLE patient's serum, for testing the viability of cultured monocytes, and for localization of histone in metaphase chromosomes.

False-positive reactions of some monoclonal anti-c reagents

Cross-reactivities of monoclonal reagents for red blood cell (RBC) typing are seen very rarely, e.g. in some reagents for testing of ABO or Lewis antigens. We report on two patients in whom we observed weak reactions using a monoclonal anti-c reagent containing clone MS35, but no reactions with anti-c reagents containing other monoclonal or polyclonal antibodies. To resolve this discrepancy, we studied RHCE exon 1 (Ex1) and 2 from genomic DNA of one patient and performed quality controls of the false-positive reacting anti-c reagent. RHCE Ex1 showed no abnormalities as well as RH Ex2 examined with primers specific for Ex2 D/C. No amplification product of RH Ex2 was received with primers specific for c. A titration study with untreated, papain-treated and sialidase-treated adult and newborn RBCs showed anti-i reactivity of the false-positive reacting reagent. This reactivity could not be removed by absorption with c-negative newborn RBCs without reducing the anti-c titre in the same way, indicating a cross-reactivity. Some manufacturers give a remark on a possible cold agglutinin activity of their monoclonal anti-c reagents in the instruction leaflet, but in order to avoid such irritation, we recommend to remove this cross-reactivity by dilution during the manufacturing process.

Characterization of anti‐D monoclonal antibody reagents based on their reactivity with the weak D phenotype

Anti-D monoclonal antibody (MoAb) reagents have improved D typing in routine tests. However, they exhibit a wide range of reactivity with the weak D phenotype depending on the characteristics of the different MoAbs used. We analyzed the reactivity of immunoglobulin (IgM) anti-D by cluster analysis to characterize MoAb that have similar reactivities with the weak D phenotype. We used 36 consecutive samples with weak D phenotype in routine testing and determined their reactivity with different IgM and IgG anti-D MoAbs. The samples were characterized as belonging to a weak D type or category using commercial molecular biology kits. The various anti-D MoAbs showed a wide grade of reactivity with the weak D samples. Similarities and dissimilarities in the behavior of the anti-D MoAbs with the weak D phenotype samples were detected with cluster analysis and the multidimensional scaling analysis. These analyses indicated different families of MoAbs characterized as having a high degree of homogeneity in their reactivity with the weak D phenotype. Between these MoAb families, the most effective at reacting with the weak D phenotype were RUM-1 and 175-2. The results show that it is possible to classify the anti-D MoAbs on the basis of their reactivity with the weak D phenotype. This provides information about different MoAbs' properties on the basis of their belonging to a given of anti-D family.

A novel KEL*1,3 allele with weak Kell antigen expression confirming the cis‐modifier effect of KEL3

KEL1 (K) is the most immunogenic red blood cell antigen of the Kell blood group system. The frequently occurring anti-KEL1 alloantibodies may cause hemolytic transfusion reactions as well as severe hemolytic disease of the fetus and newborn. So far, reports on weak KEL phenotypes are scarce. Blood samples of two unrelated Central European propositi with faint reactions in routine KEL1 typing were analyzed by extended serologic phenotyping, flow cytometry, genotyping by polymerase chain reaction with sequence-specific priming, and genomic DNA sequencing of separated parental KEL alleles. Both propositi exhibited an unusual KEL:1,2,3,4 phenotype: markedly weakened and negative reactions were observed in serologic KEL1 typing in gel and tube technique, respectively. No KEL1 epitope loss was detected using five different monoclonal anti-KEL1 reagents. KEL genotyping confirmed KEL*1/2 and KEL*3/4 (Kpa/Kpb) heterozygosity of both individuals. Importantly, DNA sequencing of the two separated parental alleles of both propositi revealed a KEL*1-specific coding nucleotide T578 and a KEL*3-specific T841 on the same allele. This novel KEL*1,3 (KKpa)allele was associated with an approximately 80 percent reduction in KEL1 expression, compared to KEL:1,2,-3,4 controls. The low KEL1 density was attributed to acis-modifier effect of KEL3, so far only reported in association with weakened expression of KEL2 (k). This is the first description of the KEL*1,3 allele encoding KEL1 and KEL3 on the same molecule. In individuals with weakened KEL1 because of KEL3 in cis, very sensitive serologic or molecular genetic techniques may be required for reliable Kell typing.

Blood group serology

Blood group serology

Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia

AbstractDrug-induced immune thrombocytopenia (DITP) is caused by drug-dependent antibodies (DDAbs) that are nonreactive in themselves but bind tightly to specific platelet membrane glycoproteins (GP) when soluble drug is present at pharmacologic concentrations. This reaction takes place without covalent linkage of drug to the target, indicating that drug does not function as a classical hapten to promote antibody binding. Studies to define other mechanism(s) responsible for this interaction have been frustrated by the polyclonal nature of human DDAbs and limited quantities of antibody usually available. We produced 2 monoclonal antibodies (mAbs), 314.1 and 314.3, from a mouse immunized with purified human GPIIb/IIIa and quinine that recognize the N terminus of the GPIIb β propeller domain only when soluble quinine is present. Both monoclonals closely mimic the behavior of antibodies from patients with quinine-induced immune thrombo-cytopenia in their reactions at various concentrations of quinine and quinine congeners. Sequencing studies showed that the 2 mAbs are closely related structurally and that mAb 314.3 probably evolved from mAb 314.1 in the course of the immune response. These monoclonal reagents are the first of their kind and should facilitate studies to define the molecular basis for drug-dependent antibody binding and platelet destruction in DITP.

Anti-peptide antibody screening: Selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique

A refined surface plasmon resonance method was developed to measure the kinetics of peptide binding to rabbit monoclonal antibodies (RabMAbs). Optimized amounts of RabMAbs were captured onto sensor chips from hybridoma supernatants followed by binding of free peptides from solution. This allowed kinetic measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies and determination of affinity constants without complications contributed by avidity considerations. Peptide-binding responses were normalized for the amount of antibody present in each sample and a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants ka and kd, and the affinity constant KD ( kd / ka), could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of peptide antigens were performed to validate the reliability of single-concentration measurements. By combining data on affinity, activity and concentration, ranking of the antibody-containing supernatants was performed, allowing selection of high quality RabMAbs for binding of peptides in solution.

Citrullination-dependent Differential Presentation of a Self-peptide by HLA-B27 Subtypes

Inflammatory processes are accompanied by the posttranslational modification of certain arginine residues within proteins to yield citrulline, although it is largely unknown how this modification influences antigen presentation. We employed crystallographic and functional studies to investigate whether the exchange of arginine to citrulline affects the display of a peptide by two human major histocompatibility antigen class I subtypes, HLA-B(*)2705 and HLA-B(*)2709. Both differ only in residue 116 within the peptide binding groove despite their differential association with ankylosing spondylitis, an inflammatory rheumatic disorder. The crystal structures described here show that a modified self-peptide, pVIPR-U5 (RRKWURWHL; U = citrulline), is presented by the two HLA-B27 molecules in distinct conformations. These binding modes differ not only drastically from each other but also from the conformations exhibited by the non-citrullinated peptide in a given subtype. The differential reactivity of HLA-B27-restricted cytotoxic T cells with modified or unmodified pVIPR supports the structural findings and shows that the presentation of citrullinated peptides has the potential to influence immune responses.

Expression of Transforming Growth Factor-β1 and Hypoxia-Inducible Factor-1α in Renal Transplantation

Chronic allograft nephropathy (CAN) includes pathologic changes of interstitial fibrosis, tubular atrophy, and fibrous intimal thickening. Transforming growth factor (TGF)-β1 is a fibrogenic cytokine involved in renal allograft fibrosis. Hypoxia-inducible factor (HIF)-1α is induced as an adaptive response to hypoxia triggering the production of fibrogenic cytokines such as TGF-β1. Between January 1995 and February 2005, we performed 71 renal allograft biopsies in 61 recipients. Immunohistochemical studies were performed with an immunoperoxidase technique using as the primary antibody either a rabbit anti-human TGF-β1 polyclonal or a mouse anti-human HIF-1α monoclonal reagent. The glomerular TGF-β1 expression in recipients diagnosed with glomerulonephritis was significantly greater than other pathologic groups ( P < .05), and the glomerular TGF-β1 expression in the heavy proteinuria group (≥2.5 g/d) was significantly greater than the low proteinuria group (<1.0 g/d; P < .05). The tubular and interstitial TGF-β1 and HIF-1α expressions in CAN were greater than in other groups ( P < .05). The tubular TGF-β1 expression among the graft loss group was significantly greater than the graft function group ( P < .05).

A monoclonal antibody marker for the exclusion-zone filaments of Trypanosoma brucei

BackgroundTrypanosoma brucei is a haemoflagellate pathogen of man, wild animals and domesticated livestock in central and southern Africa. In all life cycle stages this parasite has a single mitochondrion that contains a uniquely organised genome that is condensed into a flat disk-like structure called the kinetoplast. The kinetoplast is essential for insect form procyclic cells and therefore is a potential drug target. The kinetoplast is unique in nature because it consists of novel structural proteins and thousands of circular, interlocking, DNA molecules (kDNA). Secondly, kDNA replication is critically timed to coincide with nuclear S phase and new flagellum biogenesis. Thirdly, the kinetoplast is physically attached to the flagellum basal bodies via a structure called the tripartite attachment complex (TAC). The TAC consists of unilateral filaments (within the mitochondrion matrix), differentiated mitochondrial membranes and exclusion-zone filaments that extend from the distal end of the basal bodies. To date only one protein, p166, has been identified to be a component of the TAC.ResultsIn the work presented here we provide data based on a novel EM technique developed to label and characterise cytoskeleton structures in permeabilised cells without extraction of mitochondrion membranes. We use this protocol to provide data on a new monoclonal antibody reagent (Mab 22) and illustrate the precise localisation of basal body-mitochondrial linker proteins. Mab 22 binds to these linker proteins (exclusion-zone filaments) and provides a new tool for the characterisation of cytoskeleton mediated kinetoplast segregation.ConclusionThe antigen(s) recognised by Mab 22 are cytoskeletal, insensitive to extraction by high concentrations of non-ionic detergent, extend from the proximal region of basal bodies and bind to the outer mitochondrial membrane. This protein(s) is the first component of the TAC exclusion-zone fibres to be identified. Mab 22 will therefore be important in characterising TAC biogenesis.

Increased epithelial expression of HLA-DQ and HLA-DP molecules in salivary glands from patients with Sjogren's syndrome compared with obstructive sialadenitis

Salivary gland specimens from 10 patients with primary Sjögren's syndrome (pSS) were examined by two-colour immunofluorescence with various combinations of monoclonal and polyclonal antibody reagents of the following specificities: human leucocyte antigen (HLA) class I and II (DR, DP and DQ), CD3, CD45 (leucocyte common antigen), various cytokeratins, and factor VIII-related antigen. Tissue specimens from 10 normal glands and 10 glands with obstructive sialadenitis (no known autoimmunity) served as controls. Only some intercalated ducts and scattered acini of the normal major glands expressed HLA class II determinants (< 5% of total epithelial area); the relative proportion of positive elements indicated differential expression (DR > DP > DQ). SS glands contained substantial T cell infiltrates and increased numbers of activated (DR+) T cells; adjacent epithelium showed extensive differential expression of HLA class II determinants (DR > DP > DQ). Glands with obstructive sialadenitis showed similarly increased epithelial expression of HLA-DR but with surprisingly small amounts of concomitant HLA-DP and -DQ expression. Epithelial HLA class II expression probably depends on cytokines as an inductive event, which is not unique for SS but particularly prominent in this disorder. Our results suggest that epithelial expression of HLA-DP or -DQ, rather than -DR, might be a prerequisite for the autoimmune process of SS to develop in genetically susceptible individuals.

Donation testing

Donation testing

Prospective Study of Human Metapneumovirus Detection in Clinical Samples by Use of Light Diagnostics Direct Immunofluorescence Reagent and Real-Time PCR

A Light Diagnostics human metapneumovirus (HMPV) monoclonal antibody reagent was evaluated using cytospin-enhanced direct immunofluorescence (DFA), and the results were compared to those for TaqMan reverse transcription-PCR (RT-PCR). Of the 202 samples tested, 48 were positive by RT-PCR, and 41 (85.4%) of these were positive by DFA (P = 0.0771). The commercial availability of an HMPV DFA reagent will be of significant benefit to clinical laboratories.

Comparison of Antigenic Regions Identified on IgG1Fc Using Bioinformatics vs Pepscan Analysis

Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identified. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents.As an extension to this work we have used these peptides to evaluate the location of epitope targets of five IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294-EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and five epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epitopes in the CH2 domain (256-TPEVTCVVVDVSHED-270) and CH3 domain (418-QQGNVFSCSVMHEAL-432). Similarly, RFAb PRTS1 detected all four epitopes plus a fifth in the CH3 domain (382-ESNGQPENNYKTTPP-396). In essence there was a consensus of target epitopes identified by these rheumatoid factor antibodies. Interestingly, two epitopes (256–270, CH2 domain and 360–374, CH3 domain) were novel in that they had not been identified in previous pepscan studies. The other epitopes recognised, either overlapped or were immediately adjacent to previous epitopes detected by poly/monoclonal rheumatoid factor antibodies.Molecular modelling (PCImdad) of IgG1Fc showed that all five epitopes were exposed and surface accessible for antibody interaction. In addition, a bioinformatics analysis of the Fc region using ExPASy was employed to identify key antigenic determinants. This ‘in silico’ approach may provide a means of determining key regions without the need to develop overlapping peptides spanning the entire length of a macromolecule.

Performance of the Panleucogating protocol for CD4+T cell enumeration in an HIV dedicated laboratory facility in Barbados

To compare the Panleucogating (PLG) protocol with the routinely used four-color protocol for CD4+ T cell count enumeration. One hundred fifty-three blood samples were randomly selected from samples received at the National HIV Laboratory for routine immunological monitoring. Samples were prepared using Coulter CYTO-STAT tetraCHROME monoclonal antibodies and FlowCARE PLG CD4 reagent for four-color and PLG, respectively, and analyzed on the Beckman Coulter EPICS XL flow cytometer. The PLG protocol used a sequential gating strategy where CD4+ T cells were identified using side scatter properties of cells and CD45 staining. The four-color protocol used CD45 and CD3 to identify CD4+ T cells. Absolute CD4+ T cell counts and percentages ranged from 4 to 1,285 cells/microL and 0.9 to 46.7%, respectively. Linear regression analyses revealed good correlation of PLG with the four-color protocol (absolute counts, R2 = 0.95; percentages, R2 = 0.98) over the entire range including the clinically relevant range. Bland Altman statistics revealed no bias for CD4 counts <500 cells/microL and a slight underestimation by PLG for counts >500 cells/microL (Bias = -32.7 cells/microL; 95% agreement limits = -151.3- +86.0). CD4+ T cell percentages were the similar over the entire range (Bias = 0.6%; 95% agreement limits = -1.97 +/- 3.18). PLG is an accurate method for enumerating CD4+ T cells and has resulted in major cost savings to the Government of Barbados. This has implications for the sustainability of the National HIV containment program in Barbados and the other resource limited Caribbean countries. The PLG technique is now being routinely used in Barbados.

教育講演２ 多発性硬化症の病態はどこまでわかったか？

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Fortunately, progress has been made for patients with this devastating disorder thanks to the induction of novel treatment strategies. In the 1990s, autoimmunity against myelin-related proteins was verified in humans, while the molecular mechanisms of the pathological process resulting in CNS demyelination were also studied in depth using experimental autoimmune encephalomyelitis (EAE). In the present decade, those achievements led to clinical trials of a variety of monoclonal antibody reagents for preventing disease relapse. Although such treatment seems to be ideal, as it targets a specific harmful immune reaction on the basis of findings from EAE studies, it has yet to become a first-line strategy, because of, in part, unexpected serious adverse reactions. As a result, interferon-beta therapy, the efficacy of which was first reported in 1993. has maintained a good position among treatment options for suppressing disease activity. Interferon-beta is considered to exert its anti-inflammatory effect via a Th2 shift in immune responses. In addition to aberrant cellular immunity, recent progress in MS research has shed light on the involvement of disturbances in humoral immunity, including the presence of NMO-IgG and anti-aquaporin-4 antibodies. Thus, it is important to elucidate the pathological significance of those autoantibodies, as well as establish treatment strategies for patients who are positive for them. However, since the above-mentioned treatments have been developed only for patients with relapsing-remitting MS. it is also important to consider the pathogenesis of primary progressive MS, which constitutes 10-15% of the patient population. Neurologists cannot be indifferent to current studies on MS, as even viral etiologies long ago abandoned have been recently revisited. In this field of neurology, every step of progress may readily lead to the establishment of a new treatment options.

Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques

RhD discrepancies between current and historical results are problematic to resolve. The investigation of 10 discrepancies is reported here. Samples identified were those that reacted by automated gel technology and were negative with an FDA-approved reagent. Reactivity with a commercially available panel of monoclonal anti-D was performed. Genomic DNA was evaluated for RHD alleles with multiplex RHD exon polymerase chain reaction (PCR), weak D PCR-restriction fragment length polymorphism, and RHD exon 5 and 7 sequence analyses. The monoclonal anti-D panel identified two samples as DVa, yet possessed the DAR allele. Two weak D Type 1 samples had a similar monoclonal anti-D profile, but only one reacted directly with one of two FDA-approved anti-D. Only two of four weak D Type 2 samples reacted directly with one FDA-approved anti-D, and their D epitope profile differed. The monoclonal anti-D reagents did not distinguish between partial and weak D Types 1 and 2. Weak D Types 1 and 2 do not show consistent reactivity with FDA-approved reagents and technology. To limit anti-D alloimmunization, it is recommended that samples yielding an immediate-spin tube test cutoff score of not more than 5 (i.e., < or =1+ agglutination) or a score of not more than 8 (i.e., < or =2+ hemagglutination) by gel technology be considered D- for transfusion and Rh immune globulin prophylaxis. That tube test anti-D reagents react poorly with some Weak D Types 1 and 2 red cells is problematic, inasmuch as they should be considered D+ for transfusion and prenatal care. Molecular tests that distinguish common partial and Weak D types provide the solution to resolving D antigen discrepancies.