Immunology Laboratory Research Articles

Morphological Aspects of Intestinal Failure in Experimental Peritonitis And Correction

Our studies show that pathomorphological changes in the small intestine in acute experimental peritonitis are characterized by dystrophic, inflammatory-destructive and vascular disorders in the vascular tissue structures of the small intestine wall. In the pathogenesis of damage to the small intestine, deep inflammatory and destructive changes in the vessels of the microhemocirculatory bed, which subsequently lead to disruption of cell trophism, tissue hypoxia, damage to cellular elements, disruption of cellular metabolism, deficiency of energy and plastic materials, accumulation of perverted metabolic products in cells and tissues, are of great importance. . The use of the “Zerotox“ enterosorbent in the treatment complex for the treatment of endogenous intoxication and toxemia had a positive effect on the morphofunctional status of the small intestine, on the clinical course of the disease, on the composition of the intestinal microflora, indicators of local immunological protection, improved clinical and laboratory signs of endogenous intoxication, reduced the recovery period bowel function, reduced the number of postoperative complications

Immunological routine laboratory parameters at admission influence the improvement of positive symptoms in schizophrenia patients after pharmacological treatment.

The standard care of schizophrenia patients is based on the assessment of their psychotic behavior, using interview-based, subjective scales that measure symptoms severity. We aimed at defining easily accessible and inexpensive blood-derived clinical diagnostic parameters that might serve as objective markers in the prediction of the effects of pharmacological treatment of schizophrenia patients. A total of 40 patients with schizophrenia diagnosis according to ICD 10 during psychotic decompensation were included in the study. Blood-based biochemical parameters, BMI and interview-based medical scales of symptom severity were determined - all at admission and after 12 weeks of standard pharmacological treatment. The drops in scale values were correlated with clinical parameters. All scale changes after treatment were dependent on the value of the given scale at admission, with higher initial values leading to larger drops of the values after treatment. Models based on those correlations were significantly improved when immune and metabolism parameters were included. C4 complement and C-reactive protein (CRP) level at admission were predictive of changes in Positive and Negative Syndrome Scale (PANSS) subscales related to significant disruption of thought processes, reality testing and disorganization. The pharmacological treatment-driven changes in scales representing negative symptoms were correlated with markers of the patients' thyroid status and metabolism. We show that objective markers can be obtained by testing immune and metabolic parameters from the patients' blood and may be added at a low cost to the standard care of schizophrenia patients in order to predict the outcome of pharmacological treatment.

General knowledge of frequently applied test procedures in rheumatological and immunological diagnostics and research

Knowledge of test procedures is essential for an optimal approach in rheumatological and immunological diagnostics as well as for acorrect interpretation of the findings. In practice, they are abasis for the independent provision of diagnostic laboratory services. For scientific questions they have become indispensable tools in many areas. This article gives an overview on the most frequently used and important test methods in acomprehensive form. The advantages and performance of the different methods are addressed and the limitations and possible sources or error are discussed. Quality control increasingly plays a decisive role in the diagnostic and scientific practice, with the legal regulations applying to all test procedures in laboratory diagnostics. For the discipline of rheumatology, the rheumatological and immunological diagnostics are of particular importance as the majority of the known disease-specific markers are detectable by means of these procedures. At the same time, immunological laboratory diagnostics are a highly interesting field of activity which are expected to have a strong impact on the future developments in rheumatology.

Effect of ferritin, INR, and D-dimer immunological parameters levels as predictors of COVID-19 mortality: A strong prediction with the decision trees

Background and objectiveA hyperinflammatory environment is thought to be the distinctive characteristic of COVID-19 infection and an important mediator of morbidity. This study aimed to determine the effect of other immunological parameter levels, especially ferritin, as a predictor of COVID-19 mortality via decision-trees analysis. Material and methodThis is a retrospective study evaluating a total of 2568 patients who died (n = 232) and recovered (n = 2336) from COVID-19 in August and December 2021. Immunological laboratory data were compared between two groups that died and recovered from patients with COVID-19. In addition, decision trees from machine learning models were used to evaluate the performance of immunological parameters in the mortality of the COVID-19 disease. ResultsNon-surviving from COVID-19 had 1.75 times higher ferritin, 10.7 times higher CRP, 2.4 times higher D-dimer, 1.14 times higher international-normalized-ratio (INR), 1.1 times higher Fibrinogen, 22.9 times higher procalcitonin, 3.35 times higher troponin, 2.77 mm/h times higher erythrocyte-sedimentation-rate (ESR), 1.13sec times longer prothrombin time (PT) when compared surviving patients. In addition, our interpretable decision tree, which was constructed with only the cut-off values of ferritin, INR, and D-dimer, correctly predicted 99.7% of surviving patients and 92.7% of non-surviving patients. ConclusionsThis study perfectly predicted the mortality of COVID-19 with our interpretable decision tree constructed with INR and D-dimer, especially ferritin. For this reason, we think that it may be important to include ferritin, INR, and D-dimer parameters and their cut-off values in the scoring systems to be planned for COVID-19 mortality.

Essential Oils of Rosmarinus officinalis and Eucalyptus globulus Cultivated in the Mountainous Region of the State of Rio de Janeiro (Brazil): Chemical Profile and Antileishmanial Activity

Pharmacognosy Research,2023,15,2,297-306.DOI:10.5530/pres.15.2.032Published:February 2023Type:Original Article Authors:Rogéria Cristina Zauli, Tarcísio Martins Santos, Alberto Wisniewski Jr, Patrícia Xander, and Aislan Cristina Rheder Fagundes Pascoal Author(s) affiliations:Rogéria Cristina Zauli1, 2, 3, Tarcísio Martins Santos4, Alberto Wisniewski Jr4, Patrícia Xander1, 3, Aislan Cristina Rheder Fagundes Pascoal5,* 1Department of Pharmaceutical Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Laboratory of Cellular Immunology and Biochemistry of Fungi and Protozoa, Federal University of São Paulo, Diadema, São Paulo, BRAZIL. 2Technical Support Center for Teaching, Research and Extension (NATEPE), Federal University of São Paulo, Campus Diadema, Diadema, São Paulo, BRAZIL. 3Biology-Chemistry Graduate Program, Federal University of São Paulo, Campus Diadema, Diadema, São Paulo, BRAZIL. 4Department of Chemistry, Federal University of Sergipe, Av. Marechal Rondon, São Cristóvão, SE, BRAZIL 5Department of Basic Sciences, Multi-user Biomedical Research Laboratory, Fluminense Federal University (ISNF-UFF), Nova Friburgo, Rio de Janeiro, BRAZIL. Abstract:The present study aimed to characterize the chemical profile of essential oils of Rosmarinus officinalis (EORo) and Eucalyptus globulus (EOEg) cultivated in the mountainous region of the state of Rio de Janeiro, Brazil, and to verify the leishmanicidal potential, as well as to determine the cytotoxicity in vitro in mammalian cells. EORo had α-Pinene (37.99%), Cineol (21.48%), D-Verbenone (5.78%) and Limonene (3.28%) as major constituents. The EOEg had as major components Cineol (75.52%), Limonene (8.82%) and o-Cymeno (7.81%). The EOEg had a lower IC50 value of 14.03 ± 2.08 μg/mL, while EORo had an IC50 value of 31.12 ± 4.6 μg/mL, both during the 24-hr exposure period. In the 48-hr period, the IC50 value was almost twice as high with rosemary oil, while in eucalyptus oil there was no significant difference when exposed in this period. The selectivity of EOs was demonstrated in murine cells, showing almost 30 times less toxic to the cell than to the parasite. It can be concluded that this is a pioneering study on the potential of these essential oils against the leishmania parasite. Furthermore, the low toxicity effect on the host cell encourages future studies and in vivo applications. Keywords:cytotoxicity, E. globulus, Essential oil, Leishmanicidal, R. officinalisView:PDF (359.76 KB)

Characteristics of immunological events in Epstein-Barr virus infection in children with infectious mononucleosis.

Epstein-Barr virus (EBV) infection occurs commonly in children and may cause acute infectious mononucleosis (AIM) and various malignant diseases. Host immune responses are key players in the resistance to EBV infection. We here assessed the immunological events and laboratory indicators of EBV infection, as well as determined the clinical usefulness of evaluating the severity and efficacy of antiviral therapy in AIM patients. We enrolled 88 children with EBV infection. The immune environment was defined by immunological events such as frequencies of lymphocyte subsets, phenotypes of T cells, and their ability to secrete cytokines, and so on. This environment was analyzed in EBV-infected children with different viral loads and in children in different phases of infectious mononucleosis (IM) from disease onset to convalescence. Children with AIM had higher frequencies of CD3+ T and CD8+ T cells, but lower frequencies of CD4+ T cells and CD19+ B cells. In these children, the expression of CD62L was lower and that of CTLA-4 and PD-1 was higher on T cells. EBV exposure induced granzyme B expression, but reduced IFN-γ secretion, by CD8+ T cells, whereas NK cells exhibited reduced granzyme B expression and increased IFN-γ secretion. The frequency of CD8+ T cells was positively correlated with the EBV DNA load, whereas the frequencies of CD4+ T cells and B cells were negatively correlated. During the convalescent phase of IM, CD8+ T cell frequency and CD62L expression on T cells were restored. Moreover, patient serum levels of IL-4, IL-6, IL-10, and IFN-γ were considerably lower throughout the convalescent phase than throughout the acute phase. Robust expansion of CD8+ T cells, accompanied by CD62L downregulation, PD-1 and CTLA-4 upregulation on T cells, enhanced granzyme B production, and impaired IFN-γ secretion, is a typical characteristic of immunological events in children with AIM. Noncytolytic and cytolytic effector functions of CD8+ T cells are regulated in an oscillatory manner. Furthermore, the AST level, number of CD8+ T cells, and CD62L expression on T cells may act as markers related to IM severity and the effectiveness of antiviral treatment.

Interrelations between viral load and cellular immunity in patients with COVID-19 of varying severity

Assessment of viral load levels in various biological samples taken from the respiratory tract can be an indicator of an ongoing process of active viral replication and may be used to monitor severe respiratory viral infections. The study of the relationship between SARS-CoV-2 viral load and immunological laboratory parameters is an important step in the search for clinical markers of COVID-19.The aim of this research was to quantify viral load in patients with COVID-19 and to identify the relation-ship between viral load and changes in the parameters of the cellular component of the immune system.A laboratory examination was carried out on 74 patients diagnosed with COVID-19, they were divided into 3 groups based on the severity of the disease: mild, moderate, severe. Total viral load in clinical samples was determined by the number of SARS-CoV-2 RNA copies per 100 copies of the reference RNaseP gene. A comprehensive assessment of the cellular component of the immune system was performed using flow cytometry and direct monoclonal antibodies, and the IL-6, and C-reactive protein concentrations were determined.We revealed a relationship between the development of serious clinical conditions in the patients with COVID-19, and the levels of viral load. High levels of viral RNA in biological samples correlate with main indicators of the T cell component of the immune system associated with disease severity. In a subgroup of patients with an extremely high viral load, strong positive correlations were found between the relative numbers of cytotoxic lymphocytes (CD3+CD8+), activated T lymphocytes (CD3+HLA-DR+), as well as absolute and relative numbers of activated B lymphocytes and NK cells (CD3-CD25+).Laboratory monitoring of the cellular component of the immune system, along with the assessment of viral loads, should improve early assessment of clinical condition in the patients with COVID-19. Changes in expression levels of activation markers on immune cells can be potentially viewed as indicators of recovery during COVID-19.

Aide à l’interprétation des phénotypages lymphocytaires en cas de lymphopénie NK isolée

AbstractThe isolated decrease in “Natural Killer” (NK) lymphocytes is a rare situation that often goes unnoticed on the blood count, and is detected by immunophenotyping. The NK cell lymphopenia, isolated or not, is found in several pathological contexts, particularly in infection, neoplasia and during auto-immune diseases. The potential etiologies of isolated NK cell lymphopenia from the retrospective analysis of 2 824 immunophenotyping carried out in the Immunology laboratory at the CHU of Montpellier in 2019 will be presented here. Patients with autoimmune diseases were at the head of our series, 70 % of which were in flare of the disease, followed by patients with hemopathies and chronic inflammatory diseases. Immunosuppressive therapies, in particular corticosteroid therapy, may also induce NK cell lymphopenia.

P765 Tolerating infliximab therapeutic drug monitoring without anti-drug antibodies – an argument for drug intolerant assays?

Abstract Background Therapeutic drug monitoring (TDM) helps guide clinical decision making in IBD patients on infliximab (IFX). There are two types of IFX TDM assays, drug tolerant and intolerant, where drug tolerant assays allow the detection of anti-drug antibodies (ADA) in the presence of drug and the latter does not. Our immunology laboratory is in the process of changing from a drug tolerant to a drug intolerant IFX assay for cost reasons. We aimed to determine whether this switch would affect the management of IBD patients with therapeutic IFX levels and detectable antibodies. Methods This was a retrospective cross-sectional study in a central London tertiary hospital. Patients were included if they had a diagnosis of Crohn’s Disease (CD), Ulcerative Colitis (UC), or Indeterminate colitis, and had therapeutic IFX levels (>3mg/ml) with detectable anti-drug antibodies (ADA) (>10 AU/ml) between January 2017 to March 2022. Electronic health records were reviewed to determine clinical actions within 4 months of drug measurement. Actions were categorized into three groups by two gastroenterology physicians: no change to management, ADA dependent or independent actions. ADA dependent decisions were immunomodulator (IMM) addition or discontinuation. ADA independent decisions included switching biologic, stopping IFX, or escalation/de-escalation in IFX dosing. Results A total of 2675 IFX TDM tests were performed for IBD patients in the defined period. Of these, 237 patients had 477 TDM results with therapeutic IFX levels and detectable ADA. Baseline characteristics were 36.8% (n=87) female, mean age (y) of 30 (SD ±13), and 66.2% (n=157) had CD. At the time of measurement, 76.1% (n=363/477) of patients were on combination therapy with an IMM and 23.1% (n=110) had active disease on physicians’ global assessment. Changes to management were made in 23.5% (112/477) of clinics following TDM, but only 2.5% (12/477) of actions were ADA dependent. There was no difference in high titer ADA group (>40 AU/ml) compared to low. Variables that significantly affected clinical decisions were IFX dose (p<0.001), IFX interval (p=0.004), and clinical disease activity (p<0.001). Conclusion 1 in 40 clinical decisions made in patients with therapeutic drug levels and detectable ADAs rely on knowing the ADA level. A cost analysis, with an appropriate IFX drug level threshold under which ADAs would be performed, assessing the proposed switch to a drug intolerant TDM assay would help determine whether cost savings outweigh missed decision-making opportunities.

P799 Infliximab and immunomodulator combination therapy in inflammatory bowel disease – no longer inseparable?

Abstract Background The gold-standard for prescribing Infliximab (IFX) in IBD is to use it in combination with an immunomodulator (IMM), thereby reducing the risk of immunogenicity and improving overall outcomes. This practice, however, comes with considerable increased risk of adverse events. There is mounting evidence that withdrawal of IMMs, especially after 6 months of combination therapy (CT), does not reduce IFX trough levels or increase the risk of loss of response (Drobne et al., 2015; Mahmoud et al., 2022). We identified IBD patients on IFX CT who were eligible to stop their IMM based on defined criteria and assessed clinical outcomes of patients who had previously had their IMM withdrawn. Methods This was a retrospective cross-sectional study in a central London tertiary hospital. We included patients with a diagnosis of Crohn’s Disease (CD), Ulcerative Colitis, or Indeterminate colitis, with therapeutic IFX levels >5mg/ml, undetectable anti-drug antibodies (ADA) (<10 AU/ml) and at least 6 months of CT from induction between January 2017 and December 2021. Patients were identified on an immunology laboratory database and electronic patient records were manually assessed for baseline characteristics and clinical outcomes. The primary timepoint of interest for clinical outcomes is 4 months after measurement of IFX drug levels. Results A total of 825 IBD patients had IFX drug levels performed in the defined time-period. 296 patients had IFX levels >5mg/ml and undetectable ADAs; of these 160 (54%) had been on CT for >6 months. Baseline characteristics were 41.3% (n=66) female, mean age (y) of 29 (SD ±14) and 70.6% (n=113) had CD. In the 4 months after IFX drug assays, 140 of 160 (87.5%) patients remained on CT whereas 20 (12.5%) had previously stopped their IMM. Of those on CT, 90.7% (127/140) were in clinical remission at the time of the assay compared to 90% (18/20) of patients in the IMM withdrawal group (p=0.46). At the time of data collection, 79.3% (111/140) of CT patients remained on IFX therapy compared to 75% (15/20) who withdrew IMMs (p=0.33, median follow up 19 months). IMM withdrawal was predominantly due to side effects (n=16, 80%) with the remainder due to clinical remission (n=4, 20%). The 2/20 (10%) patients with active disease in the IMM withdrawal group were active prior to stopping IMMs. Conclusion Patients with good IFX drug levels and undetectable ADAs after at least 6 months of IFX CT did well regardless of whether they withdrew IMMs or not, supporting recent studies that suggest it is safe to do so. Given the number of our patients who could potentially benefit from stopping their IMMs, prospective controlled studies are required to determine whether this is advisable given the theoretical risk of loss of response.

A multiomic approach to autoimmunity in the immunology laboratory

In the diagnostic immunopathology laboratory, technology has permitted the detection of autoantibodies with increasing sensitivities and turn-around times. Over the last decade or so, we have pioneered a unique, research approach to understanding autoantibodies and autoantibody-associated autoimmunity by looking simultaneously at both the proteomic (autoantibody) and transcriptomic (B cell receptor) compartments of patient-derived samples. Our approach stems from the ‘bedside’, allowing our findings to have immediate clinical relevance and advances our understanding of the loss of B cell tolerance and the origins of potentially pathogenic autoantibodies.

The contribution of antinuclear antibodies in Primary Biliary Cholangitis (PBC): An experience from the immunology laboratory at University Hospital Center Hassan II, Fes, Morocco.

Patients older than 18 with chronic hepatitis or cirrhosis were recruited from September 2021 to January 2023. Clinical data were collected, including age, sex, symptoms, and biochemical parameters (ALT), (AST), (ALP), (GGT), (TB), (PT) and (PN), autoantibodies associated with hepatic autoimmune disorders: (AMA), anti-gp 210, anti-Sp100 and anti-F-actin antibodies were identified using HEp-2 cells as substrate (Fig. 1); and/or multiple Immunodot liver tests. HBsAg, anti-HCV, and HIV test results of the patients were found to be negative. 19 out of 71 had a confirmed diagnosis of PBC; 5 patients with AIH-PBC overlap (26.3%), and 14 patients with PBC (73.7%). Patients mainly were females with a 0.12-sex ratio. ANA patterns were dominated by cytoplasmic filamentous staining (AC-21) with punctuate nuclear envelope staining (AC-12). Multiple Immunodot tests were performed on all patients: AMA was present in 89.4%, anti-gp 210, and anti-Sp100 antibodies in 57.8% and 21.1%, respectively (Table 1).Patients were divided into groups depending on the positivity of AMA, anti-gp 210, and anti-Sp100 antibodies. 10.5% of PBC patients (2) had negatives AMA and positive anti-gp210. Almost half of PBC patients (11) had associated positive AMA and positive anti-gp210 and/or anti-Sp100, while 26.3% of them (5) had only positive AMA (Table 2). The findings in this work support the role of anti-gp 210 and anti-Sp100 in identifying patients with PBC and overlap syndromes. Moreover, Anti-gp 210 antibodies are found to be helpful for the diagnosis of PBC patients who are negative for AMAs.

PREVALENCE OF LATENT TUBERCULOSIS INFECTION AMONG MEDICAL WORKERS

The analysis of the results of immunological tests (skin test with tuberculosis recombinant allergen in standard dilution, hereinafter referred to as the test with ATP) and laboratory immunological test IGRA, delivered in 2019-2022 to employees of medical organizations of the Moscow Department of Health, working in four administrative districts of Moscow (Central, Western, South-Western, Troitsk and Novomoskovsk, next – CAD, WAD, SWAD/T&NAD). Among the average annual number of employees over 5 years – 43,452 people, with medical examination and exclusion of tuberculosis, the prevalence of persons with latent tuberculosis infection (LTI) was 2.6, 6.1 and 1.8%, in the CAD, WAD and SWAD/T&NAD, respectively, or 3.4% in general to these districts. At the same time, the average annual frequency of detection of cases of LTI («first detected LTI») was 0.6, 1.3 and 0.6% in these districts, respectively, and 0.75% in all districts as a whole. 95.4% of the employees were assigned a course of preventive therapy (PT), 45.8% of which the course was started. Among the latter, 85.0% successfully completed the course, 14.1% – the course was interrupted by the patient, and 0.9% – canceled by the doctor due to adverse events. The main PT regimen used (78.4%) included 3 months of taking a combination of isoniazid and pyrazinamide (INN: isoniazid 150 mg + pyrazinamide 500 mg), which showed a low proportion of adverse events – 3.1%. The identification of workers of medical organizations of LTI allows to determine a group high risk for tuberculosis and to allocate a group of persons for preventive therapy

Immunological and translational key challenges in systemic lupus erythematosus: A symposium update

The first LBMR-Tim (Toulouse Referral Medical Laboratory of Immunology) symposium convened on December 16, 2022 in Toulouse, France to address challenging questions in systemic lupus erythematosus (SLE). Special focus was put on (i) the role played by genes, sex, TLR7, and platelets on SLE pathophysiology; (ii) autoantibodies, urinary proteins, and thrombocytopenia contribution at the time of diagnosis and during follow-up; (iii) neuropsychiatric involvement, vaccine response in the COVID-19 era, and lupus nephritis management at the clinical frontline; and (iv) therapeutic perspectives in patients with lupus nephritis and the unexpected adventure of the Lupuzor/P140 peptide. The multidisciplinary panel of experts further supports the concept that a global approach including basic sciences, translational research, clinical expertise, and therapeutic development have to be prioritized in order to better understand and then improve the management of this complex syndrome.

Influence of blood sample storage and different types of anticoagulants on results of NK cytotoxicity tests.

Natural killer (NK) cells are important players in the human immune response. Impaired NK function may lead to serious, life-threatening conditions. Defects may be consequences of genetic mutations or results of secondary factors such as infections, malignancies and autoimmune diseases. The cytotoxicity test is very useful, but its accessibility is limited to special immunological laboratories. Blood samples are often transported to remote centers, which takes time and requires special conditions.The aim of this study was to compare cytotoxicity assay results between samples preserved with three different anticoagulants to standardize the diagnostic procedure. Peripheral blood from healthy donors was taken with three anticoagulants: heparin, K2EDTA and citrate. Peripheral blood mononuclear cells (PBMC) were isolated and tested directly after blood drawing and after 24-hour storage. Cytotoxic abilities of NK cells were tested in 4 h co-culture with K562. NK cytotoxicity was measured by flow cytometry. In most cases of analyzed healthy donors, cytotoxicity results were similar regardless of type of anticoagulant. However, the highest mean values were obtained in samples with citrate. There was a significant decrease in cytotoxicity after 24 hours of storage of the whole blood at ambient temperature. The mean drop in cytotoxicity results was substantial for all anticoagulants: 76% for heparin, 67% for citrate and 70% for EDTA. Results of spontaneous NK cytotoxicity seem to be affected by the anticoagulants used for blood protection. Commercial instant cytotoxicity testing and delayed analysis after blood storage gave the highest results in blood with sodium citrate.

Liver kidney microsome antibodies. Analysis of a laboratory series

ObjectivesThe objectives were to characterize the liver kidney microsome (LKM) antibody profile of a 14-month-old girl with autoimmune hepatitis and analyze the laboratory prevalence of LKM positivity. Design and methodsThis is retrospective analysis of the LKM antibody immunofluorescence tests performed by the Immunology Laboratory of Johns Hopkins Hospital from September 8, 2020 to July 31, 2022. LKM positive sera were also tested by an ELISA for LKM1 antibodies, which recognize the cytochrome P450 2D6 antigen. In silico analysis of 2D6 mRNA expression across anatomical sites was performed using Bgee and GTEx Portal databases. ResultsOf the total of 1598 patients (893 F, 705 M, ages 0.8–94 years) tested for LKM antibodies, 3 were positive, yielding a 0.2% period prevalence. The clinical diagnosis was autoimmune hepatitis in the index case, acute viral hepatitis in a 3-yo male, and hepatocellular carcinoma in a 54-yo male. LKM antibodies yielded the classical homogenous staining pattern in the liver cytosol and proximal kidney tubular cells. The first two patients were also positive for LKM1 antibodies, whereas the third was negative. 2D6 mRNA was expressed highly in the liver, moderately in the duodenum, and minimally in other tissues. ConclusionsOverall, LKM antibodies are rare. They contribute to establish a diagnosis of autoimmune hepatitis, although they are also found in other liver diseases. The cytochrome P450 2D6 is one of the antigens recognized by LKM antibodies, but other antigens are likely targeted considering that 2D6 is minimally expressed in the kidney and yet LKM antibodies bind to kidney tubuli.

An understanding of the evaluation of histocompatibility testing for a deceased-donor transplant

Dear Editor, Several obstacles have been overcome in the recent decades to make organ transplantation an effective lifesaving treatment for many patients. However, there is a tremendous disparity between the demand for organs and the number of organs necessary around the world, which is regarded as one of the major barriers, and India is not an exception.[1] Since 2013, the rate of deceased-donor organ donation increased from 0.27 per million people (pmp) to 0.65 pmp in 2018. Whereas the rate of deceased-donor renal transplant increased from 0.43 pmp to 0.86 pmp during the same period; this is roughly a 2.4-fold increase in just 5 years.,[2] Still, a lot of support and awareness for deceased-donor organ transplant programs is needed to help patients who do not have living kidney donors. There are numerous obstacles to overcome, with persuading family members being one of the most significant. Furthermore, transportation and allocation of the cadaver are also other issues associated with deceased-donor transplants. Once a cadaver is available, a set of histocompatibility testing of the potential patients is performed to determine the transplant’s suitability. Hence, a transplant immunology laboratory’s ability to accurately report the compatibility tests (to evaluate preformed human leukocyte antigen [HLA] donor-specific antibodies) on time is critical to the success of a deceased-donor transplant program. We want to emphasize the importance of histocompatibility testing evaluation in deceased-donor transplant programs. To support the deceased-donor transplant program, the laboratory must set up an efficient operational system to determine what kind of tests will be needed before, during, and after the transplant. In addition, a functioning 24-h on-call system should be in place. So far, a standalone transplant focused laboratory in North India, accredited for ISO15189: 2012, has aided pretransplant testing workup for 62 cadaveric cases (between February 2015 and December 2022) by optimizing the appropriate and effective test for anti-HLA antibodies between complement-dependent cytotoxicity (CDC) crossmatch, flow cytometric crossmatch (FCXM), and Luminex crossmatch.Both the CDC and the FCXM are time-consuming and labor-intensive, with the potential of false results, especially in the case of deceased donations where there is no time to redo the tests. Even technological advancements have contributed to a better understanding of transplant immunology in the recent years. In the identification of HLA antibodies, the advent of Luminex cross-matching technology provides more sensitivity than CDC and FCXM. Furthermore, unlike Luminex crossmatch, CDC and FCXM are manually created assays that require manual interpretation and whose processing method varies between laboratories. Furthermore, they (CDC and FCXM) both require a high number of viable lymphocytes to complete the experiment, whereas Luminex crossmatch requires a cell lysate to process. It is difficult to isolate a high number of viable lymphocytes from a cadaver; hence, cell count is a major concern in the cadaver transplant workup. The outcome may be harmed as a result of sample quality difficulties. Due to the aforementioned constraints, these approaches are less reliable during cadaver workup, and they also take a longer time than Luminex crossmatch. Even lysate can be saved for posttransplant monitoring.[3,4] Mishra et al. also support the findings of the use of Luminex crossmatch for pretransplant workup of renal transplants.[4] Later on, data published by Mathur et al. on 342 patients supported the usefulness of Luminex lysate crossmatch in deceased donor pretransplant workup.[3] Further, some more published literature[5,6] supports these findings; hence, Luminex crossmatch (lysate) should be used as the primary compatibility investigation for deceased-donor transplants. At present, no specific pretransplant compatibility workup for deceased-donor transplants is available. In routine practice, broadly, the pretransplant compatibility workup falls into two broad categories; cell-based tests (CDC and FCXM) and HLA bead-based (solid phase) tests (such as anti-HLA antibody screening and ant-HLA antibody identification). Laboratories typically utilize testing algorithms for compatibility, which are combinations of two or more tests, since no single test, whether cell-based or HLA bead-based, is 100% accurate and sensitive. To make donor lysate Luminex crossmatch a consensus technique, transplant physicians can be taught about the benefits. Recently, some pretransplant compatibility testing algorithm in the Indian setup is published,[7,8] which necessitates the development of one for better organ transplant outcomes, particularly in cadaver instances where there are numerous hurdles (both clinical and nonclinical). For deceased donor workup, expert analysis in a quicker turnaround time is critical, and hence, the laboratory should be equipped with cutting-edge advanced histocompatibility equipment to aid in the evaluation of histocompatibility testing for deceased-donor transplants. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Current issues of standardization of antinuclear antibodies’ detection by indirect immunofluorescence on HEp-2 cells

Background. Antinuclear antibodies (ANA) are a group of antibodies that target nuclear and cytoplasmic antigens. Testing for ANA using an immunofluorescence assay (IFA) on HЕp-2 cell (IFA-HEp-2) is a screening test for the diagnosis of systemic autoimmune rheumatic diseases, druginduced lupus erythematosus, autoimmune liver diseases, juvenile idiopathic arthritis. In routine ANA testing final report forms bears important, but unstandardized variables (e. g., screening and final titers of ANA, coding of glow types, the procedure for performing confirmatory tests). Aim of the study. Develop a standard report form for IFA-HEp-2 testing results.Material and methods. Survey of 10 immunological and clinical diagnostic laboratories using the ICAP questionnaire adapted by the Working Group on Standardization of the definition of ANA by the IFA-HEp-2 method (Committee on Immunology of the Association ‘Federation of Laboratory Medicine’).Results. According to the results of a survey: most of the participants use a screening dilution of serum 1:160; indicates the maximum end titer of antinuclear factor (ANF), cytoplasmic staining; identifies staining types (including AC encoding); considers it mandatory to make confirmatory tests to detect ANA to individual nuclear antigens in ANF-positive patients, but does not provide a list of this tests in the results form; describes ANF titer for each of the detected glows separately; designates ANF titers using a colon and does not give a clinical interpretation of the results. Based on interlaboratory consensus, ICAP recommendations and the requirements of ISO 15189, the standard report form for IFA-HEp-2 testing results was developed.Conclusions. To achieve high quality of interaction between clinical diagnostic/immunological laboratories and clinical departments of health care facilities, it’s necessary to introduce into practice a standard form for IFA-HEp-2 testing results, developed relying on modern clinical recommendations and interlaboratory consensus.

Comparison of two anti-histones antibodies commercial assays in an immunology laboratory and systemic lupus erythematosus

BackgroundAnti-histones antibodies (AHAs) are antibodies directed against histone proteins – structural proteins that provides scaffolding for DNA to be wrapped around. AHAs, measured in the serum, are diagnostically helpful in cases of systemic lupus erythematosus (SLE) and drug-induced lupus erythematosus (DILE). In the diagnostic laboratory, they may be measured by enzyme-linked immune-sorbent assay (ELISA) or line immunoassays (LIA); however, the performance of these have never been directly compared in the literature. MethodsWe evaluated a commonly used commercial ELISA and LIA to compare the performance in our immunology laboratory. Retrospective and prospective analyses were undertaken over a 5.5-year period. We also examined their performance in the model disease of SLE and compared their performance to disease activity and the main clinical features. ResultsOne hundred and thirty-five patients were evaluated including 65 SLE patients. Based on the quantitative cut-offs, there was only moderate agreement between the two assays (κ = 0.444). Both assays only had modest agreement with the clinical situation of the evaluated patients. When considered within the SLE context, both assays were moderately correlated with disease activity. Positive AHAs by ELISA were associated with SLE cytopaenias, and both ELISA and LIA correlated with positive anti-double stranded DNA. ConclusionsModerate agreement analytically were seen between ELISA and LIA methods in a general laboratory cohort. Both assays were comparable in their diagnostic performance. ELISA detection of AHAs appears to have some clinical use in our cohort of SLE patients. Future studies are required to explore the clinical utility of AHAs in SLE and other disorders.

Association Between Vitamin D and Minor Salivary Gland Inflammation.

Objective There has been increasing research regarding the effects of vitamin D on autoimmune disorders. There is some evidence of a correlation between vitamin D levels and sicca symptoms. We attempted to evaluate the correlation between vitamin D levels and inflammation of the minor salivary gland (MSG). Methods Data for 214 patients who had undergone MSG biopsy were reviewed. Eighteen patients with other autoimmune/neoplastic processes were excluded. Seventy-seven of 196 patients had serum Vitamin D levels available and were selected for this retrospective study. Demographic, clinical, and immunological features, extra-glandular manifestations, autoantibodies, and clinical laboratory tests were collected and compared between patient groups with a focus score (FS) of 0 and 1 or more. Results Out of 77, 29 patients had an FS of 0 while 48 had an FS of 1 or more. Mean vitamin D levels were noted to be lower in patients with an FS of 1 when compared to patients with an FS of 1 or more. Conclusion In this study, patients with an FS of 1 were noted to have low vitamin D levels but this association was not seen at a higher FS.