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Background & Aims: Sepsis is the principal cause of death in the first months of life and leads to 30- 50% of infant mortality in developing countries (1). The prevalence of sepsis is reported differently in many countries. In developed countries, the prevalence of sepsis is 1-4 per 1,000 live births, while in poor and developing countries it is almost ten times higher (2). Neonatal sepsis leads to 25.2% of all neonatal deaths in Iran (3). The clinical signs of neonatal sepsis are nonspecific and may be mixed with noninfectious disorders such as aspiration syndrome and respiratory distress syndrome (4,5). The most important factor in reducing infant mortality from sepsis is appropriate and quick treatment (6). On the other hand, the clinical signs of affected infants, including decreased neonatal reflexes, bradycardia, hypo/hyperthermia, respiratory distress, seizures, weakness and lethargy, apnea, and abdominal distension, are not sensitive enough to definitively diagnose sepsis. Blood tests and immunological tests also have less sensitive and specific compared to blood culture (7). Since sepsis is one of the most important causes of mortality and disability in the neonatal stage, with early diagnosis and treatment of the disease and improving the quality of care during delivery, neonatal survival can be increased, the clinical signs, predisposing factors, laboratory findings, treatment and outcome of neonates with the diagnosis of sepsis who admitted to Afzalipour hospital in Kerman were evaluated. Methods: This was a descriptive and cross-sectional study. The statistical population was all infants admitted to Afzalipour Hospital in Kerman from 2015 to 2017.The sample size was determined with the census method (sample size = 200 infants). Infants with Complete Blood Count (CBC), arterial gas measurement, C Reactive Protein (CRP), blood and urine cultures, and Cerebrospinal fluid (CSF) were included in the present study. All neonates with a diagnosis other than sepsis were excluded from the study. Based on clinical and laboratory findings, neonates were classified into three groups: 1- Infants who had positive blood culture sepsis in addition to clinical and laboratory findings. 2- Neonates who had only laboratory and clinical findings of sepsis but their blood culture was negative. 3- Infants who had clinical signs of sepsis but were negative for laboratory findings and blood culture. All infants were treated with antibiotics immediately after the tests. Clinical and laboratory findings as well as the type of antibiotic and maternal and neonatal risk factors were collected using a checklist. Outcomes of treatment were expressed as improvement and discharge, complications, and death. The collected data were analyzed using SPSS software version 25 (version 25, SPSS Inc., Chicago, IL). Quantitative data were presented as Mean ± SD (standard deviation) and frequency and percentage. Results: The highest age of infants was related to the birth group (91%). 59.5% of infants were male and 40.5% were female. The mean duration of admission was 9.76. 1.05 days. The most common clinical signs were tachypnea (74%), poor nutrition (36%), apnea (32%), and hypo/hyperthermia (25%) respectively. The results showed that 91.5% of participants used ampicillin, 93% of them gentamicin, 84% of them used vancomycin, 52% of them used meropenem, 18.5% of them used metronidazole, 19.5% of them used amikacin, 18 % of them took cefotaxime, 19% of them used fluconazole, 1% of them used clindamycin and 1.5% of them used ceftazidime Regarding the frequency distribution of risk factors, the results showed that 23.5% of participants had PROM (Premature rupture of Membrane), 11 % had maternal urinary tract infection, 12 % had maternal fever and 91.5% had low birth weight. The results showed that the mortality rate for participants was 78% and the rate of discharge and recovery was 22%. The results showed that the mean number of white blood cells was 16.468 ± 0.103 × و 103 and the mean CRP of the participants was 22.6 ± 2.8. The results of this study also showed that 14.5% of participants had positive blood cultures and 85.5% of them had negative blood cultures. Conclusion: Systemic and local infections are very common in infants. Sepsis is a systemic response to infection and is divided into early, late and nosocomial types (17). The results showed that 59.5% of the participants who had early sepsis were male and 40.5% of them were female, which is consistent with the results of Khalili et al. (13). While in the textbooks, the incidence of male infants is twice as high as female infants (18). In the study of Arab Mohammadi et al., 60% of patients were boys and 40% were girls. In this study, the most common clinical symptoms were tachypnea (74%), hypo/hyperthermia (25%), and tachycardia (5%), respectively, which is consistent with the results of Hengst et al. (10) while it is not similar to the results of Khalili et al. (13), Santana et al. (9) and Vergnano et al. (14). In other textbooks, the most common clinical symptoms are fever, jaundice, respiratory distress, hepatomegaly, anorexia, vomiting, and cyanosis (19). In the study of Shiva et al. (20), a total of three symptoms of tachycardia, tachypnea, hypothermia had a sensitivity of 53.6% and a specificity of 100% in identifying sepsis with positive blood culture. In the study of Arab Mohammadi et al. (21), hyperthermia, jaundice, respiratory distress, hepatomegaly, anorexia, vomiting, lethargy, and cyanosis were reported as the most common symptoms of sepsis. Investigation of 500 infants suspected of sepsis, temperature instability, and respiratory symptoms were the most common symptoms. The results showed that 14.5% of the participants had positive blood cultures and 85.5% of them had negative blood cultures. This rate (positive blood culture) was 15.4% for the study of Khan et al (22). This rate was 14% in the study of Manucha et al. (23) in India. The reason for the lack of positive blood culture is neonatal SEPSIS is one of the diseases in which the actual number of cases is much less than the number of suspected cases and laboratory-related problems also play an important role in this results (24). The prevalence of sepsis with positive blood culture has been reported in the study of Shiva et al. 25% (20), Samaei et al. 41% (25), and Hosseini et al. The results showed that the mean CRP of the participants in our study was 22.6. 2.8. According to textbooks, if CRP is performed in combination with other tests, it is effective in screening for sepsis, but if used alone as a primary test to look for infection, it helps in less than 10% of definite diagnoses (26, 27). CRP sensitivity has been reported from 46 to 74% in various studies (28). In the meta-analyzes about this subject, the first time CRP sensitivity was 39%, CPR specificity was 92.5%, positive predictive value was 53.2% and negative predictive value was 93.6%. Therefore, it seems that the serum level of CRP for the first time at the time of admission in infants suspected of early neonatal sepsis in the neonatal intensive care unit (NICU) has no value in determining sensitivity (29). In the study by Zecca et al, the negative predictive value of CRP was 100% (30 %). A study by Cetinkaya et al on 100 infants suspected of having early neonatal sepsis showed that the diagnostic power of CRP increased over time and peaked 24 to 48 hours after the onset of sepsis, which is consistent with our study (31). Therefore, if serum CRP levels remain negative 24 hours after the onset of clinical symptoms, the high negative predictive value is 99%, and antibiotic therapy can be discontinued in the absence of strong clinical suspicion of sepsis (32). [ABSTRACT FROM AUTHOR]

Background & Aims: One of the most important tests for the detection of antibodies against neutrophil cytoplasm in autoimmune vasculitis diseases is the Antineutrophil Cytoplasmic Antibodies (ANCA) test. The ANCA test is the first order in the initial diagnosis and screening of patients with suspected autoimmune diseases, including small-vessel vasculitis, as well as the follow-up of patients' treatment, which uses indirect immunofluorescence assay (IFA) to detect the presence of antibodies against antigens in the cytoplasm of neutrophils. In addition to vasculitis, the evaluation of this antibody is used in other autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and inflammatory bowel diseases (IBD). To prepare IFA slides for diagnostic purposes, neutrophils extracted from human peripheral blood are used as substrates. The IFA is a common and acceptable method in medical diagnostic laboratories for the detection of these autoantibodies. The first experience in the development of this technique back to the work of Koons and Kaplan, although today, this method has been widely developed in the field of laboratory diagnosis as well as research in basic medical sciences. A distinct advantage of the immunofluorescence assay is its applicability to fresh tissue and fixed cells. This method is based on the specific antigen binding to specific antibodies conjugated with fluorescent dyes and fluorescent light emission. There is two common types of immunofluorescence assay include direct and indirect immunofluorescence used for detection autoantibodies. In the direct immunofluorescence method, specific antibodies are conjugated to fluorescent dyes, and by binding to their specific antigen, the green fluorescent light is recognized under a fluorescent microscope. Although this procedure is faster, it is less often applied in medical diagnostic labs. In the IFA method, a specific antibody against the target antigen (called the primary antibody) binds to the target antigen recognized by a secondary antibody that is against the fixed region of the primary antibody and conjugated with fluorescent dyes, such as fluorescein isothiocyanate (FITC). In the indirect immunofluorescence assay, due to the binding of several secondary antibodies to the primary antibody, the generated fluorescence signals will be amplified, which in turn increases the fluorescence intensity and sensitivity. The aim of this study was to design and prepare slides for antibodies detection against antigens in cytoplasm neutrophils by the indirect immunofluorescence method. Furthermore, the next step by optimizing fluorescent patterns to detect different types of antibodies against neutrophil cytoplasm by indirect immunofluorescence method. Since 1980, the search for antibodies against the neutrophil cytoplasm has been recognized as one of the most effective diagnostic tools for diseases associated with small to medium-sized vasculitis. ANCA in the diagnosis and follow-up of the treatment of vasculitic diseases including granulomatosis with polyangiitis, formerly known as Wegener granulomatosis, microscopic polyangiitis, Eosinophilic granulomatosis with polyangiitis, formerly known as Churg – Strauss syndrome, is used. Most laboratories around the world use IFA for screening and early detection of ANCA. After the ANCA test is positive, the target antigens of the antibodies are evaluated by the ELISA method. The two most common targets of autoantibodies are myeloperoxidase (MPO) and proteinase-3 (PR-3) in patients with vasculitis. However, a clinical association of these vasculitis diseases with other antigens such as elastase, lactoferrin, azurocidin, lysozyme, H-Lamp-2, and other lesser-known antigens has also been observed. Antibodies against neutrophil cytoplasm have also been reported in other small vessel vasculitis diseases such as microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA), and necrotizing crescentic glomerulonephritis. Initially, the non-commercial ELISA method was used to diagnose PR3-ANCA and MPO-ANCA, but inconsistent results between laboratories showed the global need for standardization of this test . In 1998, fifteen laboratory centers around the world evaluated and standardized the standard method for measuring antibodies against neutrophil cytoplasm (ANCA) and its specific antigens, including PR3 and MPO, by indirect immunofluorescence and ELISA in patients with idiopathic systemic vasculitis. . The result of this standardization increased the detection value of ANCA by immunofluorescence and ELISA methods. One of the most important pillars of the commercialization of a product is the supply of raw materials with appropriate and sustainable quality. The first step in producing ANCA slides is the isolation of high purity and standard quality peripheral blood neutrophil cells. Peripheral blood neutrophils are short-lived, highly sensitive cells that cannot be stored for long periods of time, and it is recommended that these cells be isolated from whole blood immediately after blood sampling. To date, several methods have been proposed to isolate neutrophils from peripheral blood. In this study, it was shown that using the dextran method and subsequent separation by centrifugal gradient method with filament is one of the cost-effective methods with high purity to provide the ANCA test substrate. Methods: Using the Ficoll density gradient centrifugation and dextran solution, neutrophils were isolated from the peripheral blood of healthy individuals (without a history of any disease, especially autoimmune diseases in themselves and first-degree relatives). The purity and efficiency of the isolated cells were evaluated by Wright Giemsa staining, the neobar slide, and an automatic counting machine. Isolated neutrophils were fixed on slides using an ethanol fixative. At this stage, by selecting the best protocol, in terms of time and type of neutrophil fixation stage, they were fixed on the slide. In the next step, using positive and negative samples that have already been determined by ANCA with valid commercial kits (IVD certified), the slides containing fixed cells were fluorescent stained and compared with control samples. Results: The number and purity of neutrophils isolated were evaluated by a Sysmex automatic cell counter. Depending on the peripheral blood neutrophils, the mean(±standard deviation) of the number of isolated neutrophils was 6000(±950) cells per microliter. According to white blood cell differential count, the mean purity of isolated neutrophils was 94.1%. Also, by trypan blue staining, the percentage of viable cells was determined to be more than 85% before fixation. The results of ANCA indirect fluorescent staining on the prepared slides were completely consistent with the results of control samples. Conclusion: The results of this study showed that the method used can be applied for the commercialization and production of ANCA slides. In this regard, in addition to optimizing the steps of separation and fixing the neutrophils, it is necessary to evaluate and optimize the stability of the prepared slides during storage and production in order to produce in large numbers. [ABSTRACT FROM AUTHOR]