ANA HEp-2 Research Articles

Acute autoimmune hemolytic anemia in a patient with systemic lupus erythematosus

Introduction Autoimmune hemolytic anemia occurs due to the accelerated destruction of erythrocytes as a result of the dysfunction of immune system cells, which produce antibodies against the normal antigens of the membrane of hematopoietic cells. One of its causes is systemic lupus erythematosus. Materials and methods We present a case of a 20-year-old patient who was hospitalized with acute autoimmune hemolytic anemia, having been diagnosed with SLE at the age of 18 years. At the onset of the disease, hemolytic anemia was a differential diagnostic challenge. Results The differential diagnosis between primary and secondary autoimmune hemolytic anemia (AIHA) was an important step. The presence of antinuclear antibodies (ANA Hep2, Anti-dsDNA, Anti-cardiolipin, Anti-phospholipids, anti-Ro, Anti-Sm B) were important arguments in making the diagnosis. The relapse of AIHA was caused by inadequate treatment, due to a lack of compliance. Pulse therapy combined with methylprednisolone and cyclophosphamide successfully resolved the AIHA. Conclusions Hematological abnormalities are commonly seen in SLE patients, but hemolytic autoimmune anemia is a rare condition. A timely diagnosis of the cause of hemolytic anemia and proper treatment of lupus by correcting autoimmune disorders are crucial in disease management. Pulse therapy combined with corticosteroids and immunosuppressants is effective in acute relapses of hemolytic anemia.

E020 Pneumatosis intestinalis with spontaneous pneumoperitoneum in a patient with systemic sclerosis-myositis overlap syndrome: a case report and review of the literature

Abstract Background/Aims Pneumatosis intestinalis (PI) is a rare complication of systemic sclerosis (SSc), characterised by submucosal and/or subserosal collections of free gas, forming cystic lesions within the gastrointestinal (GI) tract. Methods A 67-year-old man with SSc-myositis overlap syndrome presented acutely unwell with worsening abdominal pain, bloating, bloody diarrhoea and vomiting for three weeks. He was afebrile and haemodynamically stable. His abdomen was distended with generalised tenderness but soft and regular bowel sounds were audible. There were no palpable organomegaly or signs of peritonism. He had raised CRP, serum amylase, serum lactate and pre-renal acute kidney injury. He tested positive for ANA Hep2 (speckled) and anti-Mi-2b. His contrast-enhanced CT abdomen and pelvis displayed extensive pneumatosis changes, moderately dilated small bowel loops and pneumoperitoneum without portal venous gas or acute thrombotic occlusion in the main-stem superior or inferior mesenteric arteries. He was managed conservatively with bowel rest, elemental diet, cycling antibiotics for small intestinal bacterial overgrowth (SIBO) and commenced on end-of-life care as surgical exploration was deemed too risky considering that he has exceedingly poor cardiac reserve. He made a surprisingly excellent recovery and his repeat CT scans at approximately 11 weeks showed a complete resolution of pneumatosis intestinalis and pneumoperitoneum. Results PI in SSc generally runs a benign course and is usually managed conservatively. The development of PI is multifactorial, involving a complex interplay between mechanical, biochemical, bacterial and drug factors. PI is usually asymptomatic but patients can present with GI and constitutional symptoms or peritonism. CT scan is the most preferred diagnostic modality for PI. Conservative management is preferable to surgical intervention after careful exclusion of acute surgical causes. The underlying cause of PI should be appropriately addressed and managed in all cases. The management of PI is based on the severity of symptoms and it includes bowel rest, elemental diet or parenteral nutrition, antibiotics, high concentration oxygen, hyperbaric oxygen therapy and/or octreotide. Our patient was managed conservatively as he did not exhibit clinical signs of peritonism or bowel ischaemia and surgical interventions were deemed too risky. He was treated with and has responded well to cycling oral antibiotics for SIBO which was thought to be contributory to his PI. Conclusion This case illustrates that PI with or without pneumoperitoneum could be a rare complication of SSc, and contrary to common belief, it is usually benign and managed conservatively with favourable outcome. An accurate interpretation of its clinical significance is crucial as acute presentations and ambiguous imaging findings can simulate acute surgical emergency leading to diagnostic dilemma and unnecessary or risky surgical interventions. However, emergency laparotomy should be considered in patients who exhibit signs of peritonism or bowel ischaemia and therefore timely involvement of surgeons and multidisciplinary team is essential. Disclosure K. Lim: None. S. Knights: None. J. Pauling: None. J. Gotto: None.

OA20 Pneumatosis intestinalis with spontaneous pneumoperitoneum in a patient with systemic sclerosis-myositis overlap syndrome: a case report and review of the literature

OA20 Pneumatosis intestinalis with spontaneous pneumoperitoneum in a patient with systemic sclerosis-myositis overlap syndrome: a case report and review of the literature

Frequency of ANA/DFS70 autoantibodies in Colombian patients with undifferentiated connective tissue disease.

The objective was to describe the clinical characteristics and the frequency of the ANA/DFS70 autoantibodies in patients affected by undifferentiated connective tissue disease (UCTD) in a tertiary hospital in Colombia. This descriptive cross-sectional study enrolled patients who fulfilled the classification criteria for UCTD. ANAHEp- 2 test and the modified assay for ANA/DFS70 autoantibodies were performed through the indirect immunofluorescence technique. Erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, and the antibodies to anti-extractable nuclear antigens, DNA, phospholipids (IgG, IgM, IgA), and cyclic citrullinated peptide were also evaluated. Fifty-three patients were studied; 42/53 (79%) tested positive for ANA and 5/42 (11.9%) for ANA/DFS70 antibodies with a dense fine speckled fluorescent pattern (AC-2) in ANA HEp-2 test that was confirmed by a modified HEp-2-DFS70 assay. Patients had arthralgia (87%, n=47), non-erosive arthritis (66%, n=34), xerostomia (64%, n=34), xerophthalmia (42%, n=22), and Raynaud's phenomenon (17%, n=9). Arthralgia, xerophthalmia, xeroderma, and absence of disease evolution to a specific disease over five years were more frequent in patients with a positive result for the anti-DFS70 antibodies. The ANA/DFS70 autoantibodies were more frequent in patients with UCTD compared to other rheumatic diseases for which they were initially evaluated. More studies are required to support the predictive role of this antibody to the absence of progression to a well-defined connective tissue disease.

The clinical significance of anti-DFS70 autoantibodies and its correlation with Vitamin D levels.

This study aims to investigate the clinical significance of anti-dense fine speckled 70 (DFS70) autoantibodies and its association with systemic autoimmune rheumatic diseases (SARD) related autoimmune markers and Vitamin D levels. The study group consisted of 281 (mean age±SD: 45.31±15.89 years; 88.3% female) anti-DFS70 autoantibody-positive patients. All patients' sera in the study group were tested by ANA HEp-2 indirect immunofluorescent antibody (IIF) and immunoblotting (IB) methods (Euroimmun AG, Lübeck, Germany). Anti-DFS70 antibody-positive patients were divided into two subgroups. Anti-DFS70 antibody-positive patients with SARD were assigned as Group 1 (n=43), anti-DFS70 antibody-positive patients without SARD were assigned as Group 2 (n=238). A control group with anti-DFS70 negative patients with SARD were assigned as Group 3 (n=49, mean age±SD: 49.86±12.08; 79.6% female). The clinical characteristics, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), neutrophil/lymphocyte ratio, thrombocyte/lymphocyte ratio (TLR), rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and 25-hydroxyvitamin D3 (25OHD3) levels were compared between three groups. The majority (61.9%) of anti-DFS70 antibody positive patients had no specific diagnosis. Other systemic diseases were detected as allergic diseases (10.0%), hematological abnormalities (5.0%), thyroid diseases (3.6%), gastrointestinal system diseases (1.8%), malignancies (1.4%), and infections (1.1%). ESR, CRP levels, and TLR were lower in Group 2 than Groups 1 and 3 (p<0.05). RF and anti-CCP positivity rates were lower in Group 2 when compared with Groups 1 and 3 (p<0.05). 25OHD3 levels did not differ between three groups (p=0.103). We observed that anti-DFS70 autoantibody may be associated with organ-specific autoimmune diseases, allergic diseases, and hematological disorders. Therefore, it is essential to evaluate these pathologies in patients positive for anti-DFS70 antibodies.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

BackgroundThe V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses.MainbodyTwenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized.ConclusionThe V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

A case of Hashimotos encephalopathy failed to respond to steroids- A case report

A 52-year-old hypothyroid presented with episodes of seizures in last ten days with altered behavior. CT scan of brain showed cerebral atrophy and MRI of brain revealed non-specific changes. In EEG, there was slow wave pattern. CSF study showed increased cell count with all lymphocytes and raised protein with normal glucose. Anti- TPO Antibody was positive. All relevant investigations like HIV, HBsAg, Anti- HCV Ab, ANA (Hep2 method), ANA profile, P-ANCA, c-ANCA, VGKC (voltage gated potassium channel) Ab, NMDA Receptor/Anti- Glutamate Antibody) Ab, VDRL were negative. She was given pulse doses of methylprednisolone followed by maintenance prednisolone. Then her GCS improved followed by sudden deterioration. Repeat CSF showed marginal decrement of protein and cell count. Then she was given IV Immunoglobulin and patient improved. At discharge her CSF study normalized. Anti-TPO Ab titre came to normal level and EEG normalized. Hashimoto’s encephalopathy(HE) should be suspected in a case of sub-acute encephalopathy with high levels of anti-thyroid antibodies may be with normal thyroid functions. Here the patient did not have improvement on steroids. The patient needed immunoglobulin to improve.

Classification of ANA HEp-2 slide images using morphological features of stained patterns

Correct diagnostics of autoimmune disorders is important for a treatment planning as many of such diseases demonstrate similar symptoms. Owing to ANA HEp-2 medical test it is possible to distinguish among such autoimmune disease as lupus erythematosus, scleroderma and Sjogren’s syndrome. It is known that manual disease classification performing based on analysis of indirect immunofluorescent images obtained in the course of this test is error-prone. Here, we present an automatic method for classification of ANA HEp-2 images that performs separation of the individual cell on pre-segmented slide images and subsequent cell and slide image classification. The method uses morphological properties of the stained patterns located inside the cell nucleus for an individual cell classification and a voting scheme for a classification of the whole ANA HEp-2 slide image.

Leptin concentration in children with juvenile idiopathic arthritis.

Leptin regulates the organism's immune response. Juvenile idiopathic arthritis (JIA) is a chronic joint disease in children, leading to chronic changes in motor organs. In children with JIA (n = 42) and healthy subjects (n = 28), leptin concentration (LEP), body mass index (BMI), haematocrit (HTC), haemoglobin (HB), morphotic elements (WBC,LYMPH), erythrocyte sedimentation rate (ESR), and ANA Hep-2 antibodies were analysed. JIA group was divided into: children with a longer (51-148 months) (IA) n = 22 and a shorter disease period (2-18 months) (IB) n = 20. Only 58.3% of the IA and 50% of the IB group had ANA Hep-2 confirmed. The ill children had higher and more diversified LYMPH and ESR levels compared to the healthy children. The highest LEP for the IA group was 37.5 ng/cm3, (Me 5.85), for IB - 40.10 ng/cm3, (Me 2.46) as compared to the IC - 3.74 ng/cm3 (Me 2.85), respectively. The average BMI value for the IA group was 16.61 kg/m2, for IC it was 18.91 kg/m2, and the median for IB was 15.89 kg/m2. Children with BMI values < 23 kg/m2 from the IA and IB group had a reduction in LEP as compared to control group (p = 0.04). The relationship between the illness and LEP diversification per BMI unit was found in both groups. Children with a shorter illness period had higher LEP differentiation per BMI unit compared to the healthy children. Children with juvenile idiopathic arthritis with BMI < 23 kg/m2 had lower leptin concentrations than healthy subjects. Ill children with a shorter-term disease had a higher diversification of leptin concentration per BMI unit as compared to healthy controls.

Interpretation of ANA indirect immunofluorescence test outside the darkroom using NOVA view compared to manual microscopy.

Objective. To evaluate NOVA View with focus on reading archived images versus microscope based manual interpretation of ANA HEp-2 slides by an experienced, certified medical technologist. Methods. 369 well defined sera from: 44 rheumatoid arthritis, 50 systemic lupus erythematosus, 35 scleroderma, 19 Sjögren's syndrome, and 10 polymyositis patients as well as 99 healthy controls were examined. In addition, 12 defined sera from the Centers for Disease Control and 100 random patient sera sent to ARUP Laboratories for ANA HEp-2 IIF testing were included. Samples were read using the archived images on NOVA View and compared to results obtained from manual reading. Results. At a 1 : 40/1 : 80 dilution the resulting comparison demonstrated 94.8%/92.9% positive, 97.4%/97.4% negative, and 96.5%/96.2% total agreements between manual IIF and NOVA View archived images. Agreement of identifiable patterns between methods was 97%, with PCNA and mixed patterns undetermined. Conclusion. Excellent agreements were obtained between reading archived images on NOVA View and manually on a fluorescent microscope. In addition, workflow benefits were observed which need to be analyzed in future studies.

ANA HEp-2 cells image classification using number, size, shape and localization of targeted cell regions

The ANA HEp-2 medical test is a powerful tool in autoimmune disease diagnostics. The last step of this test, the interpretation of immunofluorescent images by trained experts, represents a potential source of errors and could theoretically be replaced by automated methods. Here we present a fully automatic method for recognition of types of immunofluorescent images produced by the ANA HEp-2 medical test. The proposed method makes use of the difference in number, size, shape and localization of cell regions that are targeted by the antinuclear antibodies – the humoral components of immune system that bind human antigens as a result of the immune system malfunction. The method extracts morphological properties of stained cell regions using a combination of thresholding-based and thresholding-less approaches and applies a conventional machine-learning algorithm for image classification.

Avaliação clínico-laboratorial de pacientes com síndrome antifosfolípide primária segundo a frequência de anticorpos antinucleares (FAN Hep-2)

To evaluate the frequency of clinical and laboratory manifestations in patients with primary antiphospholipid syndrome (PAPS) with positive antinuclear antibodies (ANA Hep-2+) compared to those in whom this antibody is negative (ANA Hep-2-). This is a transversal study with 58 patients (82.8% females) with PAPS. Demographic and clinical data, comorbidities, medications, and antiphospholipid antibodies were evaluated. Twenty (34.5%) out of 58 patients were positive for ANA Hep-2. Comparing the group of patients ANA Hep-2+ with those that were ANA Hep-2-, it was observed that both groups of patients with APS did not show statistically significant differences regarding demographic data, as well as the duration of the disease. As for clinical and laboratorial manifestations, the ANA Hep-2+ group showed higher frequency of deep venous thrombosis (85 versus 52.6%, P = 0.04), a statistically higher frequency of anticardiolipin IgG (85 versus 52.6%, P = 0.02), and a tendency for anticardiolipin IgM (80% versus 52.6%, P = 0.05), as well as greater medians of those antibodies [33 (0-128) versus 20 (0-120) GPL, P = 0.008] and [33 (0-120) versus 18,5 (0-120) MPL, P = 0.009]. Such difference was not observed regarding other manifestations of APS, presence of comorbidities, lifestyle, and medications used. Patients with PAPS with ANA Hep-2+ have a higher frequency of deep venous thrombosis and anticardiolipin IgG and IgM.

A Screening for the Occurrence of Autoreactive Antibodies in Sera of Pregnant and Non‐pregnant Bitches

Sera of healthy pregnant (group I, n = 11) and non-pregnant (group II, n = 11) bitches were screened for autoantibodies (AAb). In both groups, blood samples were drawn every fifth day between days 5 and 55 after mating. Serum was analysed via indirect immunofluorescence (IIF) with the Canine ANA HEp-2 Screening Kit. In all animals, anticytoplasmic AAb were detected. Utilizing primate-heart substrate slides AAb against contractile proteins of the cytoplasm could be observed. The predominating fluorescence pattern in pregnant animals resembled above all desmin, which was proven via Western blot. The sera were then pre-incubated with tropomyosin, actin, vimentin, vinculin and keratin solutions, and assessed on HEp-2 slides and on human and canine fibroblasts as well. The latter substrate was used to verify whether the detected Ab were in fact AAb. Utilizing tropomyosin, revealed elimination of the cytoplasmic fluorescences on all three substrates. It is therefore assumed, that in sera of healthy dogs, AAb against contractile structure proteins of the cytoplasm are present regularly. The majority of pregnant bitches presented with higher end-point titres (EPT), than to be found in non-pregnant dogs. AAb against desmin played the key role in those patterns. In addition, sera were screened for thyroid specific AAb, namely thyroglobulin, thyroid peroxidase (TPO), T3 and T4, and for AAb against insulin by ELISA or Western blot (TPO). Only in two of the pregnant bitches a weak positive reaction (1:100) for T3-AAb was detected.

P0311 SEROLOGICAL AND HISTOPATHOLOGICAL FEATURES OF CHILDREN WITH AUTOIMMUNE HEPATITIS - OVERLAP SYNDROME

Introduction: Autoimmune hepatitis in children is chronic progressive disease, which can coexist with hepatotropic viral infection and primary sclerosing cholangitis. The specifity of circulating autoantibodies can be used to distinguish between type I (ANA< SMA) and type II (LKM). Aim of the study was to compare the antibody profile and histopathogical changes in autoimmune hepatitis, coexisting other diseases. Methods: 100 children with autoimmune hepatitis type I and 21 children with autoimmune hepatitis type II have been scored (IAHP Report 1999) according to the histologic criteria (interface hepatitis, bridging necrosis, biliary lesions, granuloma formations, Pearl’s stain for iron), serum biochemistry (ALT level, alkaline phosphatase), gamma globulin concentration including IgG, serum autoantibodies (ANA, AMA, LKM, AMA, pANCA), viral markers (antibodies against HCV, antiHbs, Hbe, Hbc, PCR for HCV). Results: At the time of diagnosis histology of the liver was characterised by necroinflammatory changes accompanied by fibrosis and/or cirrhosis. Viral infection occurred in 8 children (2 children were infected by HCV, 6 children by HBV), 6 children demonstrated features of primary sclerosing cholangitis, including 3 children with ulcerous colitis. Necroinflammatory changes in autoimmune and viral hepatitis were severe (according to Batts&Ludwig 1995); in HBV: predominantly lobular changes, lymph follicle formations; in HCV: steatosis and Fe accumulation in the hepatocytes and Kupffer cells. Histological features of biliary changes and mild cholestasis were seen in all cases of primary sclerosing cholangitis; granuloma formation in the portal tracts in 2 cases. All children were treated with Azathioprine and Prednisolone (without Interferone in 8 cases of viral hepatitis). IN PSC: mitovhondrial pattern in ANA HEP-2 = coarse granular in the cytoplasm; cytokeratin pattern = dots in the nucleus and fibrous cytokeratin structure of the cytoplasm. IN AIH type II 10 children were anti-HCV positive, but only 3 of them were RNA positive. Conclusion: Variable autoantibody level with false positivity for anti HCV occurs in autoimmune hepatitis (7 children with PCR negative for HCV) and overlapping syndrome (2 0children with PCR positive for HCV). Histopathological changes in overlap syndrome are not diagnostic, but suggestive for the aetiology. Overlap syndrome in children responded poorly to classical treatment of autoimmune hepatitis.