NEW BIORAPID PRODUCT. NEW biorapid MONONUCLEOSIS 25 Test

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1 14th June /MK/BR/20 NEW 25 est NEW BIORAPID PRODU We are happy to announce the launch of a new product of the biorapid family based on immunochromatography technology: - 25 est code his product is intended for the qualitative detection of heterophile antibodies to Infectious Mononucleosis. completes the very successful biokit range of products for IM: Monogen and olor-mono. Find enclosed complete product information that we hope helps you in the launch of the assay. Good sales and good luck!

2 Index: - General information on EBV - Diagnosis - - Assay procedure - Performance - ompetitors - Features and Benefits - onclusion - Package insert Page 2 of 13

3 Infectious Mononucleosis General information on the Epstein-Barr virus: linical symptoms he Epstein-Barr virus (EBV) was first discovered in 1964 as the cause of infectious mononucleosis. (IM). his disorder is usually an acute, benign and self-limiting lymphoproliferative condition that is usually subclinical in children and a mild condition in adults. he EBV is also the cause of nasopharyngeal carcinoma and neoplasms of the thymus, parotid gland and larynx. Burkitt s lymphoma, a malign tumour of the lymphoid tissue occurring mainly in African children, is also caused by EBV. Epstein-Barr virus is a gamma-herpesvirus (see figure 1). he EBV has a very limited host range and tissue tropism: Human B-lymphocytes and epithelial cells of the oropharynx and nasopharynx. EBV is widely disseminated. It is estimated that 95% of the world population is exposed to the virus, which makes it the most widespread virus known to man. EBV appears to be transmitted primarily by close contact with infectious oral-pharyngeal secretions, however the virus has been reported to be transmitted by blood transfusion and transplacental routes. However, under ordinary conditions, transmission of the virus through transfusion or transplacental exposure is unlikely. In addition, EBV-associated post-transplantation lymphoproliferative disease develops in 1% to 10% of organ transplant recipients. he frequency of seronegative patients is nearly 100% in early infancy, but declines more or less rapidly with increased age and depending on socio-economic conditions, to less than 10% in young adults. After primary exposure, a person is considered to be immune and generally no longer susceptible to new re-infections. In Western society, primary exposure to EBV occurs in two waves. Approximately half of the population is exposed to the virus before the age of 5 years. A second wave of seroconversion occurs during late adolescence, between 15 to 24 years of age. More than 90% of EBV-infected individuals intermittently shed the virus for life even when totally asymptomatic. hildren can acquire the virus at an early stage by sharing contaminated drinking glasses and generally undergo subclinical disease. aliva sharing between adolescents and young adults often occurs by kissing, thus the nickname of kissing disease. In these individuals the disease may go unnoticed or be present in varying degrees of severity as infectious mononucleosis (IM). he cardinal clinical features of the mononucleosis syndrome are fever, sore throat, malaise, and fatigue, accompanied by signs of tonsillopharyngitis and lymphadenopathy. here is frequently a prodromal period of 2-5 days consisting of malaise and fatigue, prior to the onset of the full syndrome. he adenopathy in mononucleosis most prominently involves the anterior and posterior cervical lymph nodes, but diffuse adenopathy also often occurs. plenomegaly develops in the first 3 weeks of illness in about 50% of cases. he adenopathy and organomegaly are often most prominent in the second to fourth weeks of illness. Other frequently observed clinical signs include: rash, usually erythematous and maculopapular, soft palate petechiae, bilateral edema of the eyelids, and jaundice. It is well established that ampicillin will induce a typical rash in virtually all patients with mononucleosis. Page 3 of 13

4 Infection with EBV has been shown to be very common in small children. By the age of 4, as many as 70% of children will have acquired the antibody. he clinical manifestations in children under 4 years of age typically entail more Patients with infectious mononucleosis typically demonstrate an absolute lymphocytosis (greater than 50%), prominent atypical lymphocytosis (often greater than 10-20%), a positive test for Paul- Bunnell heterophile antibodies, and mild to moderate elevation of liver enzymes. A diagnosis made on the basis of the clinical record and symptomatology alone is difficult. Numerous cases have been cited in which IM has been misidentified with other non-related viral and bacterial diseases. For this reason, blood and serum tests are very helpful in diagnosis. Diagnosis of Epstein-Barr virus infection Diagnosis of EBV could be divided into non-specific and specific methods. Heterophile antibodies he immunitary system of infected patients produces heterophile antibodies, most of them IgM. hese antibodies do not react directly to EBV antigens but even appear a little bit earlier than EBV specific IgM antibodies. he heterophile antibody that appears in the EBV infection also has the capability of reacting to a glycoprotein present on the surface of sheep erythrocyte antigen. his antigen was discovered by Paul-Bunnell in Heterophile antibody assays are very useful as screening tests as they detect the very early primary infection. biokit has a well known rapid test based on the Paul-Bunnell antigen, the Monogen (ode number ) and a rapid test based on slide haemagglutination, olor-mono (ode number ). Elisa tests Enzyme immunoassay tests are based on purified proteins, recombinant antigens, or synthetic peptides. here are specific tests for detection of all EBV antigens for IgG or IgM such as VA, EBNA or Early Antigens. he Elisa test provides the best combination of sensitivity, specificity and user-friendliness for confirmation and followup. Immunofluorescence assays hese are considered as a reference method for EBV diagnosis.. he assays are also based on recombinant antigens or synthetic peptides. hey use microscope slides as a support for the solid phase and anti IgG or IgM conjugate labelled with a fluorescent marker. hese methods require highly skilled staff to read and interpret the results. Immunoblot Enzyme immunoassay tests based on recombinant antigens that use a nitro-cellulose strip as a support for the solid phase. he strip incorporates all the antigens separately, thus allowing the assay to determine the presence of specific antibodies to these antigens in the patient samples. Page 4 of 13

5 is a rapid, one-step qualitative screening test for the detection of Infectious Mononucleosis heterophile antibodies in serum, plasma or whole blood. he test is able to specifically detect heterophile antibodies in the early phase of Infectious mononucleosis. is a rapid qualitative one-step immunochromatographic assay. he test device includes a sample well (), a conjugate containing colloidal gold particles coated with highly purified Paul-Bunnell antigen, and a chromatographic membrane strip where Paul-Bunnell antigen has been immobilized to act as a capture means. ensitivity has been adjusted to mach that provided by Monogen (latex based assay), a worldwide reference for IM diagnosis. Method he sample is introduced into the sample well () and allowed to migrate along the attached membrane strip. If IM heterophile antibodies are present in the sample, they will bind to the conjugate forming an immunocomplex. he complex continues to migrate further along the membrane where it will be captured by the Paul-Bunnell antigen immobilized in the est zone (), leading to the formation of a coloured line. he remaining conjugate will bind to the control zone () forming a second coloured line indicating correct performance of the test. If IM heterophile antibodies are present in the sample, two coloured lines will appear in the reading window and indicate a positive result. If IM heterophile antibodies are absent, only the ontrol line will appear and a negative result is indicated. If no lines or only the est line appears, the test is invalid. Material provided - 25 test devices. - 1 x 2 ml diluent buffer. - 1 x 1 ml positive control. - 1 x 1 ml negative control disposable pipettes. Page 5 of 13

6 ample collection can be used for serum, plasma (EDA, heparin and citrate) or Whole Blood (EDA, heparin and citrate). pecimens that contain particulate material may give inconsistent results and should be clarified before testing by high speed centrifugation (10,000 rpm) for 10 minutes. torage and stability should be stored at 2-8. he stability of biorapid MONONULEOI is that stated on the kit label if stored under the above conditions. est devices and diluent buffer can be stored separately at any temperature between Quality control he blue line at the location of the control zone is for manufacturing control purposes only and does not interfere with performance of the test. Any blue dye that may remain in the window at reading time does not interfere with the results. he ontrol line () is an internal procedural control. his line will always appear if the test has been performed correctly and the test device is functioning properly. Use the controls included in the kit to check the performance of the biorapid MONONULEOI assay. If the expected results are not obtained, do not use the kit. Page 6 of 13

7 Assay procedure FOR ERUM, PLAMA AND ONROL: - Allow test devices and samples to reach room temperature before use.. - Remove the test device from the pouch and place it on a flat surface. - Label the test device with the patient s name or identification number. - Hold the pipette in a vertical position and dispense 3 drops (75 µl) of serum or plasma into the sample well (). - For the positive and negative controls, hold the dropper in a vertical position and dispense 2 drops into the sample well (). - Immediately add 2 drops of the diluent buffer holding the dropper in a vertical position. - Read the result after 5 minutes and then discard the test device. Do not read a test result after more than 5 minutes have elapsed. FOR WHOLE BLOOD: - Allow test devices and samples to reach room temperature before use.. - Remove the test device from the pouch and place it on a flat surface. - Label the test device with the patient s name or identification number. - Hold the pipette in a vertical position and dispense 2 drops (50 µl) of blood into the sample well (). - Immediately add 3 drops of the diluent buffer holding the dropper in a vertical position. - Read the result after 5 minutes and then discard the test device. Do not read a test result after more than 5 minutes have elapsed. DO NO INERPRE HE E AFER 15 MINUE Interpretation of the results POIIVE If two coloured lines (est and ontrol) are visible, the test result is positive. Any visible colour on the est line () should be interpreted as a positive result even if it has less intensity than the ontrol line (). NEGAIVE If only the ontrol line () is visible, the test result is negative. INVALID If no distinct lines appear or if only the est line is visible, the test is invalid. he sample should be retested using another test device. Page 7 of 13

8 POIIVE NEGAIVE INVALID biokit biokit biokit Limitations of the procedure - he results should be interpreted in light of the clinical, haematological, and serological information of the patient. - Occasionally detectable levels of heterophile antibodies are late in developing in symptomatic IM patients.. If symptoms persist, the assay should be repeated within a few days. ome patients, especially children and adolescents, may remain persistently negative. It has been reported that only 80 to 90% of adults and less than 50% of young children develop heterophile antibodies. - In some individuals, detectable levels of heterophile antibodies may persist for months and, more rarely, for years. Expected values Different studies of the presence of IM heterophile antibodies in blood donors show that the incidence of the disease ranges from 0.9 to 1.7% of the population. As the presence of heterophile antibodies indicates a relatively recent infection, these results suggest that the true incidence of the disease is higher than the number of diagnosed cases. Page 8 of 13

9 Performance characteristic A total of 622 samples (296 serum samples, 261 plasma samples, and 65 whole blood samples) were evaluated both in an internal and external study using and another commercially available immunochromatographic (I) test: Genzyme OOM Mono est. he Genzyme kit is a FDA approved market leader in the United tates. All samples were confirmed as positive or negative by a commercially available latex particle agglutination test for the qualitative detection of IM heterophile antibodies. Results erum samples Genzyme Mono est * 220 (*) he discrepant sample was negative for a latex agglutination test. Plasma samples Genzyme Mono est * 208 (*) he discrepant sample was negative for a latex agglutination test. Whole blood samples Genzyme Mono est All samples Genzyme Mono est When compared to Genzyme Mono est, showed a sensitivity of 100% (142/142) and a specificity of 99.6% (478/480). he overall agreement between the two I tests was 99.7%. omparison of to Biokit Monogen Page 9 of 13

10 MONOLAEX ** 428 When compared to the Biokit MONOLAEX agglutination test, the biorapid MONONULEOI test showed a sensitivity of 100% (120/120) and a specificity of 97.9% (428/437). he overall agreement was 98.4%. Reproducibility A reproducibility evaluation of the test was conducted at 3 external laboratories where testing was performed by personnel with diverse educational backgrounds. At each site, testing was performed on randomly coded samples, 3 positive and 3 negative, for 3 days and in triplicate. he results obtained showed a 100% agreement with the expected results. Page 10 of 13

11 ompetitors ompetitors on lateral flow wo main competitors may be found in lateral flow technology: - OOM Mono est, Genzyme - learview IM, Inverness he following table summarises all the characteristics and stated performance: BIOKI GENZYME INVERNE est Name Biorapid Mononucleosis OOM Mono est learview IM No. of ests Device shape Plastic cassette Only trips Plastic cassette Negative ontrol YE YE NO Positive ontrol YE YE NO ample Diluent YE YE YE apillary tubes & bulb for NO YE NO blood collection Disposable pipettes YE NO YE Incubation time 5 min. 5 min. 5 min. erum/plasma 15 min. Whole Blood torage R: Device + Diluent 2-8, Kit controls R R Declared ensitivity 100% 100% 95.5% (blood) 98.5% (erum/plasma) Declared pecificity 99.5% vs. lateral flow 97.9% vs. Latex 95.9% 100% omments + ontrols included + harp clear bands + ontrols included - Poor readability, faint bands - trip assay: trip must be dipped in the sample tube. - More manipulation - Potential risk of sample contamination + lear bands - Does not include controls Page 11 of 13

12 Internal evaluation vs. competitors A total of 21 clinical samples, characterized as positive by biokit Monogen but with a different degree of reactivity, were evaluated using, Genzyme OOM Mono est and learview IM. Results detected 18 out of 21 samples. Genzyme OOM Mono est detected 15 out of 21 samples. learview IM detected 16 out of 21 samples. Monogen Genzyme OOM Mono est Monogen learview IM Monogen he showed the best correlation with the biokit Monogen, considered as a worldwide reference for the detection of heterophile antibodies against Infectious Mononucleosis. Page 12 of 13

13 Features and benefits Features Biokit s own Paul Bunnell antigen orrelation with Monogen Official approvals Positive and Negative ontrols included Blood Finger tip sample erum or plasma sample Easy reading Room temperature storage for test devices and diluent Long shelf-life Plastic device Benefits - he best antigen on the market as demonstrated by Monogen and olor-mono. - Excellent sensitivity - Excellent specificity - E mark - U FDA - Full confidence in the results obtained - learview does not include controls - Doctor s Office est - Easiest sample to be taken - No ample preparation - No lab tubes to collect serum or plasma - ost savings - Also suitable for the typical samples used in any laboratory - Objective results - harp bands - No background - No need for staff trained people in reading agglutination tests - No extra refrigeration equipment required - More room in refrigerators - implifying distributor and end-users maintaining a correct inventory - Easy manipulation compared to strip tube assays - trip tube assay needs sample aliquot to dip the strip in. - Potential risk of sample contamination onclusion is manufactured by one of the most expert companies in IM diagnosis. Monolatex and olor-mono are worldwide references in quality according to different international publications. As demonstrated in the evaluations, matches the performance of the Biokit reference assay, Monolatex, and the U market leader for lateral flow IM detection, the Genzyme Mono est. Apart from performance, the test shows important advantages vs. competitors: Vs. learview: biorapid includes positive and negative controls whereas learview does not. Vs. Genzyme: he biorapid assay is a practical plastic device. he Genzyme is a strip-tube assay. he strip must be dipped in the sample tube. Page 13 of 13

14 READ HIGHLIGHED HANGE REF /10

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16 Amostras de sangue total este comparativo One-step immunoassay for the detection of infectious mononucleosis heterophile antibodies in serum, plasma or whole blood. otal amostras este comparativo Ao ser comparado com um teste imunocromatográfico (I) comercial, o teste mostrou uma sensibilidade de 100% (142/142) e uma especificidade de 99,6% (478/480). A concordância total entre os dois testes foi de 99,7%. Ao ser comparado com um teste comercial de aglutinação de partículas de látex, o teste biorapid MONONULEOI mostrou uma sensibilidade de 100% (120/120) e uma especificidade de 97,9% (428/437). A concordância total entre os dois testes foi de 98,4%. Interferências Para estudar possíveis interferências foram analizadas amostras com risco potencial de produzir reações falsopositivas, como 10 amostras de soro positivas de anticorpos IgM anti-toxoplasma, 10 positivas de anticorpos IgM anti-mv e 10 positivas para fator reumatóide. odas amostras apresentaram resultados negativos. Reprodutibilidade Uma avaliação da reprodutibilidade do teste foi realizada em 3 laboratórios externos onde a prova foi realizada por pessoal com diferente nível de treinamento. Em cada laboratório, o teste se realizou com amostras rotuladas aleatoriamente, 3 positivas e 3 negativas, durante 3 dias e por triplicado. Os resultados obtidos apresentaram 100% de concordância com os resultados esperados. Bibliografia Ver References do texto inglês. ummary Infectious mononucleosis (IM) is an acute infectious disease caused by the Epstein-Barr virus (EBV). In 1932, Paul and Bunnell reported that the serum of patients with IM develop high titers of heterophile antibodies to sheep erythrocytes. Also were described agglutinins to red blood cells from other mammals. he proteins responsible for this agglutination are glycoproteins from red cell membranes called Paul-Bunnell antigen by several authors. tudies made on these glycoproteins show that those purified from bovine red blood cells are the most sensitive to IM heterophile antibodies. Heterophile antibodies to sheep erythrocytes may also be detected in sera from normal people, individuals who have received injections of serum, and others. raditionally the IM heterophile antibodies have been distinguished from other heterophile antibodies by a differential absorption test with bovine red blood cells and guinea pig kidney tissue. he use of highly purified Paul-Bunnell antigen provides a simple method with improved sensitivity for the specific detection of heterophile antibodies associated with IM. Principle is a rapid qualitative one step immunochromatographic assay. he test device includes a sample well (), a conjugate containing colloidal gold particles coated with highly purified Paul-Bunnell antigen, and a chromatographic membrane strip where Paul-Bunnell antigen has been immobilized to act as a capture. he sample is added into the sample well () and allowed to migrate along the attached membrane strip. If IM heterophile antibodies are present in the sample, they will bind to the conjugate forming an immunocomplex. he complex continues to migrate further along the membrane where it will be captured by the Paul-Bunnell antigen immobilized on the test zone (), leading to the formation of a coloured line. Remaining conjugate will bind to the control zone () forming a second coloured line indicating the proper performance of the test. If IM heterophile antibodies are present in the sample, two coloured lines will appear in the reading window and indicate a positive result. If IM heterophile antibodies are absent, only the ontrol line will appear and a negative result is indicated. If no lines or only the est line appears, the test is invalid. omponents REF E 25 test devices. - DIL 1 x 2 ml diluent buffer.ris buffer with additives. - ONROL + 1 x 1 ml positive control. Rabbit IgG anti-paul-bunnell antigen diluted in a buffer. ontains < 0.1% sodium azide. - ONROL - 1 x 1 ml negative control. Non reactive diluted human serum. ontains < 0.1% sodium azide. - PIPEE 25 disposable pipettes. 24 Precautions is intended for IN VIRO diagnostic use. odium azide may react with lead or copper pipes and plumbing creating highly explosive metal azides. Flush drains with water thoroughly after disposing of the remains of reagents. All human source material used in the preparation of this product was found to be negative for the presence of HIV- 1/HIV-2 and HV antibodies, as well as for the hepatitis B surface antigen, using a method approved by the Food and Drug Administration (UA). WARNING: POENIALLY BIOHAZARDOU MAERIAL. Because no test method can offer complete assurance of the absence of infectious agents, this product should be handled with caution. Do not use test devices if the aluminum pouch has been damaged during storage. A new pipette must be used for each sample to avoid contamination errors. Dispose all used materials in a suitable biohazardous waste container. 1

17 torage he reagents will remain stable through the expiration date shown on the label if stored between 2-8. Do not freeze. est devices and diluent buffer could be stored separately at 2 to 25. biokit biokit biokit ample collection ERUM or PLAMA: Use fresh serum or plasma (EDA, heparin, citrate). Other anticoagulants should be evaluated before use. amples can be stored at 2-8 for 2 days. For longer periods samples should be frozen (-20 ). Avoid repeated freezing and thawing. Homogenize the samples before analysis. Grossly hemolyzed, turbid, or lipemic samples should not be used. amples that contain particulated material may give inconsistent results and should be clarified by centrifugation before testing. WHOLE BLOOD: ollect whole blood samples using a tube containing anticoagulant (EDA, heparin, citrate). Other anticoagulants should be evaluated before use. Whole blood samples can be stored at 2-8 for 2 days. Quality control he blue line at the location of the control zone () is for manufacturing control purposes only and does not interfere with the performance of the test. Any blue dye that may remain in the window at the reading time, does not interfere with the results. he ontrol line () is an internal procedural control. If the test has been performed correctly and the test device is functioning properly this line will always appear. o check the performance of, use the controls included in the kit. If expected results are not obtained, do not use the kit. Procedure FOR ERUM, PLAMA, and ONROL: - Allow test devices and samples to reach room temperature before using. - Remove test device from the pouch and place it on a flat surface. - Label test device with patient name or identification number. - Hold the pipette in vertical position and dispense 3 drops (75 µl) of serum or plasma into the sample well (). - For the positive and negative controls, hold the dropper in vertical position and dispense 2 drops into the sample well (). - Immediately after, add 2 drops of the diluent buffer holding the dropper in vertical position. - Read the result at 5 minutes, then discard the test device. Do not read a test result after more than 5 minutes. FOR WHOLE BLOOD: - Allow test devices and samples to reach room temperature before using. - Remove the test device from the pouch and place it on a flat surface. - Label test device with patient name or identification number. - Hold the pipette in vertical position and dispense 2 drops (50 µl) of blood into the sample well (). - Immediately after, add 3 drops of the diluent buffer holding the dropper in vertical position. - Read the result at 5 minutes, then discard the test device. Do not read a test result after more than 5 minutes. Interpretation of the results POIIVE If two coloured lines (est and ontrol) are visible, the test result is positive. Any visible colour on the est line () should be interpreted as a positive result even if it has less intensity than the ontrol line (). NEGAIVE If only the ontrol line () is visible, the test result is negative. INVALID If no distinct lines appear or if only the est line is visible, the test is invalid. he sample should be retested using another test device. Limitações do procedimento POIIVO NEGAIVO EM VALIDADE - Os resultados devem ser interpretados tendo em consideração a informação clínica, hematológica e serológica do paciente. - Ocasionalmente níveis detectáveis de anticorpos heterófilos se desenvolvem tardiamente em pacientes sintomáticos de MI. e os sintomas persistirem se recomenda repetir o teste após uns dias. Alguns pacientes, especialmente crianças e adolescentes, podem persistir permanentemente como negativos. e publicou que apenas de 80% a 90% dos adultos e menos de 50% dos jovens desenvolvem anticorpos heterófilos. - Em alguns indivíduos podem persistir níveis detectáveis de anticorpos heterófilos durante meses e, mais raramente, durante anos. Valores previstos Diferentes estudos realizados em doadores de sangue indicam que a presença de anticorpos heterófilos de MI oscila entre 0,9 e 1,7% da população. Dado que a presença destes anticorpos indica uma infecção relativamente recente, os resultados obtidos sugerem que a incidência real da doença pode ser maior que o número de casos diagnosticados. aracterísticas funcionais Um total de 622 amostras (296 soros, 261 plasmas e 65 sangue total) foram avaliadas internamente e em uma avaliação externa utilizando o teste e outro teste imunocromatográfico (I) comercial. odas as amostras foram confirmadas como positivas ou negativas mediante um teste comercial de aglutinação de partículas de látex. Resultados Amostras de soro este comparativo (*) A amostra discrepante deu resultado negativo com um teste de aglutinação de látex. Amostras de plasma este comparativo * * 208 (*) A amostra discrepante deu resultado negativo com um teste de aglutinação de látex. 2 23

18 onservação Os reativos permanecem estáveis até a data de validade indicada na etiqueta, se forem conservados entre 2-8. Não congelar. Os dispositivos e o tampão diluente podem ser conservados em separado entre 2 e 25. biokit biokit biokit oleta da amostra ORO OU PLAMA: Usar soro fresco ou plasma (EDA, heparina, citrato). Outros anticoagulantes devem ser avaliados antes de serem utilizados. As amostras podem ser conservadas por 2 dias entre 2-8. Para guardar por um período de tempo mais longo as amostras devem ser congeladas (-20 ). Evitar congelar e descongelar as amostras repetidamente. Homogeneizar as amostras antes de serem analisadas. Não se devem utilizar amostras hemolizadas, turvas ou lipêmicas. As amostras que contêm material particulado podem dar resultados inconsistentes e necessitam ser clarificadas por centrifugação antes de realizar o teste. ANGUE OAL: oletar o sangue em tubos com anticoagulante (EDA, heparina, citrato). Outros anticoagulantes devem ser comprovados antes de serem utilizados. As amostras de sangue total podem ser conservadas durante 2 dias entre 2 e 8. ontrole de qualidade A linha azul que se observa na zona ontrole () é um controle de fabricação e não interfere no funcionamento do teste. Qualquer resto de corante azul que se pudera visualizar na janela no momento de realizar a leitura, não interfere nos resultados. A linha ontrole () é um controle interno do procedimento. e o teste foi realizado corretamente e o dispositivo funciona devidamente, esta linha sempre deve aparecer. Para comprovar o correto funcionamento do utilizar os controles incluídos no kit. e não se obtêm os resultados esperados, não utilize o kit. Procedimento PARA ORO, PLAMA E ONROLE: - Deixar que os dispositivos e as amostras atinjam a temperatura ambiente antes de utilizá-los. - Retirar o dispositivo da embalagem e colocá-lo sobre uma superfície plana. - Rotular o dispositivo com o nome do paciente ou número de identificação. - Manter a pipeta em posição vertical e dosificar 3 gotas (75 µl) de soro ou plasma no pozinho de amostra (). - Para os controles positivo e negativo, manter o conta-gotas em posição vertical e dosificar 2 gotas no pozinho de amostra (). - Imediatamente depois, acrescentar 2 gotas de tampão diluente, mantendo o conta-gotas em posição vertical. - Ler o resultado após 5 minutos e descartar o dispositivo. Não fazer a leitura dos resultados em caso de se exceder o período de tempo indicado. PARA ANGUE OAL: - Deixar que os dispositivos e as amostras atinjam a temperatura ambiente antes de utilizá-los. - Retirar o dispositivo da embalagem e colocá-lo sobre uma superfície plana. - Rotular o dispositivo com o nome do paciente ou número de identificação. - Manter a pipeta em posição vertical e dosificar 2 gotas (50 µl) de sangue no pozinho de amostra (). - Imediatamente depois, acrescentar 3 gotas de tampão diluente, mantendo o conta-gotas em posição vertical. - Ler o resultado após 5 minutos e descartar o dispositivo. Não fazer a leitura dos resultados em caso de se exceder o período de tempo indicado Interpretação dos resultados POIIVO e as duas linhas (este e ontrole) forem visíveis, o resultado será positivo. Qualquer cor que a linha de este () adquira, será indicação de resultado positivo, inclusive se a cor for menos intensa que a da linha de ontrole (). NEGAIVO Em caso de que apenas a linha de ontrole () adquira cor, o resultado será considerado negativo. EM VALIDADE e nenhuma das linhas adquire cor ou apenas a linha de este fica colorida, o resultado será considerado sem validade. Portanto, o teste deverá ser repetido utilizando-se outro dispositivo. Limitations of the procedure POIIVE NEGAIVE INVALID - he results should be interpreted in light of the clinical, hematological, and serological information of the patient. - Occasionally detectable levels of heterophile antibodies are late in developing in patients symptomatic for IM. If symptoms persist it is recommended to repeat the assay in several days. ome patients, especially children and adolescents, may remain persistently negative. It has been reported that only 80 to 90% of adults and less than 50% of young children develop heterophile antibodies. - In some individuals, detectable levels of heterophile antibodies may persist for months and, more rarely, for years. Expected values Different studies of presence of IM heterophile antibodies in blood donors show that the incidence of the disease ranges from 0.9 to 1.7% of population. As presence of heterophile antibodies indicates a relatively recent infection, these results suggest that the true incidence of the disease is higher than the number of diagnosed cases. Performance characteristics A total of 622 samples (296 serum samples, 261 plasma samples, and 65 whole blood samples) were evaluated internally and in an external study with and another commercially available immunochromatographic (I) test. All samples were confirmed as positive or negative by a commercially available latex particle agglutination test for the qualitative detection of IM heterophile antibodies. Results erum samples omparative test (*) he discrepant sample was negative by a latex agglutination test. Plasma samples * omparative test - 1* 208 (*) he discrepant sample was negative by a latex agglutination test. 22 3

19 Whole blood samples All samples omparative test omparative test When compared to a commercially available immunochromatographic (I) test, showed a sensitivity of 100% (142/142) and a specificity of 99.6% (478/480).he overall agreement between the two I tests was 99.7% When compared to a commercially available latex particle agglutination assay, the test showed a sensitivity of 100% (120/120) and a specificity of 97.9% (428/437). he overall agreement was 98.4%. Interferences o study possible interferences, samples with potential risk of producing false positive results were tested, including 10 serum samples positive for toxoplasma IgM antibody, 10 positive for MV IgM antibody and 10 positive for rheumatoid factor. All samples gave a negative result. Reproducibility A reproducibility evaluation of the test was conducted at 3 external laboratories where testing was performed by personnel with diverse educational backgrounds. At each site, testing was performed on randomly coded samples, 3 positive and 3 negative, for 3 days and by triplicate. Results obtained showed a 100% agreement with the expected results. References Biosafety in Microbiological and Biomedical Laboratories. D/NIH manual, 5th Edition, Davidsohn I. erologic diagnosis of infectious mononucleosis. JAMA 108: , Gray JJ, aldwell J, illis M. he rapid serological diagnosis of infectious mononucleosis. Journal of Infection 25: 39-46, Linderholm M, Boman J, Juto P, Linde A. omparative evaluation of nine kits for rapid diagnosis of infectious mononucleosis and Epstein-Barr virus-specific serology. J lin Microbiol 32: , Medical Devices Agency Evaluation Report: An evaluation of fourteen commercial kits used to screen for the presence of infectious mononucleosis (MDA/98/63). Medical Devices Agency Evaluation entre. UK National Health ervice. 6. Pozzetto B, Mbida AD, Bourlet, Grattard F, Bonnevial L. omparative evaluation of eight commercial tests for the diagnosis of infectious mononucleosis by Epstein-Barr virus-specific or non specific serology. erodiagn Immunother Infect Dis 8: , Virtanen. Incidence of infectious mononucleosis antibodies in blood donors. Acta Pathol Microbiol Immunol cand 56: 53-56, este de um só passo para a detecção de anticorpos heterófilos da mononucleose infecciosa em soro, plasma ou sangue total. umário A mononucleose infecciosa (MI) é uma doença infecciosa aguda causada pelo vírus de Epstein-Barr (EBV). Em 1932, Paul e Bunnell observaram que os soros de pacientes com MI continham anticorpos heterófilos contra hemácias de carneiro. Mais tarde também foram descritas aglutininas contra eritrócitos de outros mamíferos. As proteínas responsáveis por esta aglutinação são glicoproteínas de membrana dos glóbulos vermelhos, denominadas antígeno de Paul Bunnell por diversos autores. Os estudos realizados demonstram que o antigênio de Paul-Bunnell purificado de eritrócitos bovinos é mais sensível aos anticorpos heterófilos da MI que o procedente de outros mamíferos. ambém se pôde detectar anticorpos heterófilos contra eritrócitos de carneiro diferentes dos da MI em soros de pessoas saudáveis, de indivíduos que receberam injeções de soros, e outros. radicionalmente os anticorpos heterófilos da MI têm sido distinguidos de outros tipos através da uma análise de absorção diferencial realizada con hemácias bovinas e tecido renal de cobaia. O uso do antígeno de Paul-Bunnell altamente purificado neste teste proporciona um método mais simples e mais sensível para a detecção dos anticorpos heterófilos de MI. Princípio é um teste imunocromatográfico de um só passo. O dispositivo inclui um pozinho para a adição da amostra (), um conjugado de partículas de ouro coloidal sensibilizadas com antígeno de Paul-Bunnell altamente purificado, e uma membrana cromatográfica na que se imobilizou o antígeno de Paul-Bunnell, que atuará como captor. A amostra se dosifica no pozinho correspondente () e começa a migrar ao longo da membrana adjunta. e a amostra contém anticorpos heterófilos de MI, estes se unirão ao conjugado formando um imunocomplexo. O complexo continua sua migração ao longo da membrana onde será capturado pelo antígeno de Paul-Bunnell imobilizado na zona este (), dando lugar à formação de uma linha colorida. O conjugado restante se unirá à zona ontrole () formando uma segunda linha colorida, a qual indica o bom funcionamento do teste. e a amostra contém anticorpos heterófilos de MI, na janela de leitura aparecem duas linhas coloridas indicando um resultado positivo. e a amostra não contém anticorpos heterófilos de MI, só aparecerá a linha ontrole indicando um resultado negativo. e não aparece nenhuma linha ou se só aparece a linha este, o resultado não se considera válido. omponentes REF E 25 dispositivos. - DIL 1 x 2 ml tampão diluente. ampão ris com aditivos. - ONROL + 1 x 1 ml controle positivo. IgG de coelho contra o antígeno de Paul-Bunnell diluído em um tampão. ontém azida sódica < 0,1%. - ONROL - 1 x 1 ml controle negativo. oro humano negativo diluído. ontém azida sódica < 0,1%. - PIPEE 25 pipetas descartáveis. Precauções é para o diagnóstico IN VIRO. A azida sódica pode reagir com os encanamentos e ralos de chumbo ou cobre, originando azidas metálicas altamente explosivas. Ao descartar os restos de reativos, fazê-lo em abundante volume de água. odos os materiais de origem humana utilizados na preparação deste produto foram testados e apresentaram resultados negativos em relação à presença de anticorpos contra os vírus HIV-1/HIV-2 e HV, assim como para o antígeno de superfície da hepatite B, utilizando um método aprovado pela Food and Drug Administration (UA). AENÇÃO: MAERIAL DE RIO BIOLÓGIO. Dado que nenhum método pode oferecer a total segurança da ausência de agentes infecciosos, este produto deve ser manipulado com precaução. Não utilizar os dispositivos se a embalagem de alumínio sofreu danos durante sua conservação. Para evitar erros de contaminação, deve-se utilizar uma nova pipeta para cada teste. Descartar todos os materiais usados em recipientes adequados para material bio-contaminante. 4 21

20 ampioni di sangue totale otale campioni est comparativo est comparativo Quando è stato confrontato con un test immunocromatografico (I) commerciale, il ha dimostrato una sensibilità del 100% (142/142) e una specificità del 99,6% (478/480). La concordanza totale tra i due test è stata del 99,7%. Quando è stato confrontato con un test commerciale di agglutinazione di particelle di lattice, il biorapid MONONULEOI ha dimostrato una sensibilità del 100% (120/120) e una specificità del 97,9% (428/437). La concordanza totale tra i due test è stata del 98,4%. Interferenze Per studiare eventuali interferenze, sono stati analizzati campioni con rischio potenziale di dare falsi risultati positivi, includendo 10 campioni di sieri positivi per gli anticorpi IgM anti-toxoplasma, 10 positivi per gli anticorpi IgM anti-mv e 10 positivi per il fattore reumatoide. utti i campioni hanno dato risultati negativo. Riproducibilità Della valutazione della riproducibilità del test si sono incaricati 3 laboratori esterni in cui il test è stato realizzato da personale con un diverso livello di addestramento. In ogni centro, il test è stato eseguito con campioni etichettati aleatoriamente, 3 positivi e 3 negativi, per 3 giorni e tre volte. I risultati ottenuti presentavano una concordanza del 100% con i risultati previsti. Bibliografia Vedere References nel testo inglese Inmunoensayo de un solo paso para la detección de anticuerpos heterófilos de mononucleosis infecciosa en suero, plasma o sangre total. umario La mononucleosis infecciosa (MI) es una enfermedad infecciosa aguda causada por el virus de Epstein-Barr (EBV). In 1932, Paul y Bunnell observaron que los sueros de pacientes que padecían MI contenían anticuerpos heterófilos contra hematíes de cordero. Más tarde fueron también descritas aglutininas contra eritrocitos de otros mamíferos. Las proteínas responsables de esta aglutinación son glicoproteínas de membrana de los glóbulos rojos denominadas antígeno de Paul-Bunnell por varios autores. Estudios realizados muestran que el antígeno de Paul-Bunnell purificado de eritrocitos bovinos es más sensible a los anticuerpos heterófilos de MI que el procedente de otros mamíferos. ambién se pueden detectar anticuerpos heterófilos contra eritrocitos de cordero, diferentes de los de MI, en sueros de personas sanas, en individuos que han recibido inyecciones de suero y otros. radicionalmente los anticuerpos heterófilos de MI se han diferenciado de los otros tipos mediante una prueba de absorción diferencial consistente en la observación del distinto comportamiento de los mismos frente a los absorbentes de riñón de cobaya o de glóbulos de buey. El uso del antígeno de Paul-Bunnell altamente purificado en este test, proporciona un método más simple y más sensible para la detección de los anticuerpos heterófilos de MI. Principio es un test inmunocromatográfico de un solo paso. El dispositivo incluye un pocillo para la adición de la muestra (), un conjugado de partículas de oro coloidal sensibilizadas con antígeno de Paul-Bunnell altamente purificado, y una membrana cromatográfica en la que se ha inmobilizado antígeno de Paul-Bunnell que actuará como captura. La muestra se dosifica en el pocillo correspondiente () y empieza a migrar a lo largo de la membrana adjunta. i la muestra contiene anticuerpos heterófilos de MI, éstos se unirán al conjugado formando un inmunocomplejo. El complejo continúa su migración a lo largo de la membrana donde será capturado por el antígeno de Paul-Bunnell inmobilizado en la zona test (), dando lugar a la formación de una línea coloreada. El conjugado sobrante se unirá a la zona control () formando una segunda línea coloreada, lo que indica el buen funcionamiento del test. i la muestra contiene anticuerpos heterófilos de MI, en la ventana de lectura aparecerán dos líneas coloreadas indicando un resultado positivo. i la muestra no contiene anticuerpos heterófilos de MI, sólo aparecerá la línea ontrol indicando un resultado negativo. i no aparece ninguna línea o si sólo aparece la línea est, el resultado no es válido. omponentes REF E 25 dispositivos. - DIL 1 x 2 ml tampón diluyente. ampón ris con aditivos. - ONROL + 1 x 1 ml control positivo. IgG de conejo contra el antígeno de Paul-Bunnell diluido en un tampón. ontiene azida sódica < 0,1%. - ONROL - 1 x 1 ml control negativo. uero humano negativo diluido. ontiene azida sódica < 0,1%. - PIPEE 25 pipetas desechables. Precauciones es sólo para el diagnóstico IN VIRO. La azida sódica puede reaccionar con tuberías y desagües de plomo o cobre dando lugar a azidas metálicas altamente explosivas. Al desechar los restos de reactivos, deje correr agua abundante. odo el material de origen humano utilizado en la preparación de este producto ha sido encontrado negativo a la presencia de anticuerpos de los virus HIV-1/HIV-2 y HV, así como a la del antígeno de superficie de la hepatitis B, utilizando un método aprobado por la Food and Drug Administration (UA). AENIÓN: MAERIAL DE RIEGO BIOLÓGIO. Ya que ningún método puede ofrecer la total seguridad de la ausencia de agentes infecciosos, este producto debe manejarse con precaución. No utilizar los dispositivos si la bolsa de aluminio ha sido dañada durante su conservación. Para evitar errores de contaminación, debe utilizarse una nueva pipeta en cada ensayo. Depositar todos los materiales usados en recipientes adecuados para material biocontaminante. 20 5

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