Cepario autóctono de leptospiras en la prueba de micro - aglutinación / Micro - agglutination test of Autogenous leptospirosis

Introducción: leptospirosis. Zoonosis más frecuente en Cuba, como enfermedad infecto-contagiosa. Objetivos: identificar las cepas de leptospiras aisladas localmente, para ser empleadas en la prueba de microaglutinación. Apreciar su comportamiento serológico frente a las cepas de referencia. Método: estudio experimental, de sueros de pacientes presuntivos de padecer Leptospirosis, con 60 muestras, en el Instituto Carlos J. Finlay, de La Habana. Identificación de 40 sueros reactores a la hemaglutinación pasiva. Identificación de 34 cepas, de un total de 250 aislamientos, a partir de hemocultivos, de pacientes con antisueros policlonales a serogrupos y anticuerpos monoclonales a serovar. Se conformaron cinco cepasrepresentativas de los serovares: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi e Icterohaemorrhagiae Copenhageni, como las de mayor circulación en la región. Se realizó la prueba de microaglutinación con las cepas aisladas localmente, y las de referencia de forma paralela. Resultados: mayor reactividad con las cepas locales, y conformación de cinco cepas representantes de los serovares: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi e Icterohaemorrhagiae Copenhageni, como las de mayor circulación en la región. Se encontró una concordancia de 86,36% en la reactividad. Conclusiones: los resultados mostraron mayor reactividad con las cepas locales que con las de referencia, elevada concordancia e incremento de los títulos de anticuerpos de los sueros reactores usando cepas locales, y disminución del promedio de las reacciones cruzadas cuando se utilizaron las cepas locales. Se demostró su utilidad en la evaluación de estudios inmunológicos.

Introduction: leptospirosis, a contagious infectious disease considered the most common zoonosis in Cuba. Objectives: to identify locally isolated leptospirosis strains for micro- agglutination test. To evaluate their serological behavior against the reference strains. Method: an experimental study of 60 presumptive Leptospirosis patients' serum samples. Forty sera reactive to passive hemagglutination. To isolate 250 blood cultures of patients. To show 34 strains identified at the Carlos J. Finlay Institute, with polyclonal antisera to serogroups and monoclonal antibodies to serovar. Five strains represented by the serovars: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi and Icterohaemorrhagiae Copenhageni, were the most circulating strains in the region. The microagglutination test was performed in parallel form to locally isolated strains and reference strains Results: greater reactivity with the local strains. Five strains representing the serovars: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi and Icterohaemorrhagiae copenhageni as the most circulating in the region. The reactivity showed 86.36% concordance. Conclusions: there was greater reactivity with the local strains than the reference strains. A higher concordance increased the titers of antibodies of the reactive sera, by the use of local strains. The average of cross reactions decreased when the local strains were used. The evaluation of immunological studies was demonstrated.

Human Neutrophil Alloantigen-3a Alloimmunization among Pregnant Korean Women / 대한수혈학회지

BACKGROUND: Alloantibodies against human neutrophil alloantigen (HNA)-3a are associated with severe and fatal transfusion related acute lung injury (TRALI). HNA-3 genotyping and HNA-3a antibody (Ab) identification are essential to diagnosis and prevention of TRALI caused by HNA-3a Ab. However there had been no laboratory for HNA-3a Ab identification in Korea. The aims of this study were to establish the HNA-3a Ab test in Korea and to estimate the incidence of HNA-3a alloimmunization among pregnant Korean women. METHODS: HNA-3a homozygotes and HNA-3b homozygotes were identified by HNA-3 genotyping. Three HNA-3a homozygotes and three HNA-3b homozygotes are included in the granulocytes panel, which consisted of 10 donors for granulocytes. Sera from 650 pregnant Korean women were tested for granulocyte Ab using a mixed passive hemagglutination assay (MPHA). When a HNA-3a Ab was detected, the woman's HNA-3 was typed to support her HNA-3a alloimmunization. RESULTS: MPHA showed positive reactions in the sera from 26 women (4.0%, 26/650). HLA Abs were detected in 18 women (2.8%, 18/650), among whom HNA Abs were identified simultaneously in 7 women. Granulocyte Abs were detected in sera from 15 women (2.3%, 15/650). The incidence of HNA-3a, HNA-1b, HNA-1a, HNA-2a, and unidentified HNA Abs among pregnant Korean women was 0.77% (5/650), 0.77% (5/650), 0.62% (4/650), 0.15 (1/650), and 0.31% (2/650), respectively. CONCLUSION: In this study, we established the HNA-3a Ab test using MPHA for diagnosis and prevention of TRALI caused by HNA-3a Ab. The incidence of HNA-3a Ab in pregnant Korean women was 0.77% (5/650).

Establishment of Platelet Antigen and Antibody Tests by Mixed Passive Hemagglutination with Frozen-stored Platelets / 대한수혈학회지

BACKGROUND: Platelet antigen and antibody tests have been used in platelet immunological disorders, such as neonatal alloimmune thrombocytopenia (NAIT) and post-transfusion purpura (PTP). Mixed passive hemagglutination (MPHA) method has several advantages, including frozen preservation of platelets, ability to differentiate between anti-HLA and platelet-specific antibodies, and quick and easy interpretation without expensive equipment. In this study, we intended to develop the MPHA method using indicator cells of anti-Rh(D) sensitized group O, Rh+ RBCs. METHODS: We made indicator cells sensitized with anti-Rh(D) with various strengths (1:32 to 1:256) and determined the optimal strength. We determined the sensitivity of the MPHA and compared the results using flow cytometry. We observed the changes of the reaction according to the storage time of indicator cells. RESULTS: The optimal sensitization strengths of the indicator cells were 1:192 and 1:256. MPHA showed strong positive results with 1:8,192 diluted positive control, while the detection limit of flow cytometry was 1:128. Until the second week (mean 16 days), the indicator cells showed good results comparable to those of fresh ones. CONCLUSION: We developed the MPHA method using indicator cells of anti-Rh(D) sensitized group O, Rh+ RBCs. We produced the indicator cells in our own laboratory and obtained platelet panels with rare antigen typing using frozen-stored platelets. This technology will be used effectively for detection of platelet antigens and identification of platelet antibodies and also for platelet crossmatching.

Comparison of Nine Different Qualitative HBsAg Assay Kits / 대한진단검사의학회지

BACKGROUND: Qualitative hepatitis B surface antigen (HBsAg) assay kits are still commonly used in Korea where hepatitis B virus (HBV) infection is endemic. The accurate determination of HBsAg plays a crucial role in the diagnosis and prevention of HBV infection, especially in endemic areas. The aim of this study was to compare the detection sensitivities of 9 qualitative HBsAg assay kits. METHODS: Seven pooled sera with HBsAg concentration ranging from 0.14 IU/mL to 29.96 IU/mL were prepared. The HBsAg concentration of each pooled serum was determined by a quantitative HBsAg assay, Architect HBsAg (Abbott Laboratories, Ireland). The fully automated immunoassay kits included Elecsys HBsAg (Roche Diagnostics, Germany) and Immulite 2000 HBsAg (DPC, USA) and the rapid tests included 5 immunochromatographic assay (ICA) kits and 2 reverse passive hemagglutination assay (RPHA) kits. RESULTS: Elecsys HBsAg (Roche Diagnostics) showed positive result in pooled serum with HBsAg concentration of 0.14 IU/mL, but Immulite 2000 HBsAg (DPC) showed negative result in the same concentration. Although ICA kits showed variable results among different assay kits, all of them showed negative results in pooled sera with HBsAg concentration of < or =1.89 IU/mL. Two RPHA kits showed negative results in pooled sera with HBsAg concentration of < or =7.98 IU/mL. CONCLUSIONS: Although ICAs were more sensitive than RPHAs, they had variable sensitivities for HBsAg and were less sensitive than the automated immunoassay kits. Therefore, ICAs and RPHAs should be used with caution in the screening tests for HBsAg and their sensitivities need to be improved.

Establishment of Mixed Passive Hemagglutination Assay Using In-House Anti-Rh(D) Sensitized Human O RBCs for the Detection of Granulocyte Specific Antibodies / 대한수혈학회지

BACKGROUND: Granulocyte specific antibodies are associated with several clinical conditions including febrile transfusion reaction and transfusion-related acute lung injury as well as immune neutropenias. The identification of granulocyte specific antibodies is important for the diagnosis of these disorders. However, there have been rarely confirmed clinical reports in Korea since the testing techniques are complicated and difficult to maintain. In this study, development of in-house indicator cells and renewedly establishment of the mixed passive hemagglutination assay (MPHA) as a serologic test to detect and identify granulocyte specific antibodies were conducted. METHODS: The in-house indicator cells for MPHA were made by sensitizing human Rh(D) positive O RBCs with human IgG anti-Rh(D) (DiaMed AG, Switzerland) and then combining with AHG anti-IgG (Immucor Inc., USA). To determine the optimal conditions, various combinations of anti-Rh(D) IgG sensitization strengths of indicator cells, microwell coated antigens (intact granulocyte vs. extracted granulocyte) and reaction conditions were compared. RESULTS: The best test conditions for MPHA were as follows: optimal results were obtained with the anti-Rh(D) sensitization dilutions of 1/64-1/192 and the reaction condition of 4 hours incubation at room temperature in humid chamber. Extracted granulocytes coated at the plate showed better results than intact granulocytes. HLA antigens were completely removed from extracted granulocyte antigens after acidified chloroquine treatment. CONCLUSION: Granulocyte MPHA using in-house anti-Rh(D) sensitized indicator cells was developed for the first time in Korea. The newly established MPHA would be effectively used for the diagnosis and treatment of disorders associated with granulocyte specific antigen-antibody reactions in Korea.

Comparison of Passive Hemagglutination and Indirect Immunofluorescence Assay for the Diagnosis of Scrub typhus / 감염과화학요법

The gold standard for the serologic detection of scrub typhus is the indirect immunofluorescence assay (IFA), however the passive hemagglutination assay (PHA) is commonly used method in Korea because of its availability, easy handling. We compared the sensitivity of PHA and IFA to compare their effectiveness in diagnosing scrub typhus. Out of 35 patients who were eventually diagnosed of scrub typhus by positive IFA, 19 patients (54%) were also tested positive for PHA during the acute phase (titer=1:80). Whereas 30 patients (85%) were positive in IFA during the acute phase. During the recovery phase 30 (85%) patients were positive in PHA, whereas all patients were found to be positive in IFA. Patients with 4-fold or greater rise of antibody were 12 (34.3%) in PHA, and 19 (54.3%) in IFA. We note that IFA has better sensitivity over PHA in detecting scrub typhus.

Comparison of Passive Hemagglutination and Indirect Immunofluorescence Assay for the Diagnosis of Scrub typhus / 감염과화학요법

The gold standard for the serologic detection of scrub typhus is the indirect immunofluorescence assay (IFA), however the passive hemagglutination assay (PHA) is commonly used method in Korea because of its availability, easy handling. We compared the sensitivity of PHA and IFA to compare their effectiveness in diagnosing scrub typhus. Out of 35 patients who were eventually diagnosed of scrub typhus by positive IFA, 19 patients (54%) were also tested positive for PHA during the acute phase (titer=1:80). Whereas 30 patients (85%) were positive in IFA during the acute phase. During the recovery phase 30 (85%) patients were positive in PHA, whereas all patients were found to be positive in IFA. Patients with 4-fold or greater rise of antibody were 12 (34.3%) in PHA, and 19 (54.3%) in IFA. We note that IFA has better sensitivity over PHA in detecting scrub typhus.

Comparison of Methods for the Detection of Anti-HBs for Hepatitis B Vaccination Program in Korea / 예방의학회지

OBJECTIVES: The purpose of this study was to suggest a proper method for the detection of heaptitis B surface antibody(anti-HBs) in a screening program for hepatitis B vaccination. METHODS: Sensivitity, specificity and predictive values were compared between Immunochromatographic assay (ICA) and passive hemagglutination(PHA) in 978 subjects(565 males, 413 females, 19-78 years ranging in age, mean 46.5 years old). EIA was used as a standard method for the detection of HBsAb. RESULTS: Sensitivity in the detection of anti-HBs of PHA and ICA was 88.7% and 94.9%, specificity was 94.3% and 96.6%, negative predictive value was 96.5% and 98.0%, and positive predictive value was 82.3% and 91.3%, respectively. False negative rate(11.3%) of PHA was higher than that(5.1%) of ICA. The higher the titer of anti-HBs in EIA was, the lower the false negative rate was. There was no false negative result in the cases with 101mIU/ml or more in EIA. CONCLUSION: We suggest that ICA should be the choice of screening method in the detection of anti-HBs in Hepatitis B vaccination program.

Diagnosis of human filariasis by passive-hemagglutination and soluble antigen fluorescent antibody technic (SAFA)

In the Philippines, passive haemagglutination and soluble antigen fluorescent antibody tests using Dirofilaria immitis antigens were carried out on the sera of 33 patients infected with Wuchereria bancrofti and on the sera of 24 uninfected persons. False positives were as high as 70.8% indicating that in areas where the incidence of D. immitis in dogs is high, W. bancrofti antigens must be used. D.A.Cz.

Development of Homemade Indicator Cells for Platelet Antibody Test by Mixed Passive Hemagglutination / 대한임상병리학회지

BACKGROUND: Platelet antibody test has been used in the diagnosis and management of immunological platelet disorders and platelet crossmatch. Mixed passive hemagglutination (MPHA) test is a cost-effective, reproducible, easy to perform and convenient method. Anti-IgG coated indicator red cells used for MPHA test have not been made in Korea and those cells have been exclusively donated by Dr. Shibata in Japan. This study was designed to produce domestic indicator cells and to determine its acceptability by comparing to the results obtained with Dr. Shibata's indicator cells. We produced homemade indicator cells by coating human RhD-positive O RBC with human IgG anti-D (check cell) and then coated with rabbit anti-human IgG. METHODS: Sixty three sera from healthy male donors, 58 sera tested positive in platelet suspension immunofluorescence test (PSIFT), and 61 sera tested negative in PSIFT were evaluated by MPHA employing both homemade and Dr. Shibata's indicator cells. RESULTS: The concordance rate between PSIFT and homemade MPHA was 74%. Test results of MPHA with homemade indicator cell showed excellent correlation with Shibata's indicator cell (P=0.002). All of 63 sera from healthy volunteer male blood donors without history of transfusion were tested negative with homemade MPHA method. Test results of MPHA employing homemade indicator cells showed excellent correlation with those of PSIFT (P=2.67x10-8). CONCLUSIONS: Homemade indicator cells we developed in this study were able to replace Dr. Shibata's indicator cells for the MPHA test.

Avaliação da prova de hemaglutinação passiva no diagnóstico da leptospirose humana em diversos períodos evolutivos da doença / Evaluation of passive hemagglutination test in the diagnosis of human leptospirosis in different evolutive periods of the illness

Foram realizadas provas de hemaglutinação passiva em amostras séricas de 189 pacientes com leptospirose, distribuídas quanto ao período decorrido após início dos sintomas. Os resultados foram comparados aos obtidos com a prova de referência, a soroaglutinação microscópica. A hemaglutinação passiva revelou, na primeira amostra examinada, maior número de resultados positivos, estatisticamente significante, a nível de 1%, no intervalo de 6 a 10 dias de doença, quando comparada à soroaglutinação. Nos demais intervalos, os valores diagnósticos das provas foram aceitos como iguais, Os resultados sugerem que a prova de hernaglutinação passiva pode ser empregada no acompanhamen to sorológico, como subsídio no estabelecimento do estágio da infecção leptospirótica (AU).

Avaliação da prova de hemaglutinação passiva no diagnóstico da leptospirose humana / Evaluation of passive hemagglutination test for human leptospirosis

A prova de hemaglutinação passiva foi avaliada em relação à soroaglutínação microscópica em 437 amostras de soros provenientes de 192 pacientes com quadro clínico sugestivo de leptospirose, internados no Hospital de Isolamento "Emílio Ribas", em São Paulo. A prova de hemaglutinação passiva apresentou associação significativa com a soroaglutinação microscópica, revelando-se, pois, uma prova alternativa de elevado valor diagnóstico (AU).

Imunodiagnóstico da leptospirose humana. 1. Antígeno polissacarídico para a prova de hemaglutinação passiva / Immunodiagnosis of human leptospirosis. 1. Polysaccharide antigen for passive hemagglutination test

Antígenos polissacarídicos F. foram extraídos de leptospiras dos sorotipos icterohaemorrhagiae e pato c, cuja relação proteína/carboidrato foi de 1,24 e 0,54, respectivamente. Sua especificidade sorológica foi avaliada na reação de hemaglutinação passiva para o diagnóstico da leptospirose humana. Para tanto, hemácias de carneiro previamente fixadas com glutaraldeído foram sensibilizadas com ambas as preparações. Esta prova foi experimentada em 91 soros obtidos de 35 pacientes com leptospirose, causada presumivelmente pelos sorotipos icterohae-morrhagiae, copenhageni, grippothyphosa e pomona. Verificou-se que ambas as frações F. comportaram-se de maneira semelhante. A reação de hemaglutinação passiva foi gênero específica, bastante sensível, positivando-se precocemente na fase aguda da doença. Foram salientadas as vantagens do emprego do antígeno F. patoc, por se tratar de sorotipo não patogênico (AU).

Imunodiagnóstico da leptospirose humana. 2. Estudo comparativo das reações de soroaglutinação microscópica, hemaglutinação passiva e imunofluorescência indireta / Immunodiagnosis of human leptospirosis. 2. Comparative study of microscopic agglutination passive hemagglutination and indirect immunofluorescence tests

Foram realizadas provas de hemaglutinação pa.ssiva e de imunefluorescêncía indireta em 98 soros obtidos de 38 pacientes com leptospirose causada presumivelmente pelos soro tipos icterohaemorrhagiae, copenhageni, grippotyphosa ou pomona. Hemácias de carneiro. sensibilizadas com antígeno polissacarfdico F" obtido de leptospira do. sorotípo patoc, foram utilizadas na hemaglutinação passiva. Para a prova de imunofluorescência indireta foram empregados antígenoa íntegros do. mesmo. sorotipo. Os resultados foram comparados com os obtidos através da prova de aglutinação microscópíca, na qual foram empregados .10 soro tipos patogênicos. As reações de hemaglutinação passiva e de imunofluorescência indireta mostraram ser gênero. específicas, sendo. que a primeira caracterizou-se por ser a mais indicada para o. diagnóstico. precoce da leptospirose humana aguda (AU).