Chronic granulomatous disease presenting with small bone osteomyelitis in a young child: A case report

Chronic granulomatous disease (CGD) is a life threatening inherited disorder with varied clinical presentations often characterized by recurrent bacterial and fungal infections along with widespread granulomatous tissue response. The disease results from phagocytic defects characterized by deficiencies in oxidative burst of neutrophils. Nitroblue tetrazolium reduction test (NBT) and Dihydrorhodamine (DHR) with PMA stimulation by flow cytometry are quick, simple, sensitive and specific laboratory tests that help establish early and reliable diagnosis of CGD with an overall improvement in survival and disease prognosis. We report a case of 2-year old child who presented with small bone osteomyelitis involving bilateral feet and was later diagnosed to have autosomal recessive CGD due to mutation in NCF1 gene.

Comparação entre duas metodologias para a realização do teste de redução do tetrazólio nitroazul (NBT) em caprinos / Comparison among two methodologys at realization nitroblue tetrazolium reduction test (NBT) in caprines

O teste de redução do tetrazólio nitroazul (NBT) é um dos métodos mais utilizados para a avaliação do metabolismo oxidativo dos neutrófilos. Porém, o custo por amostra e o tempo de utilização do corante são desvantagens do método. O objetivo deste trabalho foi comparar duas metodologias para realização do teste do NBT a fim de maximizar a sua utilização e diminuir o custo por amostra processada. Foram utilizadas 10 cabras, adultas clinicamente sadias e colhidas amostras de sangue por meio de venipunção jugular em tubos de 10mL sendo retirada uma alíquota de 500µL em tubo "eppendorff" contendo 2µL de heparina. Através desta alíquota foram realizadas três metodologias para o teste do NBT: A) técnica original Park & Good (1970); B) redução de 50% do volume do corante; C) redução de 50% e armazenamento do corante a -20°C por 180 dias. Foram confeccionados e corados esfregaços sanguíneos (May Grünwald-Giemsa) das amostras com as metodologias propostas e realizada contagem de 100 neutrófilos em microscopia óptica para determinar o percentual de neutrófilos reativos ao NBT. A análise estatística pelo método de Análise de Medidas Repetida (ANOVA) demonstrou diferenças significativas (P<0,05) entre os dois métodos e o original (A=14,4±4,6; B=1,9±1,4; C=1,1±0,9), porém apresentou alta correlação pelo teste de Spearman entre os métodos (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0,93 (AxC)). Conclui-se que o teste de Redução do Tetrazólio Nitroazul (NBT) segundo a metodologia descrita por Park & Good (1970) e a redução do corante em 50% do seu volume e a sua redução associada ao congelamento por 180 dias a -20ºC podem ser realizadas em caprinos, porém os valores devem ser comparados com as respectivas metodologias utilizadas.(AU)

The nitroblue tetrazolium reduction test (NBT) is one of the most used methods to evaluate the oxidative metabolism of neutrophils. However, the cost for each sample and dye's life-time are disadvantages of the method. This paper aim was to compare two NBT test methodologies in order to maximize its use and decrease the cost for each sample. It was made using 10 adult and healthy goats and blood samples were taken through venipuncion in jugular vein, using 10mL tubes, and then a 500µL sample was taken in eppendoff tubes with 2µL of heparin. Using this sample were made three methodologies for the NBT test: A) Park & Good (1970) original technique; B) dye reduction in 50% of the volume; C) 50% reduction and dye storage at -20°C for 180 days. It was made and dyed the blood smear (May Grünwald-Giemsa) from the samples, using the proposed methodologies and then made the counting of 100 neutrophils in optic microscopy, to determinate the percentage of NBT reactive neutrophils. The statistic analysis by the method of Repeated Measuring Analysis (ANOVA) evinced significant differences (P<0,05) between the two methods and the original (A=14.4±4.6; B=1.9±1.4; C=1.1±0.9), however it showed high correlation by the Spearman test between the methods (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0.93 (AxC)). It was concluded that the original Park & Good (1970) method and two methodologies for dye reduction in 50% and it's reduction associated with NBT dye freezing for 180 days can be used in caprines, but the values should be compared with their respective methodologies.(AU)

Comparação entre duas metodologias para a realização do teste de redução do tetrazólio nitroazul (NBT) em caprinos

Abstract: The nitroblue tetrazolium reduction test (NBT) is one of the most used methods to evaluate the oxidative metabolism of neutrophils. However, the cost for each sample and dye's life-time are disadvantages of the method. This paper aim was to compare two NBT test methodologies in order to maximize its use and decrease the cost for each sample. It was made using 10 adult and healthy goats and blood samples were taken through venipuncion in jugular vein, using 10mL tubes, and then a 500L sample was taken in eppendoff tubes with 2L of heparin. Using this sample were made three methodologies for the NBT test: A) Park & Good (1970) original technique; B) dye reduction in 50% of the volume; C) 50% reduction and dye storage at -20°C for 180 days. It was made and dyed the blood smear (May Grünwald-Giemsa) from the samples, using the proposed methodologies and then made the counting of 100 neutrophils in optic microscopy, to determinate the percentage of NBT reactive neutrophils. The statistic analysis by the method of Repeated Measuring Analysis (ANOVA) evinced significant differences (P 0,05) between the two methods and the original (A=14.4±4.6; B=1.9±1.4; C=1.1±0.9), however it showed high correlation by the Spearman test between the methods (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0.93 (AxC)). It was concluded that the original Park & Good (1970) method and two methodologies for dye reduction in 50% and it's reduction associated with NBT dye freezing for 180 days can be used in caprines, but the values should be compared with their respective methodologies.

Resumo: O teste de redução do tetrazólio nitroazul (NBT) é um dos métodos mais utilizados para a avaliação do metabolismo oxidativo dos neutrófilos. Porém, o custo por amostra e o tempo de utilização do corante são desvantagens do método. O objetivo deste trabalho foi comparar duas metodologias para realização do teste do NBT a fim de maximizar a sua utilização e diminuir o custo por amostra processada. Foram utilizadas 10 cabras, adultas clinicamente sadias e colhidas amostras de sangue por meio de venipunção jugular em tubos de 10mL sendo retirada uma alíquota de 500L em tubo "eppendorff" contendo 2L de heparina. Através desta alíquota foram realizadas três metodologias para o teste do NBT: A) técnica original Park & Good (1970); B) redução de 50% do volume do corante; C) redução de 50% e armazenamento do corante a -20°C por 180 dias. Foram confeccionados e corados esfregaços sanguíneos (May Grünwald-Giemsa) das amostras com as metodologias propostas e realizada contagem de 100 neutrófilos em microscopia óptica para determinar o percentual de neutrófilos reativos ao NBT. A análise estatística pelo método de Análise de Medidas Repetida (ANOVA) demonstrou diferenças significativas (P 0,05) entre os dois métodos e o original (A=14,4±4,6; B=1,9±1,4; C=1,1±0,9), porém apresentou alta correlação pelo teste de Spearman entre os métodos (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0,93 (AxC)). Conclui-se que o teste de Redução do Tetrazólio Nitroazul (NBT) segundo a metodologia descrita por Park & Good (1970) e a redução do corante em 50% do seu volume e a sua redução associada ao congelamento por 180 dias a -20ºC podem ser realizadas em caprinos, porém os valores devem ser comparados com as respectivas metodologias utilizadas.

Leucograma e metabolismo oxidativo de neutrófilos em cabras da raça Saanen nos períodos de gestação, parto e pós-parto / Leukocyte count and neutrophil oxidative burst in Saanen goats in the pregnancy, parturition and postpartum periods

O presente estudo teve como objetivo a avaliação do leucograma e do metabolismo oxidativo de neutrófilos em fêmeas caprinas da raça Saanen, nos períodos de gestação, parto e pós-parto. Amostras de sangue foram colhidas de 20 fêmeas nulíparas da raça Saanen, clinicamente sadias por venipunção jugular aos 49 (M1), 42 (M2), 35 (M3), 28 (M4), 21 (M5), 14 (M6), sete (M7), três (M8) dias antes do parto, no dia do parto (M9), três (M10) e sete (M11) dias após o parto, para a realização do leucograma e dosagens séricas de cortisol, estradiol e progesterona. A partir de 28 dias (M4) antes do parto até sete dias do pós-parto (M11) foram colhidas amostras de sangue para a avaliação do metabolismo oxidativo de neutrófilos por meio do teste de redução do tetrazólio nitroazul (NBT). Os resultados demonstraram que no dia do parto houve aumento nas concentrações séricas de cortisol e estradiol, e diminuição da progesterona, leucocitose por neutrofilia e desvio à esquerda leve, diminuição dos linfócitos, aumento da relação neutrófilo:linfócito, eosinopenia, monocitose e basofilia. No sétimo dia do pós-parto houve leucocitose por neutrofilia e aumento da relação neutrófilo:linfócito. Não houve nos períodos de gestação, parto e pós-parto alterações significativas no metabolismo oxidativo dos neutrófilos. Conclui-se que o parto determina elevação da concentração sérica de cortisol e estradiol, e diminuição da progesterona determinando quadro de leucocitose por neutrofilia e desvio à esquerda leve, com diminuição dos linfócitos, aumento da relação neutróflo:linfócito, eosinopenia, monocitose e basofilia. Ao sétimo dia do pós-parto há leucocitose por neutrofilia, aumento da relação neutrófilo:linfócito e do fibrinogênio. A gestação, o parto e o período do pós-parto não alteram o metabolismo oxidativo de neutrófilos avaliado por meio do teste de redução do NBT.

The purpose of this study was to evaluate the leukocyte count and the oxidative metabolism of neutrophil in Saanen goats during periods of pregnancy, parturition and postpartum. Were used 20 Saanen goats, clinically healthy and serologically negative for caprine arthritis encephalitis virus (CAEV). Blood samples were collected by jugular venipuncture 49 (M1), 42 (M2), 35 (M3), 28 (M4), 21 (M5), 14 (M6), seven (M7), three (M8) days before the parturition, on the day of birth (M9), three (M10) and seven (M11) days postpartum, for the leukocyte count, and serum for cortisol, estradiol and progesterone determination. From 28 days (M4) before parturition until seven days postpartum (M11) blood samples were collected for evaluation of oxidative metabolism of neutrophils by the nitroblue tetrazolium reduction test (NBT). The results showed that at parturition day there were an increase in cortisol and estradiol levels and a decrease in progesterone serum, neutrophilic leukocytosis and left shift slight, decrease of lymphocytes, increase in the neutrophil: lymphocyte, eosinopenia, monocytosis and basophilia. There was a neutrophilic leukocytosis and an increase in the neutrophil: lymphocyte on the seventh day postpartum. There were not significant alterations in oxidative metabolism of neutrophils during pregnancy, parturition and postpartum. It was concluded that parturition causes an elevation in cortisol and estradiol levels and a decrease in progesterone serum determining a neutrophilic leukocytosis and left shift slight, with a reduction of lymphocytes, increase in the neutrophil:lymphocyte, eosinopenia, monocytosis and basophilia. Neutrophilic leukocytosis, increase in the neutrophil: lymphocyte and fibrinogen are detected on the seventh day postpartum. Pregnancy, parturition and the postpartum do not change the oxidative metabolism of neutrophils evaluated by NBT reduction test.

Efeito inibidor do soro urêmico sobre o metabolismo oxidativo dos neutrófilos de cães / Inhibitory effect of uremic serum on neutrophils oxidative metabolism from dogs

Foi testada a hipótese de que, à semelhança do que ocorre em humanos, os componentes do soro urêmico inibem o metabolismo oxidativo dos neutrófilos de cães. Para isto, o sangue total de 10 cães foi incubado com soro homólogo urêmico e não urêmico e posteriormente comparado quanto à produção neutrofílica de superóxido estimada pelo método citoquímico de redução do tetrazólio nitroazul (NBT). A produção de superóxido gerada pelo metabolismo oxidativo dos neutrófilos tratados com soro urêmico apresentou significante redução (P<0,05) em relação aos tratados com plasma autólogo e homólogo com níveis normais de ureia. Concluiu-se que os componentes presentes no soro urêmico inibem ex vivo o metabolismo oxidativo dos neutrófilos de cães portadores de insuficiência renal e que, portanto, têm sua imunidade inata comprometida.

The hypothesis that the uremic compounds decrease the neutrophil oxidative metabolism in dogs, as occurs in humans was tested. Whole blood from 10 dogs was incubated with uremic and non-uremic homologous sera and the superoxide production by activated neutrophils was quantified by the nitroblue tetrazolium reduction test (NBT). The superoxide production generated by neutrophils in the presence of uremic serum was significantly decreased (P<0.05) in comparison with autologous and homologous sera with normal urea levels. It was concluded that the presence of components of uremic serum inhibits the ex vivo neutrophil oxidative metabolism in dogs, suggesting that, similarly to humans, dogs with renal failure may have the innate immunity response compromised.

Nitroblue Tetrazolium(NBT) Test as a Screening Test of Urinary Tract Infection in Children

PURPOSE: It has been reported that the Nitroblue Tetrazolium(NBT) test is more accurate than the urine pH, leukocyte esterase and nitrite test as a screening test of urinary tract infection (UTI). The purpose of this study is to compare the NBT test with other screening tests and evaluate the clinical usefulness of the NBT test as a screening test. METHODS: We selected 298 results out of the 304 urine cultures which were performed from March, 1999 to July, 1999 and compared them with screening tests such as NBT, urine pH, leukocyte esterase and nitrite tests. We interpreted those results as the urinary tract infection when the screening results were urine NBT(+), pH(>6.5), leukocyte esterase(>or=++) and nitrite(+). RESULTS: Urine NBT, pH and leukocyte esterase tests showed the statistical significance in comparison with the urine culture results(chi-square tests; P<0.001, <0.05, <0.001), while urine nitrite tests did not show statistical significance. As time passing, the sensitivity of 10 min, 30 min, 60 min NBT test was increased to 35%, 72%, 80% respectively but the sensitivity of urine pH and leukocyte esterase was as low as 33%, 16% respectively. But, the specificity of NBT test was reduced from 93% to 53% as the time went by, while the specificity of urine pH and leukocyte esterase tests were as high as each 79%, 96% respectively. Urine NBT tests at 10 min and 30 min showed a higher positive and negative predictive value than those of the other screening tests. CONCLUSION: Urine NBT test as a screening test for UTI was more accurate than the urine pH, nitrite and leukocyte esterase tests. But we think that we should develop a more quick and precise screening test in the future, because of the long time it requires to perform it.

Reducción del nitroazul de tetrazolio por leucocitos polimorfonucleares neutrófilos del recién nacido

Se realizó la prueba de reducción del nitroazul de tetrazolio (NBT) en granulocitos obtenidos de sangre del cordón umbilical, de 46 recién nacidos a término y de más de 2 500 g. El grupo control estuvo conformado por 80 adultos sanos, donantes de sangre voluntarios. Para valorar los efectos de diferentes niveles de complemento se utilizaron como fuentes de éste, suero de adulto normal, suero humano inactivado a 56 oC o suero fresco de ratón. El porcentaje de leucocitos polimorfonucleares neutrófilos (PMN) reductores fue significativamente inferior en el recién nacido (16,7 ± 3,6 %) en relación con el adulto (25,5 ± 4,2 %) cuando se utilizó suero humano inactivado como fuente de opsoninas, y no se hallaron diferencias significativas al utilizar suero fresco de ratón o suero adulto activo. Se destaca además el incremento de la capacidad reductora del leucocito PMN del recién nacido (58 y 95 %) de incremento al utilizar suero de adulto activo o ratón respectivamente contra el adulto (7,8 y 39,2 %) en iguales condiciones.

The test for reducing nitroblue tetrazolium in granulocytes obtained from blood of the umbilical cord of 46 at term newborns with more than 2 500 g was performed. The control group was composed of 80 sound adults, who were volunteer blood donors. Normal adult serum, inactivated human serum at 56 oC, or mouse's fresh serum were used as sources to assess the different complement levels. The percentage of reducing neutrophil polymorphonuclear leucocytes (PMN) was remarkably inferior in the newborn (16,7 ± 3,6 %) compared with the adult (25.5 ± 4,2 %) when inactivated human serum was used as a source of opsonins. No significant differences were found on utilizing mouse's fresh serum or active adult serum. It is also stressed the reducing capacity of the PMN leucocyte in the newborn (58 and 95 %) on using active adult serum or the mouse's fresh serum, respectively, against the adult (7.8 and 39.2 %) under equal conditions.

Effect of high glucose or high glucose and high insulin level on phagocytotic function and ultrastructure of activated rat alveolar macrophages / 中国病理生理杂志

AIM: The phagocytotic function and morphological changes of pulmonary alveolar macrophages (AM) of rat in high glucose or high glucose and high insulin conditions were studied. METHODS: AM were harvested from Wistar rats by bronchoalveolar lavage and were activated by Bacille Calmette - Guerin (BCG), interferon a - 2b (IFNa - 2b)or BCG and IFNa - 2b. The adherent rate, nitroblue retrazolium (NBT). reduction function and the concentrations of NO and TNF-a in culture rat AM were evaluated. The ultrastructure of AM was Observed by using transmission elects microscopy and scanning electron microscopy. RESULTS: In high glucose or high glucose glucose and high insulin conditions, the adherence of AM postponed(P＜ 0.01 ), NBT reduction of AM significantly decreased(P＜0.01); the volume of NO and TNF-a produced by AM after stimulation with BCG and IFNa-2b + BCG was significantly lower in high glucose groups than in the controls (P ＜ 0.01); their surface pseudopodia was reduced and shortened and the numbers of Golgi apparatus and rough endoplasmic reticulum decreased. CONCLUSIONS:The impaired phagocytotic function and ultrastructure of activated rat AM were much the same regardless of high glucose or high glucose and high insulin level in a short time. Diabetic subjects were predisposed to infection of the lung, which was frequently recurrent or even fatal.