Observation on the effect of modified buffy coat centrifugal canning method on improving the quality of platelet concentrates / 中国输血杂志

【Objective】 To evaluate the quality of platelet concentrates (PCs) prepared by traditional and modified buffy coat centrifugal canning methods, and to explore the effect of modified buffy coat centrifugal canning method on improving the quality of PCs. 【Methods】 The buffy coat centrifugal canning methods was divided into traditional group and modified group. In the traditional group, the buffy coat component bag and empty bag were directly combined layer by layer and vertically canned for light centrifugation. In the modified group, a foam spacer was added between the buffy coat component bag and the empty bag for light centrifugation layer by layer. The effects of the two groups of centrifugal canning methods on the preparation quality of PCs were observed. 【Results】 The platelet content in PCs prepared by the modified buffy coat centrifugal canning method was significantly higher than that in the traditional group, and the mixed amount of red blood cells was lower than that in the traditional group (P0.05), the qualified rates of platelet content and mixed red blood cell in the modified group were higher than those in the traditional group (P<0.05). 【Conclusion】 The modified centrifugal canning method with buffy coat can improve the quality of PCs, which is convenient and cheap, and is worth popularizing.

Quality evaluation of cryoprecipitate coagulation factors from buffy coat-derived plasma at different time periods / 中国输血杂志

【Objective】 To investigate the quality of cryoprecipitates prepared from buffy coat-derived plasma of fresh whole blood at room temperature 20℃-24℃ isolated at different time periods, explore the optimal time for preparing cryoprecipitates, so as to improve the utilization rate of blood. 【Methods】 A total of 250 bags of whole blood collected by CPDA-1 and stored at 20℃-24℃ from October 2020 to December 2020 were randomly selected as the experimental group, and divided into groups A1 (0-8 h), A2 (8-10 h), A3 (10-12 h), A4 (12-14 h) and A5 (14-16 h) (with 50 bags in each group) according to the preparation time point. The upper-buff-coat plasma was separated and quickly frozen as the source for cryoprecipitates. Meanwhile, another 50 bags of fresh frozen plasma prepared within 0-16h after routine storage at 2℃-6℃ were randomly selected as the control group (group B), which was used as the raw plasma to make cryoprecipitate. Coagulation factor Ⅷ (Ⅷ factor) and fibrinogen (FIB) were detected, and the effect of different preparation time and different storage temperature on the content of factor Ⅷ and FIB and the pass rate were compared. 【Results】 In comparison to the control group, the Ⅷ factor content of groups A4 and A5 was significantly decreased, and the differences between groups A4, A5 and B were statistically significant (P0.05). The Factor Ⅷ content ≥60 IU/ bag prepared from buffy coat-derived plasma accounted for 96.4% (1.5 U) in the experimental group. 【Conclusion】 The buffy coat-derived plasma prepared within 12 h at 20℃-24℃ is suitable for preparing 2 U cryoprecipitate coagulation factor, while that prepared within 12-16 h is suitable for preparing 1.5 U cryoprecipitate coagulation factor.

Determinación de la viabilidad de los concentrados plaquetarios obtenidos de capa leucocitaria (buffy coat) durante siete días de almacenamiento / Monitoring of the Viability of Platelet Concentrates Obtained from Buffy Coat during Seven Days of Storage / Determinação da viabilidade de concentrados de plaquetas obtidos de camada leucocitária (buffy coat) durante sete dias de armazenamento

Introducción: los concentrados plaquetarios (CPG) son hemocomponentes lábiles afectados por varios factores, desde el método de obtención hasta las condiciones de almacenamiento, que provocan una paulatina pérdida de funcionalidad, por lo que es necesario evaluar parámetros de calidad que garanticen la viabilidad de las plaquetas durante los días de almacenamiento, con el propósito de monitorear el mantenimiento de las características funcionales de las plaquetas. Materiales y métodos: estudio descriptivo transversal, con un tamaño muestral de 64 cpq, evaluados a los 3, 5 y 7 días de almacenamiento. Los parámetros monitoreados fueron físicos, de almacenamiento y porcentaje de la activación plaquetaria mediante la medición de P-selectina (cd62) por citometría de flujo. Se aplicó el estadístico de chi cuadrado, Anova de un factor, Kruskall-Wallis y correlación de Pearson. Resultados: existen diferencias significativas al séptimo día con relación al tercer y quinto día de almacenamiento, especialmente en el parámetro de formación de remolino plaquetario o swirling (p < 0.005) y agregados plaquetarios (p = 0.001). La activación plaquetaria aumentó significativamente (p = 0.001) desde el quinto día. Conclusiones: la viabilidad de los cpq difiere con los días de almacenamiento, por lo que es necesario evaluar pH, formación de remo-linos y agregados a todos los cpq antes de ser transfundidos como indicativos de activación plaquetaria y disminución de su funcionalidad

Introduction: Platelet concentrates (cpq) are labile blood components affected by several factors from the method of production to storage conditions that cause a gradual loss of functionality. For this reason, it is necessary to evaluate the platelet quality parameters that guarantee the viability during the storage days, with the purpose of monitoring the maintenance of the functional characteristics of the platelets. Materials and methods: This cross-sectional descriptive study had a sample size of 64 platelet concen-trates, evaluated at 3, 5, and 7 days of storage. The monitored parameters were the physical storage parameters and percentage of platelet activation by measuring P-selectin (cd62) via flow cytometry. The chi-square statistic, one-way Anova, Kruskal­Wallis test, and Pearson correlation were applied. Results: Significant differences were observed on the 7th day in relation to the 3rd and 5th day of storage, espe-cially in the swirling parameter (p < 0.005) and platelet aggregates (p = 0.001). The platelet activation increased significantly (p = 0.001) on the 5th day. Conclusions: Based on the findings of this study, the viability of the platelet concentrates differs with the days of storage. For this reason, it is necessary to evaluate the swirling, pH, and aggregates to all platelet concentrates before being transfused as an indication of platelet activation and decreased functionality

Introdução: os concentrados de plaquetas (cpq) são hemocomponentes lábeis afetados por diversos fato-res, desde o método de obtenção até as condições de armazenamento que ocasionam uma perda grada-tiva de funcionalidade, sendo necessário avaliar os parâmetros de qualidade que garantem a viabilidade das plaquetas ao longo dos dias de armazenamento, a fim de monitorar a manutenção das características funcionais das plaquetas. Materiais e métodos: estudo descritivo transversal, com tamanho de amostra de 64 cpq, avaliados aos três, cinco e sete dias de armazenamento. Os parâmetros monitorados foram físicos, de armazenamento e porcentagem de ativação plaquetária pela dosagem de P-selectin (cd62) por citometria de fluxo. Os testes estatísticos aplicados incluíram o teste qui-quadrado, anovade um fator, teste de Kruskall-Wallis e correlação de Pearson. Resultados: há diferenças significativas no sétimo dia em relação ao terceiro e quinto dia de armazenamento, principalmente no parâmetro formação de rede-moinhos ou swirling de plaquetas (p < 0.005) e agregados plaquetários (p = 0.001). A ativação plaquetária aumentou significativamente (p = 0.001) a partir do quinto dia. Conclusões: a viabilidade dos concentra-dos de plaquetas difere com os dias de armazenamento, por isso é necessário avaliar o pH, a formação de redemoinhos e agregados a todos os concentrados de plaquetas antes de serem transfundidos como indicativo de ativação plaquetária e diminuição de sua funcionalidade

An optimized BRCA1/2 next-generation sequencing for different clinical sample types / 부인종양

Comparison of optical microscopy and quantitative polymerase chain reaction for estimating parasitaemia in patients with kala-azar and modelling infectiousness to the vector Lutzomyia longipalpis

Currently, the only method for identifying infective hosts with Leishmania infantum to the vector Lutzomyia longipalpis is xenodiagnosis. More recently, quantitative polymerase chain reaction (qPCR) has been used to model human reservoir competence by assuming that detection of parasite DNA indicates the presence of viable parasites for infecting vectors. Since this assumption has not been proven, this study aimed to verify this hypothesis. The concentration of amastigotes in the peripheral blood of 30 patients with kala-azar was microscopically verified by leukoconcentration and was compared to qPCR estimates. Parasites were identified in 4.8 mL of peripheral blood from 67% of the patients, at a very low concentration (average 0.3 parasites/mL). However, qPCR showed 93% sensitivity and the estimated parasitaemia was over a thousand times greater, both in blood and plasma, with higher levels in plasma than in blood. Furthermore, the microscopic count of circulating parasites and the qPCR parasitaemia estimates were not mathematically compatible with the published proportions of infected sandflies in xenodiagnostic studies. These findings suggest that qPCR does not measure the concentration of circulating parasites, but rather measures DNA from other sites, and that blood might not be the main source of infection for vectors.

Comparison of Different Methods for Diagnosis of Malarial Parasites.

The study aims to evaluate the sensitivity and specificity of different methods for diagnosis of malarial infection. Total 200 blood samples were collected in ethylenediaminetetraacetic acid (EDTA) Vacutainer tube from clinically suspected malaria patients. Each sample was processed as thick and thin smear stained with Leishman’s stain for light microscopic examination, quantitative buffy coat test and rapid malarial antigen (HRP II and pLDH) test. The detection rate of malarial parasites by microscopic examination was 13.5%, quantitative buffy coat test was 18% and rapid malarial antigen (HRP II and pLDH) test was 20%. Thus, findings of microscopic examination must be compared with other more sensitive methods for confirmation of malaria. This will help early detection, proper diagnosis and treatment of malaria.

Usefulness of a centrifuged buffy coat smear examination for diagnosis of malaria.

Purpose: Malaria continues to be a global public health challenge. Microscopic examination of peripheral blood smear (PBS) is the standard method for malaria diagnosis, which is easily available and has low cost but its reliability is questionable at low level of parasitaemia. The present study was undertaken to assess the usefulness of a modifi ed centrifuged buffy coat smear (CBCS) technique for diagnosis of malaria and to compare it with conventional PBS examination and antigen detection test. Materials and Methods: The study was carried out over a 6-month period from July to December 2011. Blood samples (2–3 ml per patient) collected in EDTAvials from patients with a clinical suspicion of malaria were subjected to all three tests, that is PBS, CBCS and antigen test and results were compared with antigen test as the gold standard. Result: Of 1655 samples received, 394 (23.8%) samples were positive for infection with malaria parasites. All the three tests detected malaria infection equally in 279 samples, and gave varied results in the remaining 115 samples. Addition of centrifugation (i.e. CBCS) to the conventional method of PBS enabled detection of 80 more cases of plasmodia infection, especially (43, 53.7%) at low levels of parasitaemia (<200 parasites/μl). While both PBS and CBCS had excellent specifi city (99.7% and 99.2%, respectively), PBS examination had low sensitivity (72.9%) in detecting malaria parasites in comparison to CBCS. The sensitivity of CBCS in detecting malaria parasites was 91.9%. Conclusion: The development of easy, rapid and accurate tests for the reliable detection of plasmodia infection is highly desirable. The CBCS technique fulfi ls most of these criteria and may be adopted for rapid and reliable diagnosis of malaria in resource-limited settings.

Incidence, severity, prognostic significance of thrombocytopenia in malaria.

Background: Malaria is an infectious disease caused by plasmodium parasite. P. falciparum account for majority of morbidity and mortality. Thrombocytopenia and anaemia are the most frequently associated hematological complications in malaria. The low platelet count together with acute febrile syndrome emerged as the strongest predictor of malaria a finding that is frequent and present even before anemia and splenomegaly sets in. Severe thrombocytopenia is a good predictor of poor prognosis than mild and moderate thrombocytopenia. The aim is to study the incidence, severity, prognostic significance of thrombocytopenia in malaria. Methods: This was an observational and prospective study. The study enrolled 100 patients with thrombocytopenia and fever who were proven to have malaria either by peripheral smear or Quantitative Buffy Coat (QBC) test or malarial antigen assay were included in the study and patients with thrombocytopenia due to other causes were excluded from the study. Platelet count was estimated on a fully automated quantitative analyzer. All the 100 patients were followed during the hospital stay and upto discharge or till the outcome. Results: The incidence of thrombocytopenia was 73% indicating a common association in malaria. Complicated malaria was observed in 58.80% of P. falciparum infection whereas 66% of P. vivax infection was associated with uncomplicated malaria. Severe thrombocytopenia showed positive correlation with severity of malaria. Thrombocytopenic patients with effective anti-malarial treatment showed 95.90% recovery and 3 patients 4.10% had mortality. Patients with severe thrombocytopenia were 8.5 times more likely to have complicated malaria with P <0.001 according to student „t‟ test. Conclusion: Thrombocytopenia is the most common hematological finding in malaria. Severe thrombocytopenia showed positive correlation with complicated malaria and a good predictor of poor prognosis. Patients with classical malarial fever and thrombocytopenia who were negative for malaria parasite were not included in the study.

Citoinclusão de capa leucocitária e medula óssea de cães: padronização da técnica / Cell block of canine buffy coat and bone marrow in paraffin blocks: technique standardization

A técnica de citoinclusão é amplamente utilizada e reconhecida por profissionais da área da saúde, em particular médicos patologistas, porém as informações sobre a aplicação desse método em medicina veterinária ainda são raras. Análises citológicas de medula óssea e da capa leucocitária (porção do sangue formada por concentrados de leucócitos) são amplamente utilizadas no diagnóstico de patologias de origem neoplásica e não neoplásica e de agentes infecciosos. Embora a importância do uso da técnica seja amplamente reconhecida, não há registro da utilização de amostras citológicas de medula óssea e capa leucocitária na confecção de citoinclusões em blocos de parafina, como meio de diagnóstico no segmento médico veterinário. Assim, este trabalho tem como objetivo elaborar um protocolo de citoinclusão em parafina para amostras citológicas de medula óssea e capa leucocitária de cães. Foram submetidas à técnica de citoinclusão 110 amostras de capa leucocitária e 44 de medula óssea de cães portadores ou não de enfermidade sistêmica, sendo que em 68% destas, tanto para a capa leucocitária quanto para a medula óssea, as amostras se mostraram viáveis. A utilização do álcool 95% como fixador e de etapas no processamento histológico de 20 minutos (álcool absoluto, xilol e parafina, três banhos cada) foi crucial para a qualidade dos cortes histológicos e para a análise microscópica dos espécimes corados pela hematoxilina-eosina. A separação mecânica da capa leucocitária e a centrifugação do aspirado de medula óssea foram eficientes e de baixo custo no preparo das citoinclusões. Ressalta-se a importância na padronização da técnica de citoinclusão, em particular para amostras de capa leucocitária e medula óssea, visando à obtenção de espécimes de qualidade independentemente das limitações de equipamentos...

The cell block technique is widely used and recognized by health professionals, but lacks in information regarding the specific contribution of this method to veterinary medicine. The cytology of bone marrow and buffy coat (cellular portion of the hole blood) are widely used in the diagnosis of neoplastic and non-neoplastic diseases, and also for the detection of infectious agents. Although the importance of these samples is widely recognized, there is no information about the use of buffy coat and bone marrow samples in the cell block procedure among the research material used for this paper. This work aims for the cell block standardization for canine buffy coat and bone marrow samples. We collected 110 buffy coat samples and 44 bone marrow samples for the cell block preparation, and 68.2% of buffy coat and bone marrow proved to be viable at the end of the procedure. The 95% ethanol fixatives along with the 20 minute processing steps (absolute ethanol, xilol and paraffin, 3 of each) were crucial for the quality of the material both in microtomy and optical microscopy. Mechanical separation of the buffy coat proved to be easy and cheap and was used to compose the cell block technique. In this research we emphasized the importance of cell block standardization in order to develop and easy, inexpensive and reproducible method, regardless of any of the professionals' limitations...

Platelet Additive Solutions / 대한수혈학회지

Storage of platelet concentrates in platelet additive solution (PAS) with plasma removal has many advantages, including reduction of allergic reactions, contributing to the available plasma pool for fractionation or transfusion, and employment of pathogen reduction technology. In order to decrease platelet activation for improvement of in vivo viability, PAS should be designed for optimization of aerobic metabolism using compounds such as glucose, acetate, citrate, phosphate, and electrolytes. After a thorough discussion, particularly on the efficacy and regulations, use of the buffy coat method as well as application of a new generation of PAS may likely be the future direction of platelet storage in Korea.

Use of buffy coat thick films in detecting malaria parasites in patients with negative conventional thick films

Objective: To determine the frequency of malaria parasite detection from the buffy coat blood ilms by using capillary tube in falciparum malaria patients with negative conventional thick ilms. Methods: Thirty six uncomplicated falciparum malaria patients confirmed by conventional thick and thin films were included in the study. The patients were treated with artemisinin combination therapy at Hospital for Tropical Diseases, Bangkok, Thailand for 28 day. Fingerpricks for conventional blood films were conducted every 6 hours until negative parasitemia, then daily fingerpricks for parasite checks were conducted until the patients were discharged from hospital. Blood samples were also concurrently collected in 3 heparinized capillary tubes at the same time of fingerpricks for conventional blood films when the prior parasitemia was negative on thin films and parasitemia was lower than 50 parasites/200 white blood cells by thick film. The first negative conventional thick films were compared with buffy coat thick films for parasite identification.Results:Out of 36 patients with thick films showing negative for asexual forms of parasites, buffy coat films could detect remaining 10 patients (27.8%) with asexual forms of Plasmodium falciparum. Conclusions: The study shows that buffy coat thick films are useful and can detect malarial parasites in 27.8% of patients whose conventional thick films show negative parasitemia.

Comparison of cytokine levels and metabolic parameters of stored platelet concentrates of the Fundação Hemominas, Belo Horizonte, Brazil

BACKGROUND: Prolonged storage of platelets could improve availability and logistical management and decrease wastage. Immunobiochemical methods can be used to guarantee the quality of platelets after prolonged storage. OBJECTIVE: The aim of this study was to compare storage-related changes in buffy coat-derived platelet concentrations versus platelet-rich plasma. METHODS: Units of whole blood were drawn using a quadruple-bag blood container system. Platelet-rich plasma and buffy coat prepared from whole blood following standard methods were stored for 9 days. During this period test samples were aseptically collected for analysis on Days 1, 2, 3, 5, 7 and 9. RESULTS: The highest CD42b expression was greater than 95%. The percentage of CD62p was significantly lower than the CD42b expression. The pH remained fairly stable during storage. Measurement of pO2 and pCO2 showed that oxygen levels were significantly higher than carbon dioxide levels. There were no significant differences in bicarbonate levels, glucose consumption and lactate production between the groups. The swirling effect with platelet-rich plasma samples decreased after 5 days of storage and after 7 days of storage for buffy coat samples. There was a significant twenty-fold increase in the mean IL-1β after 5 days of storage for both groups. Slight increases in IL-6 and IL-8 levels were seen at 5 days. CONCLUSION: The quality of platelet concentrates remained acceptable during 7 days of storage in respect to the swirling effect, pH and platelet activation. There were no significant differences between buffy coat-derived platelets and platelet-rich plasma in this study.

Comparative Study of Peripheral Blood Smear and Quantitative Buffy Coat in Malaria Diagnosis.

A rapid test for diagnosis of malaria based on acridine orange staining of centrifuged blood samples in a microhaematocrit tube (QBC) was compared with Leishman stained thin peripheral blood smear in 287 samples. Malaria was diagnosed in 44 patients by Leishman staining technique and in 65 patients by QBC method. The QBC method allowed detection of an additional 21 cases. Thus the prevalence rate of malaria during the study was 22.65%. In 222 Patients who were negative by the QBC technique, the Leishman stained smears were also negative for malarial parasite. Although QBC method was superior to the smear for malarial parasite detection, species identification was difficult by this technique. The QBC method provides a reliable, quick, easily mastered, accurate method for diagnosis of malaria. The QBC system can also be used in the diagnosis of other parasitic diseases from blood (Filariasis). However, Leishman stained thin blood film still appear superior for species identification.

Sensibilidad y Especificidad de la Procalcitonina y Tinción de Gram de Buffy Coat para el Diagnóstico Temprano de Sepsis en Pacientes Pediátricos / Sensitivity and specificity of procalcitonin and gram stain of buffy coat for early diagnosis of sepsis in pediatric patients

Objetivos: Determinar la sensibilidad y especificidad de la procalcitonina y tinción gram de Buffy coat en el diagnóstico temprano de sepsis. Métodos: Estudio descriptivo de corte transversal, en el que se estudiaron 41 pacientes menores de 5 años que ingresaron con el diagnóstico presuntivo de sepsis al Hospital del Niñ@ Manuel Ascencio Villarroel entre octubre de 2008 a Febrero de 2009. Se midieron procalcitonina, proteína C reactiva, biometría hemática y se realizaron hemocultivo y tinción de gram de Buffy coat. Se calculo la sensibilidad, especificidad y valores predictivos de los marcadores de infección estudiados. Se consideró como criterio referencia el cumplimiento de la definición operacional de sepsis. Resultados: La procalcitonina mostró una sensibilidad de 96,77% y especificidad del 40%. La proteína C reactiva tuvo una sensibilidad de 51,61%. La tinción gram de Buffy coat en la que se observó tanto cocos gram positivos intra y extracelulares, presentó una sensibilidad de 100% y 0% de especificidad, mientras que si se considera sólo bacterias intracelulares su especificidad aumenta a 80% y la sensibilidad disminuye a 35%, teniendo mejor valor predictivo positivo de 84,62%. Conclusiones: La procalcitonina es un marcador de mayor especificidad que la proteína C reactiva en la detección de sepsis temprana. La tinción gram de buffy coat requiere de mayor estudio para su validación.

Objectives: To determine the sensitivity and specificity of procalcitonin and Buffy coat Gram stain in the early diagnosis of sepsis. Methods: Cross sectional study in which 41 patients were studied under 5 years admitted with a presumptive diagnosis of sepsis at Hospital Manuel Ascencio Niñ@ Villarroel from October 2008 to February 2009. We measured procalcitonin, CRP, blood count and blood culture were gram stain and Buffy coat. We calculate the sensitivity, specificity and predictive values of markers studied.The criterion reference compliance with the operational definition of sepsis. Results: The procalcitonin showed a sensitivity of 96.77% and a specificity of 40%. C-reactive protein had a sensitivity of 51.61%. The Buffy coat Gram stain was observed in both gram-positive cocci intra-and extracellular, had a sensitivity of 100% and 0% specificity, while considering only its specific intracellular bacteria increased to 80% and the sensitivity decreases 35%, having a better positive predictive value of 84.62%. Conclusions: Procalcitonin is a marker of greater specificity than CRP in the detection of early sepsis. Gram staining of Buffy coat requires further study for validation.

Feasible choices in diagnostic methods of malaria .

Background & objectives: The present study was undertaken to find out a new easy method in the diagnosis of malaria by centrifuged buffy coat smear, which was found to be a feasible and reasonable procedure. Methods: Blood samples collected from 120 patients suspected of malaria were subjected to all three diagnostic modalities—peripheral blood smear (PS), centrifuged buffy coat smear (CBCS) and antigen detection test using pLDH and aldolase (AG). Results: The results of various methods were compared. It was seen that addition of centrifugation (i.e. CBCS) to conventional method of PS (i.e. thick and thin smears) improved its sensitivity from 85 to 93.3%. Antigen detection and CBCS were found superior to PS in sensitivity. CBCS gives combined sensitivity and specificity of both antigen and PS. Conclusion: CBCS is as sensitive as antigen test and as specific as PS in species identification. It is a reasonable and feasible procedure too.

Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples.

Purpose: Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target. Material and Methods: A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid. Results: Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community. Conclusions: The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Bone Marrow Cellularity Measurement by Myelocrit / 대한진단검사의학회지

BACKGROUND: The bone marrow (BM) cellularity has been used as a major index for the evaluation of hematologic diseases and malignancies. However, the microscopic evaluation lacks objectivity because of considerable variations among different observers. We measured myelocrit cellularity as an objective method to evaluate the cellularity. METHODS: Between November 2007 and January 2008, 489 consecutive BM aspirates including 25 cases of AML D7 marrow (7 days after initiation of chemotherapy) were examined. The conventional BM cellularity was evaluated using BM needle biopsies after hematoxylin-eosin stain. EDTA-anticoagulated BM aspirates were put into the Wintrobe tubes, centrifuged and the thickness of 5 layers from the bottom was measured: RBC, buffy coat (BC), plasma, BM particle, and fat layers. The myelocrit cellularity was defined as the ratio of BC to the BC plus fat layers. RESULTS: Both of the thickness of BC layer (r=0.721, P=0.000) and the myelocrit cellularity (r=0.735, P=0.000) correlated well with the conventional cellularity. However, the AML D7 BM cellularity correlated with BC layer (r=0.589, P=0.002), but not with the myelocrit cellularity (r=0.281, P=0.231). The cellularity of the BM other than AML D7 marrow showed a better correlation with the myelocrit cellularity (r=0.826, P=0.000) than the BC layer (r=0.713, P=0.000). CONCLUSIONS: The myelocrit is a simple, reproducible and objective method to determine the BM cellularity. For accurate assessment of BM cellularity, measurement of the thickness of BC layer in AML D7 BM and of the myelocrit cellularity in other BM samples has better be used.

Quality Assessment of Red Blood Cells, Pooled Platelets and Frozen Plasma Processed by the Buffy Coat Method / 대한수혈학회지

BACKGROUND: Buffy coat method is one of the blood components processing methods widely used in many countries including Europe and Canada. For the first time in Korea, we evaluated the qualities of blood components manufactured by buffy coat method. METHODS: We collected 400 mL whole bloods using the quadruple top and bottom blood bag from thirty-five donors. Whole bloods were processed into leukoreduced RBC, leukoreduced pooled platelet, and 24 hr frozen plasma by the buffy coat method with blood bags and instruments of Fenwal and Fresenius. The qualities of each blood component were analyzed at each scheduled day, and compared with the standard guidelines of quality control in Korean Red Cross. RESULTS: The volume and hemoglobin of RBCs were lower than the standard guidelines. Comparing with the standard of apheresis platelets, leukoreduced pooled platelets showed higher total platelet yield with the median 3.70x10(11)/unit. Frozen plasma showed increased volume recovery than the standard guideline, but the activity of factor VIII at Day 35 was decreased to 0.66+/-0.14 IU/mL. CONCLUSION: We have found that the yields of pooled platelet and the frozen plasma processed by buffy coat method were higher than the standard guidelines. To introduce the buffy coat method to routine blood component separation process in Korea, further evaluations about the cost-effectiveness of buffy coat method are required.

Assessment of DNA Viability in Long Term-Stored Buffy Coat Species for the Korean Multicenter Cancer Cohort / 예방의학회지

OBJECTIVES: Peripheral blood-buffy coat fractions (N = 14, 956) have been stored at -70degrees C in the headquarter of the Korean Multicenter Cancer Cohort (KMCC), since 1993. To study the future molecular etiology of cancers using specimens of the cohort, properly stored specimens are necessary. Therefore, the DNA-viability of the buffy coat samples was investigated. METHODS: Buffy coat fraction samples were randomly selected from various collection areas and years (N = 100). The DNA viability was evaluate from the UV-absorbent ratios at 260/280nm and the PCR for beta-globin was performed with genomic DNA isolated from the buffy coat. RESULTS: PCR products were obtained from 85 and 98% of the C and H area-samples, respectively, using 50 or 100mul of the buffy coat. There were significant differences in the yields of the PCR-amplifications from the C and H areas (p < 0.05), which was due to differences in the homogenization of the buffy coat fractions available as aliquots. The PCR-products were obtained from all of the samples (N = 7) stored at the C area-local center, but the other aliquots stored at the headquarter were not PCR-amplified. Therefore, the PCR products in almost all the samples, even including the DNA-degraded samples, were obtained. In addition, an improvement in the DNA isolation, i.e. approx. 1.6 fold, was found after using extra RBC lysis buffer. CONCLUSIONS: PCR products for beta-globin were obtained from nearly all of the samples. The regional differences in the PCR amplifications were thought to have originated from the different sample-preparation and homogenization performance. Therefore, the long term-stored buffy coat species at the KMCC can be used for future molecular studies.