Applicability of papain solutions in immunohematology / Aplicabilidade de soluções de papaína em imuno-hematologia

ABSTRACT Objective: To compare the enzyme activity of different presentations of papain solution to validate in-house preparations. Methods: Two papain solutions were prepared, and the third presentation was a commercial solution. Tests were carried out with samples of red cells typed as weak RhD. Results: In-house prepared papain solutions showed similar enzyme reactivity, and statistically no differences compared to the enzyme activity of the commercial solution. Conclusion: Evaluating the cost-benefit ratio, the in-house prepared papain solutions present more economic advantages, and can be incorporated into immunohematological routines as a way to cope with periods of financial crisis and cost-containment policies.

RESUMO Objetivo: Comparar a atividade enzimática de diferentes apresentações de solução de papaína para validação de preparados in-house. Métodos: Foram preparadas duas soluções de papaína, e a terceira apresentação tratou-se de uma solução comercial. Os testes comparativos das reações enzimáticas foram realizados com amostras de hemácias tipadas como RhD fraco. Resultados: As soluções de papaína preparadas in-house apresentaram reatividade enzimática semelhante e estatisticamente sem diferenças em comparação com a atividade enzimática da solução comercial. Conclusão: Avaliando-se a relação entre custo e benefício, as soluções de papaína preparadas in-house são economicamente vantajosas, podendo ser incorporadas às rotinas imuno-hematológicas como forma de enfrentamento em períodos de crise financeira e em políticas de retenção de gastos.

Haematological values in a healthy adult population in Yaoundé, Cameroon

Background: Haematological values derived from local populations are useful in laboratories to improve diagnoses for local patients. In Cameroon, these data are not yet available. Moreover, there is great variation in baseline parameters pertaining to full blood cell count among medical laboratories.Objectives: This study aimed to determine values for the complete blood cell count of a healthy adult Cameroonian population for use in locally derived ranges in our medical laboratories.Methods: A cross-sectional study was conducted among blood donors attending three blood banks in Yaoundé from November 2015 to September 2016. We expected to obtain at least 120 venous blood samples from both men and women. Tests were performed for (1) HIV, (2) complete blood cell count, (3) hepatitis B virus, (4) malaria, (5) syphilis, (6) C-reactive protein and (7) hepatitis C virus.Results: We enrolled 294 healthy participants (161 men, 133 women) aged 18 to 55 years. The median haemoglobin concentration was 135 g/L in men and 114 g/L in women (p < 0.001). The median reticulocyte count was 60 × 109/L in men and 40 × 109/L in women (p < 0.001).Significant variation by sex was observed for the platelet count. The median white blood cell count was 4.1 × 109/L in men and 4.6 × 109/L in women (p = 0.008). Conclusion: This study provides locally derived ranges for complete blood cell and reticulocyte counts for a healthy adult population in Yaoundé, Cameroon. These results can be used pending larger studies

Reference Intervals of Hematology and Clinical Chemistry Analytes for 1-Year-Old Korean Children

BACKGROUND: Reference intervals need to be established according to age. We established reference intervals of hematology and chemistry from community-based healthy 1-yr-old children and analyzed their iron status according to the feeding methods during the first six months after birth. METHODS: A total of 887 children who received a medical check-up between 2010 and 2014 at Boramae Hospital (Seoul, Korea) were enrolled. A total of 534 children (247 boys and 287 girls) were enrolled as reference individuals after the exclusion of data obtained from children with suspected iron deficiency. Hematology and clinical chemistry analytes were measured, and the reference value of each analyte was estimated by using parametric (mean±2 SD) or nonparametric methods (2.5-97.5th percentile). Iron, total iron-binding capacity, and ferritin were measured, and transferrin saturation was calculated. RESULTS: As there were no differences in the mean values between boys and girls, we established the reference intervals for 1-yr-old children regardless of sex. The analysis of serum iron status according to feeding methods during the first six months revealed higher iron, ferritin, and transferrin saturation levels in children exclusively or mainly fed formula than in children exclusively or mainly fed breast milk. CONCLUSIONS: We established reference intervals of hematology and clinical chemistry analytes from community-based healthy children at one year of age. These reference intervals will be useful for interpreting results of medical check-ups at one year of age.

El hemograma: nueva clasificación y perspectivas / Hemogram: new classification and perspectives

A través del tiempo, el surgimiento y desarrollo de nuevos métodos analíticos se han visto unidos a la introducción de nuevas tecnologías y a su incorporación al trabajo diagnóstico e investigativo del laboratorio clínico. En los últimos años, los indicadores de exactitud y precisión de los procedimientos en el laboratorio de hematología se han elevado extraordinariamente gracias a la llegada de los sistemas automatizados de conteo y caracterización de células sanguíneas.Desde principios de la segunda mitad del pasado siglo, la continúa fabricación de diversos modelos de contadores hematológicos por parte de las compañías proveedoras y firmas comerciales, ha nutrido el mercado internacional de distintas series de estos equipos. La combinación de principios de detección tales como la impedancia eléctrica, la radiofrecuencia, las medidas de dispersión y absorción de la luz halógena o láser en diversos ángulos y la citometría de flujo, como bases del conteo y caracterización de las poblaciones celulares hemáticas, ha hecho posible el surgimiento de autoanalizadores hematológicos de mayor costo y complejidad.Como fruto de los avances de esta tecnología y su aplicación al laboratorio de hematología, el hemograma o biometría hemática, como indicación de primera línea en la evaluación clínica de los desórdenes y respuestas del sistema hematopoyético, es hoy día una de las pruebas más accesibles y solicitadas al laboratorio clínico.1,2Esta técnica ha sido objeto de infinidad de variaciones en aspectos tales como la forma automatizada o manual de su realización, el número de parámetros que la componen, la manera de interpretarlos y la elevación de los indicadores de exactitud y precisión de sus..

Avaliação do desempenho analítico do analisador hematológico Sysmex® XE-2100D / Performance evaluation of the Sysmex XE-2100D automated hematology analyzer

Introduction: The Sysmex® XE-2100D is a multiparameter hematology analyzer designed for hematology testing in samples with ethylenediamine tetraacetic acid (EDTA). Objectives: Considering the importance of this hematology analyzer for clinical and laboratory practice, the objective of this study was to evaluate its analytical performance, comparing the obtained results with quality specifications described in literature. Material and method: In the evaluation of analytical performance, according to recommendations of the document H26-A2 of the Clinical and Laboratory Standards Institute (CLSI), intra-run imprecision, inter-run imprecision, linearity, carryover, autosampler evaluation, clinical sensitivity of the atypical lymphocytes flag (n = 400 samples) were included, as well as the comparison between automated and manual leukocyte differential count (n = 400 samples), based on an adaptation of the document H20-A2 of CLSI. Results: Repeatability, reproducibility, linearity and carryover were satisfactory according to the manufacturer's specifications. The clinical sensitivity of the atypical lymphocytes flag showed efficiency, sensitivity and specificity of 92.5%, 65.2% and 94.1% respectively. The correlation coefficients between the automated and manual differential counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils were 0.991, 0.99, 0.872, 0.974 and 0.557, respectively. Conclusions: The results were in accordance with quality specifications described in literature, indicating reliability in Sysmex® XE-2100D. This fact ensures certainty to both laboratory professionals and medical staff. We conclude that the Sysmex® XE-2100D showed excellent analytical performance, and is useful to provide reliable hematology data...

Introdução: O Sysmex® XE-2100D é um analisador hematológico multiparamétrico destinado à realização de testes hematológicos em sangue anticoagulado com ácido etilenodiamino tetra-acético (EDTA). Objetivos: Considerando a sua importância na prática clínica e laboratorial, o objetivo deste estudo foi avaliar seu desempenho analítico, comparando os resultados obtidos com especificações de qualidade descritas na literatura. Material e método: Na avaliação de desempenho analítico, conforme recomendações do documento H26-A2 do Clinical and Laboratory Standards Institute (CLSI), foram incluídos ensaios de verificação da imprecisão intraensaio ou repetitividade, imprecisão entre ensaios ou reprodutibilidade, linearidade, carryover (arraste), avaliação do mecanismo homogeneizador de amostras, sensibilidade clínica do alerta morfológico (flag) de linfócitos atípicos (n = 400 amostras) e a comparação entre a contagem diferencial de leucócitos automatizada e a manual (n = 400 amostras), baseada em uma adaptação do documento H20-A2 do CLSI. Resultados: Os ensaios de verificação da repetitividade, reprodutibilidade, linearidade, carryover (arraste) foram satisfatórios conforme especificações do fabricante. O ensaio de sensibilidade clínica do alerta morfológico (flag) de linfócitos atípicos mostrou eficiência, sensibilidade e especificidade de 92,5%; 65,2% e 94,1% respectivamente. Os coeficientes de correlação entre as contagens diferenciais automatizadas e manuais de neutrófilos, linfócitos, monócitos, eosinófilos e basófilos foram de 0,991; 0,99; 0,872; 0,974 e 0,557 respectivamente...

Técnica de cuantificación de la agregación eritrocitaria por análisis digital de imágenes / Erythrocyte aggregation quantification technique using digital image analysis / Técnica de quantificagáo do agregado eritrocitário por análise digital de imagens

La caracterización de agregados eritrocitarios es importante para analizar las posibles alteraciones en la microcirculación observadas en ciertas patologías vasculares como la hipertensión y la diabetes. El objetivo de este trabajo fue estandarizar una técnica que pueda ser utilizada por cualquier operador con un equipamiento similar. Para ello se prepararon distintas suspensiones de glóbulos rojos de dadores sanos en plasma autólogo, que fueron observadas con un microscopio óptico invertido. Se registraron para su análisis las imágenes de los agregados con una cámara digital. Se realizaron los recuentos de células individuales, agregados de 2 a 4 células, agregados de 5 ó más células y amas (redes de agregados de gran tamaño). Se midió el perímetro y el área, obteniéndose un parámetro de forma (ASP) de cada agregado de 5 ó más células. Los resultados obtenidos permitieron estandarizar el protocolo de trabajo concluyendo que la dilución óptima de glóbulos rojos en plasma autólogo para esta técnica es 0,5%.

Characterization of erythrocyte aggregates is important in the analysis of the possible alterations observed in the microcirculation of certain vascular pathologies such as hypertension and diabetes. The objective of this work was to standardize a technique that can be used by any operator having similar equipment. For that purpose, different suspensions of red blood cells from healthy donors were prepared in autologous plasma and then observed with an inverted light microscope. The images of the aggregates were recorded with a digital camera in order to be later analyzed. Individual cell count was carried out, as well as 2 to 4 cell- aggregates, 5 or more cell- aggregate and amas (big aggregate networks). Measurement of perimeter and area of each of the aggregates made up of 5 or more cells was performed, getting a shape parameter (ASP). Due to the results obtained, this working protocol has been standardized and it can be concluded that the optimal dilution of red blood cells in autologous plasma is 0.5% for this particular technique.

A caracterizagáo de agregados eritrocitários é importante para analisar as possíveis alteragóes na microcirculagáo observadas em certas patologias vasculares como a hipertensáo e a diabetes. O objetivo deste trabalho foi padronizar uma técnica que possa ser utilizada por qualquer operador com um equipamento similar. Para isso foram preparadas diversas suspensóes de glóbulos vermelhos de doadores saudáveis em plasma autólogo, que foram observadas com um microscópio óptico invertido. Foram registradas para a sua análise as imagens dos agregados com uma camera digital. Realizaramse as recontagens de células individuais, agregados de 2 a 4 células, agregados de 5 ou mais células e amas (redes de agregados de grande tamanho). Foi medido o perímetro e a área, obtendo um parametro de forma (ASP) de cada agregado de 5 ou mais células. Os resultados obtidos permitiram padronizar o protocolo de trabalho concluindo que a diluigáo ótima de glóbulos vermelhos em plasma autólogo para esta técnica é de 0,5%.

Usefulness of Frozen-thawed-deglycerolized Red Blood Cells as Quality Control Materials for Red Blood Cell Deformability Test / 대한진단검사의학회지

BACKGROUND: The red blood cell (RBC) deformability test is a useful method for measuring the ability of RBCs to adapt their shape to the flow conditions. Using this test, several investigators have shown the relationship between RBC deformability and numerous clinical conditions. For the quality control (QC) of RBC deformability test, we evaluated whether frozen-thawed-deglycerolized RBCs can be used as QC materials. METHODS: Packed RBCs were frozen with 40% (wt/vol) glycerol and stored at -80degrees C for 3 months. For 10 different frozen RBC panels, RBCs were thawed, deglycerolized and stored at 4degrees C for 4 weeks. Using microfluidic ektacytometer, we measured RBC deformability of the thawed RBCs. The stability of thawed RBCs was tested once a day for 28 days of storage time and was analyzed by simple regression analysis. The precision of the test using thawed RBCs was analyzed for 7 days of storage time by calculation of CV values of intra-assay (10 measurements/assay) and between-day measurements. RESULTS: Frozen-thawed-deglycerolized RBCs were stable for 1 week. Within-run and between-day precisions of the RBC deformability test during 7 days of storage of thawed RBCs were 1.4-2.9%, and 1.9-2.8%, respectively. CONCLUSIONS: Frozen-thawed-deglycerolized RBCs used in RBC deformability test showed satisfactory within-run and between-run precisions and stability for one week after thawing, and may be used as QC materials for this test.

Impact of external haematology proficiency testing programme on quality of laboratories.

BACKGROUND & OBJECTIVE: A reliable and reproducible report from a laboratory needs internal quality control within the laboratory and participation in external proficiency testing programmes (EPTP). This study conducted at the Department of Haematology, All India Institute of Medical Sciences (AIIMS), New Delhi, which has been conducting an EPTP since 1992, was undertaken to assess the efficacy of this programme in improving the performance of participating laboratories in reporting test samples sent for Hb, total leucocyte count (TLC), reticulocyte count and assessment of peripheral blood smear (PBS). METHODS: The samples were prepared in our laboratory according to the International Standards Organization (ISO) guidelines. The performance of individual laboratories was assessed using robust Z score, which is an indicator of acceptability of the test result. RESULTS: An improvement in the overall percentage of laboratories with acceptable reports was seen during the study period. It has increased from 38,40,40 per cent in 1992 to 85, 90,94.7 per cent in 2006 for Hb, TLC, reticulocyte count, respectively. However, the results for peripheral smear assessment improved only marginally. INTERPRETATION & CONCLUSION: The external haematology proficiency testing programme run by our department for Hb, TLC, reticulocyte count, and peripheral blood smear assessment, has helped in improving the reporting standards of these parameters in Indian laboratories.

Temperature regulation and standardization practices of clinical laboratories in Karachi

To examine the temperature regulation and standardization practices of clinical laboratories in Karachi. Forty five clinical laboratories in Karachi were examined for observing the standard protocols for running a lab with particular reference to temperature regulation. A questionnaire to the effect was filled. Among the 45 labs included, the mean complete blood count performed per day was 52 +/- 47. Only 5 [11%] labs had a temperature reader. Thirty [66.7%] labs had an air-conditioner installed, of which only 24 were found in working condition. Maintenance of instruments was carried out every 67.5 +/- .30.6 days. The mean number of haemolyzed samples was 2.3 +/- 1.7. Control was available in 24[53.3%] labs, which was used daily in only 10 labs. Quantity of blood was the same in all the tubes in only 33[73.3%] labs. Commercial laboratories should be properly registered and their quality standardized

Problems on national and locale external quality assurance schema (NEQAS and LEQAS); based on Japanese experience.

The quality and stability of surveillance materials is a critical issue to perform good external quality assessment schema. In the case of nationwide surveillance, more stable materials are required than those in the case of locale external quality control schema. For locale surveillance, fresh blood, plasma or serum materials are prepared for hematology and chemistry tests. On the other hand, dried materials are used for the chemistry test and semi-fixed blood is used for hematology tests in the national surveillance. However to evaluate automated white cell differential in hematology, there has been no good materials. We therefore prepared a mixture of EDTA and ACD solution for our locale surveillance. It gave us excellent results on the differential when we used it within 3 days after blood collection.

Second national external quality assessment scheme in hematology: the Philippine experience.

The Philippine Council for Quality Assurance In Clinical Laboratories has conducted two National External Quality Assessment Schemes (NEQAS) in Hematology. The first survey was conducted in December 1999 and the second in August 2000, with 95 and 187 laboratories, using mostly automated analyzers, participating respectively. Control materials were distributed during a two-week period by human network, and analyzed over a six to eight week period. For the first survey, only 36 laboratories (38.0%) submitted results. Data was divided into 4 peer groups based on the manufacturer. Since most of the samples were hemolysed upon analysis, only WBC and HGB parameters were evaluated. No outliers were detected in each peer group after analysis by the 'Peer Group Mean and SDI' method. Using the clinical laboratory improvement act of 1988 proficiency testing criteria (CLIA'88), only 5 results (13.9%) were unsatisfactory for WBC, and all results were satisfactory for HGB. For the second survey, 87 laboratories (47%) responded. Data was divided into 5 peer groups. There were few incidents of sample deterioration. Although majority of the coefficient of variations were acceptable, about 23 (12.6%) participants showed abnormality in at least one parameter after analysis by the 'Peer Group Mean and SDI'. Using CLIA'88, 5 WBC (6.5%), 6 RBC (7.6%), 8 HGB (9.7%), 15 HCT (19.0%), and 7 PLT (8.0%) results were unsatisfactory. In summary, the first NEQAS study served as a pilot study. Valuable lessons were learned for the improvement of the second NEQAS. The second NEQAS study was marked by a much larger sample size and better results.

Icsh activities and Asia.

The International Council for Standardization in Haematology (ICSH), an international organization promoting international agreement on hematological testing, is now restructuring to strengthen its activities. In Asia, a diversity of testing methods exists and the resulting testing levels make it difficult to compare test results internationally among Asian countries. Fortunately, the ICSH is considering regionalizing its organization to 5 sub-societies to increase its activity, and we have been able to establish a new society, ICSH-Asia, under the ICSH umbrella.

Country reports from Japan: external quality assessment scheme in Japan.

Several external quality assessment schemes (EQAS) have been conducted in Japan. Results obtained from nation-scale EQAS reveal the current quality of laboratory testing in each laboratory. The largest nation-scale EQAS in Japan is that conducted by the Japan Medical Association. The numbers of participants and of items evaluated have increased in EQAS by JMA over its history of 32 years. Improvement in inter-laboratory differences has been observed for most items in EQAS in recent decades. In 1998, about 2,500 laboratories from throughout the country participated in this surveillance, and 47 items were evaluated. The coefficient of variations for the group of all participants was less than 5% for about one third of all test items. On the other hand, very high variations over 20% were observed for 6 items. Also, inter-method differences exist for many items, which may be or may not be related to matrix effects. Retrospective evaluation of all EQAS data suggests that there is still room for improvement in inter-laboratory differences.

Quality control in a manual and an automated leukocyte differential count.

Quality control (QC) has been introduced in laboratories, and QC surveys in leukocyte differential count to enhance quality have been performed by College of American Pathologists, Japanese Association of Medical Technologists, Osaka Medical Association and manufacturers. The results of QC survey in a manual leukocyte differential count indicated problems on the differentiation of segmented neutrophils and band neutrophils and the detection of pathological blood cells on blood smear. While the results of QC survey in an automated leukocyte differential count performed by same manufacturer with an automated blood cell counter were satisfactory, however, there was a difference in leukocyte differential cell counts among laboratories with other manufacturer's instruments because the synthetic blood material used in QC is an exclusive item for an instrument. It is necessary to further reeducate the medical technologists in order to improve morphological performance, and to standardize the synthetic blood material for compatibility with various automated blood cell counters.

Hematology laboratory standardization: a plan for harmonization in Asia.

Hematology laboratory is generally required in the hospital. At the macroscale, hematology laboratories have served a large number of population. In Asia, more than 3,000 million people are potentially to use the hematology laboratory service, particularly the complete blood count. Since 1970s, automated technology has been introduced to Asia and as years passed by, technology diversity is increasing. However, there are considerable number of hematology laboratories that have no automated machine. They are still relied on manual technology which is still variable in spectrophotometer for hemoglobin determination, centrifuge for hematocrit and diluting pipet for cell counting. In particular, blood smear preparation and interpretation are very difficult to control for standardization from person to person and laboratory to laboratory. Different methodology and a large population in the huge geographical area in Asia, the agreement of standard criteria is greatly important. This report has shown strategy and action plan to reach the goal of hematology laboratory standardization in Asia.

A diversity of blood smear examination system of Thai public hospital.

A good laboratory practice is the heart of clinical laboratory quality. One must establish a standard system in order to achieve the quality. However, standard system is not only the technical but also a state of the art. The socioeconomic and culture are the influence factors. At present, technique of reporting the blood smear examination is still a nation controversy. We surveyed the blood smear examination reporting system of the public hospital in Thailand. There were 77 hospitals participated in this study. A questionnaire comprised of 23 questions was distributed to 105 clinical laboratory staffs of the public health hospitals. Results showed that there was a diversity of blood smear examination reporting system. Generally, there were 2 ways of blood smear review. Every smeared slide and only abnormal slides were re-examined by the conventional technique. When an abnormal white blood cell blood picture was observed. The presence of either blast cell or atypical lymphocyte has clinical significance. A majority of laboratory would report in form "counting number of abnormal cell within 100% of the differential count". For other abnormalities of white blood cell; i.e. the neutrophils with toxic granules, most of laboratory reported as "presence". Interestingly, the red blood cell reporting system varied from laboratory to laboratory. There was a total of nine reporting patterns. Results indicated that pattern 4 (few or some, 1+,2+,3+,4+ are 5-10%, 11-25%, 26-50%, 51-75% and 51-100% cell/oil field, respectively) was the most popular one in reporting anisocytosis and poikilocytosis. However, the reporting pattern on red blood cell staining was difference. Pattern 4 and pattern 7 (grading by the size of central pallor) obtained a same popularity on the consideration of hypochromia. But the pattern 9 (few or some, 1+,2+,3+ are 0-1, 1-3, 4-6 and >6 cell/oil field, respectively) was the most frequently used in reporting of polychromasia. Fortunately we found that the reporting system for platelet was not complicated. A majority of laboratory chose the qualitative pattern (reporting as adequate, increased and decreased). Our study indicated that Thai clinical laboratory encounter a diversity of blood smear reporting system. Every clinical laboratory should pay a great attention to this circumstance. Since a good laboratory practice is a knot of the knot-bolt system of healthcare service, therefore, a nation standard system must be established in the near future.

Standardization on laboratory diagnosis of thalassemia and abnormal hemoglobin.

Thalassemia is one of the most common single gene disorders. The geographic distribution of thalassemia and abnormal hemoglobin has been known for many years. A worldwide significant spread of these abnormal genes, especially from Southeast Asia, occurred in the last two decades. This has resulted in a dramatic increase of Hb E disorders and various Southeast Asian thalassemia genotypes, which means that requests for hemoglobinopathy investigations are likely to increase in many laboratories worldwide. Hemoglobinopathy screening and diagnosis may need to be undertaken antenatally, neonatally and in certain hematological situations. The introduction of automation for hemoglobinopathy screening, including the automated cell counting and HPLC system, is an important advance in technology for hematology laboratories. The instruments need to be calibrated and standardized to get an accurate data for interpretation. Internal and external control samples are also needed. Combination of test results is usually required to achieve a proper diagnosis, which in turn, provide a self-check for each laboratory test.