A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

An assessment of methods used in the investigation of iron status: findings in a population of young British South Asian children.

AIMS: To assess the value of laboratory tests available for the investigation of iron status in a population of young British South Asian children. METHODS: Blood count, red cell distribution width (RDW), percentage hypochromic red cells (%hypo), concentrations of C-reactive protein (CRP), zinc protoporphyrin (ZPP), ferritin, soluble transferrin receptor, plasma iron measurements and incidence of deletional forms of α-thalassaemia were determined. RESULTS: Haemoglobin, mean cell haemoglobin (MCH), ferritin and CRP values classified iron status in 151/205 (73.6%) consecutive children aged 4-43 months. Fifty-four could not be classified: 12 were anaemic with findings, other than normal CRP values, indistinguishable from those with anaemia of inflammation and 42 were non-anaemic with reduced MCH values. All 42 had normal ferritin concentration and 8 of 36 successfully tested had deletional α-thalassaemia trait. Despite apparent iron sufficiency the RDW, %hypo and ZPP values of these 42 were not significantly different from the 32 children classified with iron-deficient erythropoiesis. The gene frequency of deletional α-thalassaemia trait in the entire group was 8.6%. CONCLUSIONS: Among 205 British South Asian children aged 4-43 months with high incidences of anaemia, iron deficiency, infection and α-thalassaemia, 151 (73.6%) were classified using haemoglobin, MCH, ferritin and CRP values. In 42 non-anaemic, iron-sufficient children with subnormal MCH values, that is with a phenotype of α-thalassaemia trait, RDW, %hypo and ZPP values did not differ significantly from those with iron-deficient erythropoiesis. Raised RDW, %hypo and ZPP values should be interpreted with caution in non-anaemic young British South Asian children with microcytosis.

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Optofluidic device for label-free cell classification from whole blood.

A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 µL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy.

Evaluation of derived Coulter red blood cell parameters for the assessment of iron deficiency in adults with congenital heart disease.

INTRODUCTION: The aim of our study was to evaluate derived red blood cell parameters in determining the presence of iron depletion and iron-deficient erythropoiesis, as states that precede iron deficiency anemia, in adults with congenital heart disease. METHODS: Eighty-eight adults who were diagnosed with congenital heart disease were divided into two groups (cyanotic and acyanotic). In both groups, congenital heart disease patients were then divided into three subgroups: with iron depletion, with iron-deficient erythropoiesis, and a control group. The following parameters were measured: complete blood count, reticulocytes, ferritin, soluble transferrin receptor, haptoglobin, lactate dehydrogenase, and calculated parameters: low hemoglobin density (LHD), red cell size factor (RSF), and microcytic anemia factor (MAF). RESULTS: Discriminant analysis indicated statistically significant differences in the first discriminant function: Function 1 - body iron, LHD, MAF, sTfR, and RSF (P < 0.001) in patients with acyanotic congenital heart disease and significant differences in both discriminant functions in patients with cyanotic congenital heart disease: Function 1 - body iron, soluble transferrin receptor, LHD, RSF, MAF, lactate dehydrogenase, and haptoglobin (P = 0.008) and Function 2 - reticulocytes (#), immature reticulocyte fraction and reticulocytes (%) (P = 0.049). CONCLUSIONS: Beside parameters that describe iron metabolism dynamics (body iron and soluble transferrin receptor), LHD, indicator of hypochromia, have the highest potential to differentiate and classify iron deficiency in patients with congenital heart disease.

Correlación y conversión entre valores de colinesterasa eritrocitaria medida con las técnicas de Michel y EQM® / Correlation and interconversion between erythrocyte cholinesterase values obtained by the Michel and the EQM® techniques

Introducción. Las técnicas para medir colinesterasa eritrocitaria son muchas y es útil tener un modelo matemático que permita interconvertir valores. Objetivo. A partir de datos autóctonos de valores de referencia de actividad colinesterásica medida por las técnicas de Michel y EQM® hallar una ecuación para transformar unas unidades en otras. Materiales y métodos. Diseño descriptivo, transversal y prospectivo, con sendas muestras representativas de poblaciones laborales adultas, de 18 a 75 años de edad, no expuestas a plaguicidas inhibidores de colinesterasa, vinculadas al Seguro Social y situadas en el valle de Aburrá y en el cercano oriente antioqueño (Antioquia, Colombia). Resultados. En las 827 personas, las pruebas eritrocitarias cuantitativas (Michel y EQM®) tienen coeficiente ®r¼ entre 0,666 y coeficiente R2 de 44 por ciento. La correlación es mayor en Aburrá que en Oriente. El modelo lineal en los 827 sujetos es: EQM® en U/g oxihemoglobina = 9,57509 U/g oxihemoglobina + 29,7914 (Michel en deltas de pH/hora), Michel en deltas de pH/hora = 0,33120 deltas de pH/hora + 0,01487 (EQM® en U/g oxihemoglobina). Tanto las intersecciones (coeficiente a) como las pendientes (coeficiente b) son significativas en el modelo. En las ecuaciones ajustadas, al excluir doce datos extremos (1,5 por ciento de 827), el coeficiente r sube de 0,666 a más de 0,718 y ellas son: EQM U/g oxihemoglobina = 8,18840 U/g oxihemoglobina + 31,3920 (Michel deltas de pH/hora), Michel deltas de pH/hora = 0,292510 deltas de pH/hora + 0,016101 (EQM U/g oxihemoglobina). Conclusión. Este sistema de ecuaciones lineales permitirá convertir unidades Michel (delta PH/hora) en unidades EQM (U/g oxihemoglobina) y viceversa, lo cual facilitará el trabajo en ambientes clínicos y epidemiológicos.

Introduction. Several techniques are available to measure red cell cholinesterase; therefore, evaluations with several methods provide a measure of concordance. Objective. An equation was formulated to transform native data of reference values to reference units of cholinesterase activity as measured by Michel and EQM® tests. Materials and methods. The experimental design was descriptive, transversal and prospective. The group sampled was a representative adult working population, aged 18-75, without previous exposure to cholinesterase inhibitors pesticides. The individuals were affiliated to the Social Security System and resided in Valle de Aburrá and Cercano Oriente Antioqueño (Antioquia Province, northwestern Colombia). Results. Of 827 individuals, quantitative erythrocytes (Michel y EQM®) tests exhibited “r” coefficients between 0.67 and R2 coefficient of 44%.,This indicated that one test explained the results in other test in 44% of the cases. The corelation was higher in Aburrá than in Oriente. The linear model for the 827 individuals was as follows: EQM U/g oxy-hemoglobin = 9.575 U/ g oxy-hemoglobin + 29.791 (Michel delta pH/hour). Michel delta pH/hr = 0.3312 delta pH/hour + 0.0149 (EQM U/g oxy-hemoglobin), where EQM® was expressed in U/g oxy-hemoglobin and Michel pH change/hr. Inter-sections (coefficient a) and inclines (coefficient b) were significant in this model. In the adjusted equations, after exclusion of 12 extreme data (1.5% of 827), the r coefficient increased from 0.67 to 0.72 The adjusted equations were as follows: EQM U/g oxy-hemoglobin = 8.1884 U/g oxy-hemoglobin + 31.3920 (Michel delta pH/hour); Michel delta pH/hr = 0.2925 delta pH/hr + 0.0161 (EQM U/g oxy-hemoglobin). Conclusion. This system of linear equations permitted the transformation of Michel (delta PH/ hr) units to EQM (U/g oxy-hemoglobin) units and vice versa. This will facilitate data comparisons by clinicians and epidemiologists who are using these methods of cholinesterase...

[Clinical signification of a modified dilution method (MDM) for blood loss assessment in the cases of nonvariceal upper gastrointestinal bleeding].

The aim of this study was to compare the modified dilution method (MDM) for in vivo bloodless assessment with the accepted in the clinical practice methods. We measured 148 blood samples from 134 patients with nonvariceal upper gastrointestinal bleeding (NUGB) and 21 blood samples from healthy persons as a negative control. In the randomized group of 53 patients with NUGB we compared accuracy of the blood loss determination by means of erythrocyte mass loss (estimated with MDM), Allgower-Burri index and American College of Surgeon Index (ACSI). The obtained results give us a reason to recommend a combination between American College of Surgeon classification for blood loss in patients with NUGB and a parallel measurement of the MDM values.

Regression-based reference limits: determination of sufficient sample size.

Regression analysis is the method of choice for the production of covariate-dependent reference limits. There are currently no recommendations on what sample size should be used when regression-based reference limits and confidence intervals are calculated. In this study we used Monte Carlo simulation to study a reference sample group of 374 age-dependent hemoglobin values. From this sample, 5000 random subsamples, with replacement, were constructed with 10-220 observations per sample. Regression analysis was used to estimate age-dependent 95% reference intervals for hemoglobin concentrations and erythrocyte counts. The maximum difference between mean values of the root mean square error and original values for hemoglobin was 0.05 g/L when the sample size was > or = 60. The parameter estimators and width of reference intervals changed negligibly from the values calculated from the original sample regardless of what sample size was used. SDs and CVs for these factors changed rapidly up to a sample size of 30; after that changes were smaller. The largest and smallest absolute differences in root mean square error and width of reference interval between sample values and values calculated from the original sample were also evaluated. As expected, differences were largest in small sample sizes, and as sample size increased differences decreased. To obtain appropriate reference limits and confidence intervals, we propose the following scheme: (a) check whether the assumptions of regression analysis can be fulfilled with/without transformation of data; (b) check that the value of v, which describes how the covariate value is situated in relation to both the mean value and the spread of the covariate values, does not exceed 0.1 at minimum and maximum covariate positions; and (c) if steps 1 and 2 can be accepted, the reference limits with confidence intervals can be produced by regression analysis, and the minimum acceptable sample size will be approximately 70.

Comparative assessment of phase contrast microscopy and Coulter counter measurements in localising the site of haematuria.

Haematuria is a common diagnostic problem in clinical practice and is seen in a large number of renal, urological and systemic disorders. The routine use of dipstick methods has increased the frequency of detection of microscopic haematuria. It is desirable to develop a simple, non-invasive, easily reproducible, quantitative technique that would help to determine the site of bleeding and thereby direct the patient either to the nephrologist or to the urologist. This study compares the accuracy of phase contrast microscopy with Coulter counter measurement of red cell distribution volumes as a means of distinguishing glomerular haematuria from non-glomerular haematuria. Of 20 patients with biopsy-proven glomerulonephritis, 13 (65%) were correctly identified by phase contrast microscopy and 19 (95%) by the Coulter counter method. In 18 patients with pathological conditions affecting the lower urinary tract phase contrast microscopy was correct in 6 (33%) and Coulter counter measurement in 17 (94%). Coulter counter measurement is the simpler and more accurate method of screening for the source of haematuria, but is inaccurate when the red cell count in the urine is less than 0.02 x 10(12).

The basics of blood cell counter.

The time has come when a traditional hematologist of a developing country needs to change his frame of mind from time consuming, error prone, not so precise manual methodologies to economical, safe and precise automated procedures. More important is his role as a guide and teacher for his juniors who are exposed to automated laboratories from the beginning of their residency. At this juncture, one needs to be thorough with the principles of instrumentation, different models available, their merits and demerits and how and where these fit with the requirements of the laboratory. The haematologists also must be aware that automation has its own problems, limitations, disadvantages and interfering elements. The article discusses the present state of art.