Manual Microscopy Research Articles

Comparison on the results of Mindray EH-2080B fully automatic urinary sediment analysis system made in China and manual microscopy

Comparison on the results of Mindray EH-2080B fully automatic urinary sediment analysis system made in China and manual microscopy

Temperature dependent embryonic development of Trichuris suis eggs in a medicinal raw material

The therapeutic potential of infective pig whipworm eggs, Trichuris suis ova (TSO), is currently tested in several clinical trials on immune-mediated diseases. This paper studied the embryonic development of TSO in a medicinal raw product, where the parasite eggs were suspended in sulphuric acid (pH1). Unembryonated T. suis egg batches were stored at 5, 10, 15, 20, 25, 30, and 40°C (±1°C) and examined at 2, 4, 8, and 14 weeks. Subsequently, sub-batches from each temperature were allowed to embryonate for additional 14 weeks at 25°C, and selected samples were tested for infectivity in Göttingen minipigs.Both male and female pigs were used to evaluate eventual gender specific infectivity. Storage at 30°C up to 14 weeks and subsequent embryonation for 14 weeks at 25°C did not significantly reduce the overall larval establishment in minipigs, as compared to storage at 5°C and subsequent embryonation at 25°C. As marked impairment of egg development was observed during storage at 40°C, a second set of unembryonated egg batches were incubated at 30, 32, 34, 36, 38, and 40°C (±1°C) for 1–8 weeks. The development of the eggs was repeatedly examined by manual light microscopy, multispectral analysis (OvaSpec), and an egg hatching assay prior to the final testing in minipigs (Trial 1). These methods showed that the development started earlier at higher temperatures, but the long-term storage at higher temperature affected the egg development. The present study further documents tolerance of the TSO to storage at temperature 5–15°C, at which temperature development of larvae is not initiated.

High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells

Chlamydiae are obligate intracellular bacteria developing in an intracytoplasmic niche, the inclusion. Chlamydia growth measurement by inclusion counting is a key task in the development of novel antichlamydial antibiotics and in vaccine studies. Most of the current counting methods rely on the immunofluorescent staining of the inclusions and either manual or automatic microscopy detection and enumeration. The manual method is highly labor intensive, while the automatic methods are either medium-throughput or require automatic microscopy. The sensitive and specific PCR technology could be an effective method for growth related chlamydial DNA detection; however the currently described PCR approaches have a major limitation, the requirement of purification of DNA or RNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of Chlamydia trachomatis DNA directly from the infected HeLa cells. With our method we were able to detect the bacterial growth in a 4 log scale (multiplicity of infection (MOI): 64 to 0.0039), with high correlation between the biological and technical replicates. As a further proof of the method, we applied the direct qPCR for antibiotic minimum inhibitory concentration (MIC) measurements. The measured MICs of moxifloxacin, tetracycline, clarithromycin and compound PCC00213 were 0.031μg/ml, 0.031μg/ml, 0.0039μg/ml and 6.2μg/ml respectively, identical or close to the already published MIC values. Our direct qPCR method for chlamydial growth and antibiotic MIC determination is less time-consuming, more objective and more sensitive than the currently applied manual or automatic fluorescent microscopy- based methods.

Performance Evaluation of Three URiSCAN Devices for Routine Urinalysis.

This study compares the diagnostic performance (in routine urinalysis) of three URiSCAN devices and three Roche analyzers to manual microscopy and quantitative assays. We analyzed eight dipstick tests using three URiSCAN devices. The results were compared to those of the tests performed using three Roche analyzers. The results of leukocyte and erythrocyte screens were compared to those obtained using manual microscopy. Protein, glucose, pH, and specific gravity (SG) assays performed on the URiSCAN devices were compared with the results of corresponding quantitative assays. The rates of agreement within one grade difference were found to be more than 94.3%. When compared with manual microscopy, the Optima provided better diagnostic performance for the detection of leukocytes compared with the Urisys 1100. Compared to the Urisys 2400, the Super plus provided better diagnostic performance with regard to both leukocytes and erythrocytes. There was good correlation between the three URiSCAN devices and each quantitative assay, except for SG detection. There were well correlated results between those of the three URiSCAN devices and those obtained using the corresponding Roche analyzers, quantitative assays, and manual microscopy. URiSCAN series devices are therefore suitable for routine urinalysis in clinical laboratories.

Clustered Red Blood Cells Splitting via Boundary Analysis in Microscopic Thin Blood Smear Digital Images

Clustered Red Blood Cells are observed very frequently in the thin blood smear digital images. Separating clustered Red Blood Cells from the single Red Blood Cells and splitting of clustered Red Blood Cells into single Red Blood Cells is a challenging job in the computer-assisted diagnosis of blood for any disorder in many diseases like Complete Blood Count Test, Anemia, Leukemia and Malaria etc. The mentioned problems are highly laborious in manual microscopy for the hematologists. Many techniques currently existing for the solution suffer from both under- and over- splitting problems when highly complex clusters of Red Blood Cells occur. In addition, the existing techniques are not computationally efficient. In this paper, we address the aforementioned problems, firstly by considering the boundaries of the convex hulls of clustered Red Blood Cells and secondly, by splitting the boundaries according to the number of Red Blood Cells in relation to distance measures. Furthermore, we draw circles using a mid-point circle algorithm at each boundary cleavage to give an illusion of the Red Blood Cells. The test results of the proposed technique on a standard online dataset are presented in two ways. Statistically first of all by achieving an average recall of 0.964 and precision of 0.970 while their F-measure achieved is 0.962 as well as secondly through ground truth data with visual inspections.

Evaluation of Mindray BC-5000 hematology analyzer: a new miniature 5-part WBC differential instrument.

The Mindray BC-5000 automated hematology analyzer is a miniature, automated hematology analyzer, and 5-part leukocyte differential counter for in vitro diagnostic use in clinical laboratories. Precision, linearity, carryover, and method comparison were carried out. The analyzer was evaluated and compared with the Sysmex XE-2100 hematology analyzer and manual microscopic in the hematology laboratory of a tertiary hospital in Chinese. There were minimal carryover (<0.25%), and excellent linearity for white blood cell, and platelet counts (r > 0.99). Within-run precision was good at all levels for the routine cell blood count parameters (CV < 3.5%). Between-run precision was acceptable at all levels for the analysis parameters (CV < 5%) except for eosinophil and basophil (CV% >10%). BC-5000 displayed very good correlation (r > 0.94) with the XE-2100 for cell blood count and cell differential parameters except for basophil (r = 0.72). The comparison of 258 leukocyte differential count results analyzed in parallel with manual microscopy, BC-5000 analyzer showed perfect specificity (93%) and negative predictive value (90%) on abnormal blood samples. It is concluded that the overall performance of the BC-5000 is acceptable. The miniature analyzer is suitable for use in small- to medium-size laboratories.

Imaging flow cytometry for the study of erythroid cell biology and pathology

Erythroid cell maturation and diseases affecting erythrocytes are frequently accompanied by morphologic and immunophenotypic changes to these cells. In the past, these changes have been assessed primarily through the use of manual microscopy, which substantially limits the statistical rigor, throughput, and objectivity of these studies. Imaging flow cytometry provides a technology to examine both the morphology of cells as well as to quantify the staining intensity and signal distribution of numerous fluorescent markers on a cell-by-cell basis with high throughput in a statistically robust manner, and thus is ideally suited to studying erythroid cell biology. To date imaging flow cytometry has been used to study erythrocytes in three areas: 1) erythroid cell maturation, 2) sickle cell disease, and 3) infectious diseases such as malaria. In the maturation studies, imaging flow cytometry can closely recapitulate known stages of maturation and has led to the identification of a new population of erythroid cell precursors. In sickle cell disease, imaging flow cytometry provides a robust method to quantify sickled erythrocytes and to identify cellular aggregates linked to morbidities, and in malaria, imaging flow cytometry has been used to screen for new chemotherapeutic agents. These studies have demonstrated the value of imaging flow cytometry for investigations of erythrocyte biology and pathology.

Evaluation of an automated morphological analysis system in white blood cell identificaton

Objective To evaluate the clinical performance of an automated image analysis systems named CellaVision DM96 in classifying White Blood Cells. Methods A total of 2267 peripheral blood samples (male 1 235, female 1 034, average age 46) were obtained from outpatient and inpatient in Peking Union Medical College Hospital (PUMCH). These samples were selected to evaluate the precision, sensitivity, specificity and the analytical error of the system. We first evaluated the coincidence rate of reclassification and manual microscopy. On the base of favourable coincidence rate, we then evaluated the correlations between the pre–classification and reclassification of segmented neutrophil, band neutrophil, lymphocyte, monocyte, eosinophile, basophile, blast cell, promyelocyte, myelocyte, metamyelocyte, plasma cell and reactive lymphocyte. Results The sensitivity and specificity of pre–classification of White Blood Cell were 46%–100% and 24%–92%, respectively. When studied on the cell level, the total coincidence rate of pre–classification was 88%. And the coincidence rates of pre–classification and reclassification of White Blood Cell were 6%–95% and 25%–100%, respectively. When assessed on the sample level, the coincidence rates of pre–classification and reclassification of leukocytes were 64%–98% and 84%–100%, respectively. The correlations of pre–classification and reclassification of leukocytes in order from high to low were: lymphocyte, segmented neutrophil, eosinophile, band neutrophil, monocyte, basophile, when r were 0.943 9, 0.915 2, 0.785 4, 0.775 6, 0.676 2 and 0.289 1, respectively. The correlations between reclassification and manual microscopy of White Blood Cell were higher than those between pre–classification and manual microscopy. Order from high to low was: eosinophile, segmented neutrophil, lymphocyte, monocyte, band neutrophil, basophile. And r were 0.972 1, 0.968 5, 0.957 0, 0.831 9, 0.800 6 and 0.648 7, respectively. The ability of this automated image analysis systems at pre–classification in distinguishing between band cell and segment cell, atypical lymphocyte and normal lymphocyte was not good. Conclusion The performance of reclassification was better than pre–classification. The reclassification can be substitute for the microscopy inspection, and be used in the Clinical practice. (Chin J Lab Med, 2015, 38: 168–172) Key words: Leukocyte count; Cell shape; Automation, laboratory

ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features

These guidelines provide information on how to reliably and consistently report abnormal red blood cells, white blood cells and platelets using manual microscopy. Grading of abnormal cells, nomenclature and a brief description of the cells are provided. It is important that all countries in the world use consistent reporting of blood cells. An international group of morphology experts have decided on these guidelines using consensus opinion. For some red blood cell abnormalities, it was decided that parameters produced by the automated haematology analyser might be more accurate and less subjective than grading using microscopy or automated image analysis and laboratories might like to investigate this further. A link is provided to show examples of many of the cells discussed in this guideline.

Automated urine screening devices make urine sediment microscopy in diagnostic laboratories economically viable

Automated urinalysis devices are reproducible, accurate and faster than the standard manual microscopy. Economic analysis has shown that decreases in turn-around-time and labour cost savings offered by these devices make them more economic than manual microscopy.

Performance of the CellaVision® DM96 system for detecting red blood cell morphologic abnormalities

Background: Red blood cell (RBC) analysis is a key feature in the evaluation of hematological disorders. The gold standard light microscopy technique has high sensitivity, but is a relativity time-consuming and labor intensive procedure. This study tested the sensitivity and specificity of gold standard light microscopy manual differential to the CellaVision® DM96 (CCS; CellaVision, Lund, Sweden) automated image analysis system, which takes digital images of samples at high magnification and compares these images with an artificial neural network based on a database of cells and preclassified according to RBC morphology. Methods: In this study, 212 abnormal peripheral blood smears within the Calgary Laboratory Services network of hospital laboratories were selected and assessed for 15 different RBC morphologic abnormalities by manual microscopy. The same samples were reassessed as a manual addition from the instrument screen using the CellaVision® DM96 system with 8 microscope high power fields (×100 objective and a 22 mm ocular). The results of the investigation were then used to calculate the sensitivity and specificity of the CellaVision® DM96 system in reference to light microscopy. Results: The sensitivity ranged from a low of 33% (RBC agglutination) to a high of 100% (sickle cells, stomatocytes). The remainder of the RBC abnormalities tested somewhere between these two extremes. The specificity ranged from 84% (schistocytes) to 99.5% (sickle cells, stomatocytes). Conclusions: Our results showed generally high specificities but variable sensitivities for RBC morphologic abnormalities.

Clinical relevance and contemporary methods for counting blood cells in body fluids suspected of inflammatory disease.

In many inflammatory diseases, the cellular components in body fluids [cerebrospinal fluid (CSF), serous fluids] are increased, rendering essential diagnostic information. The diagnostic value of the total white blood cell count (WBC) and differential count has been evaluated extensively over the years, and a remarkable amount of knowledge has been gained; yet, there is a great deal of clinical uncertainty whether the diagnosis should be based solely on these variables. In some diseases, such as peritonitis, the total WBC and differential count has high sensitivity; whereas, in differentiating pleural effusions, it lacks the sensitivity required to be clinically useful. Nevertheless, many guidelines consider these tests as cornerstone parameters, and in combination with clinical variables, they can successfully guide clinical decision making in initiating or postponing a treatment course for infection and/or inflammatory diseases while awaiting culture results. Although other methods are available for detecting and differentiating WBCs in body fluids, manual microscopy is still considered the gold standard despite its many limitations. During the last decade, automated analyzers have become a popular method for first line screening. Continued progress in their design has led to major improvements including their speed, improved accuracy and lower variability compared with microscopy. Disadvantages of this method include high imprecision in low ranges (depending on the method) and interfering factors. In a time where automation is at the front line in clinical laboratories, it is essential the results obtained are precise, accurate and reproducible. This review provides an overview of the relevance for cell counting in a variety of diagnostic body fluids, and highlights the current technologies used.

Characterization of TiN, TiC and Ti(C,N) in titanium-alloyed ferritic chromium steels focusing on the significance of different particle morphologies

Titanium-alloyed ferritic chromium steels are a competitive option to classical austenitic stainless steels owing to their similar corrosion resistance. The addition of titanium significantly influences their final steel cleanliness.The present contribution focuses on the detailed metallographic characterization of titanium nitrides, titanium carbides and titanium carbonitrides with regard to their size, morphology and composition. The methods used are manual and automated Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy as well as optical microscopy. Additional thermodynamic calculations are performed to explain the precipitation procedure of the analyzed titanium nitrides.The analyses showed that homogeneous nucleation is decisive at an early process stage after the addition of titanium. Heterogeneous nucleation gets crucial with ongoing process time and essentially influences the final inclusion size of titanium nitrides. A detailed investigation of the nuclei for heterogeneous nucleation with automated Scanning Electron Microscopy proved to be difficult due to their small size. Manual Scanning Electron Microscopy and optical microscopy have to be applied. Furthermore, it was found that during solidification an additional layer around an existing titanium nitride can be formed which changes the final inclusion morphology significantly. These layers are also characterized in detail.Based on these different inclusion morphologies, in combination with thermodynamic results, tendencies regarding the formation and modification time of titanium containing inclusions in ferritic chromium steels are derived.

Manual urine microscopy versus automated urine analyzer microscopy in patients with acute kidney injury.

To examine whether a significant difference exists between the reported ranges of granular and muddy brown casts in urine specimens using manual microscopy compared with an automated urine analyzer in a cohort of patients with acute kidney injury (AKI). Freshly voided urine specimens from 25 consecutive patients who were under evaluation by the Department of Nephrology for AKI were simultaneously examined using the iQ200 automated microscopy system and manual microscopy performed by a trained observer. We coded the results according to the number of pathological casts identified and performed a 3 × 2 Freeman-Halton extension of the Fisher exact probability test. Overall, the number of casts identified via manual microscopy differed significantly (P <.001) from the number identified via the automated microscopy system. This study provides evidence of the importance of performing a manual microscopic examination of urine sediment in patients with AKI. Further studies are needed to assess whether manual microscopy provides prognostic implications regarding renal recovery, hemodialysis dependency, and mortality.

Body fluid cellular analysis using the Sysmex XN‐2000 automatic hematology analyzer: focusing on malignant samples

The majority of previous studies on body fluid (BF) mode of automatic hematology analyzer used nonmalignant BF samples. Here, we evaluated the BF mode on the recently launched Sysmex XN for counting blood cells, especially for malignant samples. A total of 405 BF specimens including 125 malignant samples were analyzed using both the automated method and manual microscopy. In non-cerebrospinal fluids (CSF) samples, there was an agreement between two methods for WBC, RBC, polymorphonuclear, and mononuclear cell counts (R(2) =0.96, 0.94, 0.88, and 0.88, respectively). CSF samples showed slightly poorer correlations than other fluids. Exclusion of malignant samples significantly improved correlations in non-CSF samples, but not in CSF samples. High fluorescence-BF (HF-BF) cells were identified significantly more frequently in malignant samples compared to benign samples (17.8 and 4.15/100 WBC, respectively; P<0.001). Receiver operating characteristic curve analysis demonstrated an HF-BF cell AUC of 0.791 using a cutoff value of 6.9/100 WBC for detecting malignant samples. The BF mode on the Sysmex XN could be an alternative method for the manual counts in the BF analysis with a few drawbacks. However, if a concentration of HF-BF cells is greater than the given threshold, microscopic examination should be subsequently performed.

A fast-response/recovery ZnO hierarchical nanostructure based gas sensor with ultra-high room-temperature output response

In this paper, a ZnO hierarchical nanostructure based gas sensor is presented. The proposed implementation features short response/recovery time and ultra-high output response at room temperature (RT). In order to take the advantages of complementary-metal-oxide-semiconductor (CMOS) process in terms of miniaturization and cost-effectiveness, a novel fabrication recipe, consisting of CMOS-compatible techniques, is proposed to form a patterned triple-layer metal, which functions as both interconnection electrodes and catalyst for our reported ZnO hierarchical nanostructure. This enables rapid and local growth of ZnO hierarchical nanostructure directly on a single silicon chip. Reported peak RT output response of 32 (20ppm NO2) provides a significant 28-fold improvement over the traditional widely adopted nanowire-based gas sensor. Meanwhile, a time efficient gas sensor is also validated by the presented temporal performance with a response and recovery time of 72s and 69s, respectively. In addition, compared with the previously demonstrated gas sensors operating at 200–300°C, the proposed RT sensing completely removes the power-hungry heater and eliminates the related thermal reliability issues. Moreover, the demonstrated process flow well addresses the challenging issues of the traditional mainstream “drop-cast” method, including poor yield, non-uniformity of device performance and low efficiency caused by inevitable manual microscope inspection.

Automated urinalysis: First experiences and comparison of automated urinalysis system and manual microscopy

Background: Urinary tract infection is a common condition which needs laboratory evaluation of urine to substantiate the clinical diagnosis and initiate treatment. The conventional urinalysis consists of using a test strip for chemical examination to identify the various urine sediments after which visual microscopy is done. We evaluate the analytical performance of automated microscopic technique (UF 500i) and compare results with those from manual microscopy. Materials and Methods: A total of 382 urine specimens were collected during a period of one month out of which 128 samples which had abnormal cell counts were analyzed for cells and particles by manual and automated microscopy by UF-500i flow cytometer. Results: The concordance of UF 500i and the manual microscopy which is considered to be the gold standard for urine microscopic examination was 90.6% for white blood cells, red blood cell, epithelial cells, cast and bacterial count. Conclusion: Automated urine sediment analyzer, UF 500i was considered reliable in the measurement of white blood cells, red blood cells, epithelial cells, cast and bacteria. Automation will surely reduce the work load, increase accuracy and reliability, and increase the throughput and turn-around time of the laboratory DOI: http://dx.doi.org/10.3126/jpn.v4i7.10316 Journal of Pathology of Nepal (2014) Vol. 4, 576-579

Relativity of Platelet Count with Automated Cell Counter and Manual Haemocytometer using Peripheral Blood Smear

Background: Obtaining accurate, precise, and reliable platelet counts as a reference for the calibration of hematology analyzers has been a continuing problem due to varying results. We proposed the relativity of platelet count by Automated Cell Counter and Manual Haemocytometer with Peripheral smear for focusing the accuracy and & cost effectiveness, without compromising the accuracy of test results. Methods: Seventy-Five blood samples in EDTA-anticoagulant vacutainer tubes were obtained and randomly divided into 3 group; Group I-normal control (n = 25), Group II-mild thrombocytopenia (n = 25) and Group III-severe thrombocytopenia (n = 25). Each blood sample had two peripheral blood smears made and stained on an automatic slide stainer. Peripheral blood smears were analyzed for platelet count by manual microscopy, an automated hematology analyzer Beckman Counter Diff 2 in all groups. The agreement between the two methodologies was assessed using the paired t-test and correlation coefficient analyses. Results: The coefficient of variation values significantly differed in Group C and was high for automated cell counter. High positive correlation was also observed between Automated cell counter values and manual method (r=0.82, p

Interpretation of ANA indirect immunofluorescence test outside the darkroom using NOVA view compared to manual microscopy.

Objective. To evaluate NOVA View with focus on reading archived images versus microscope based manual interpretation of ANA HEp-2 slides by an experienced, certified medical technologist. Methods. 369 well defined sera from: 44 rheumatoid arthritis, 50 systemic lupus erythematosus, 35 scleroderma, 19 Sjögren's syndrome, and 10 polymyositis patients as well as 99 healthy controls were examined. In addition, 12 defined sera from the Centers for Disease Control and 100 random patient sera sent to ARUP Laboratories for ANA HEp-2 IIF testing were included. Samples were read using the archived images on NOVA View and compared to results obtained from manual reading. Results. At a 1 : 40/1 : 80 dilution the resulting comparison demonstrated 94.8%/92.9% positive, 97.4%/97.4% negative, and 96.5%/96.2% total agreements between manual IIF and NOVA View archived images. Agreement of identifiable patterns between methods was 97%, with PCNA and mixed patterns undetermined. Conclusion. Excellent agreements were obtained between reading archived images on NOVA View and manually on a fluorescent microscope. In addition, workflow benefits were observed which need to be analyzed in future studies.

An automated new technique for scoring the in vivo micronucleus assay with image analysis

The mammalian erythrocyte micronucleus assay is frequently used to assess chemical-induced damage to the chromosomes or the mitotic apparatus of erythroblasts. Because quantitative analysis of micronuclei by microscopy is time consuming and laborious, several automatic scoring methodologies with image analysis have been reported. However, there have been cases in which it was difficult to examine the proportion of polychromatic erythrocytes (PCEs) among total erythrocytes as an index for bone marrow (BM) toxicity, and sample slide preparation has proven to be laborious with existing automatic methods. We developed an automatic scoring system with image analysis for the rodent erythrocyte micronucleus assay using 12-well plates employing high-content screening analyser. In our method, micronucleated PCEs (MNPCEs), PCEs and erythrocytes were identified from three kinds of images: bright field image, fluorescence image with Hoechst 33342, and fluorescence image with propidium iodide. The frequencies of MNPCEs and PCEs were subsequently calculated. A comparison of automatic and manual scoring was carried out using BM and peripheral blood (PB) obtained from mice treated with stepwise doses of mitomycin C. The scores obtained by automatic analysis corresponded to those obtained by manual scoring; the frequencies of MNPCEs in BM and PB obtained by automatic scoring were 132 and 113%, respectively, of those obtained by manual scoring, and the corresponding frequencies of PCEs were 95 and 120%, respectively. Furthermore, we performed five repeats of the examinations of mouse BM and PB treated with mitomycin C or vinblastine sulphate and showed that automatic scoring was equivalent to manual scoring in reproducibility. Meanwhile, the scoring data obtained by manual scoring tended to vary among observers. These results suggest that our automatic scoring system with image analysis is superior to manual microscopy scoring in terms of speed and objectivity, comparable in reproducibility and useful for the in vivo micronucleus assay.