Evaluation and comparison of automated hematology analyzer, flow cytometry, and digital morphology analyzer for monocyte counting.

INTRODUCTION: This study was aimed to evaluate monocyte counts on Sysmex XN-9000, Sysmex CyFlow Space System, and Sysmex DI60 and compare the performance of these systems with the reference optical microscopy (OM) assessment. METHODS: In all, 55 peripheral blood samples, collected in K3 EDTA tubes, were analyzed with XN-9000, CyFlow System (FlowDiff1 and 2), DI60, and OM. Within-run imprecision was carried out using normal samples. Data comparison was performed with Passing-Bablok regression and Bland-Altman plots. RESULTS: The within-run imprecision of monocyte count on XN, FlowDiff, OM, and DI60 ranged between 1.9% for FlowDiff 2 and 22.1% for DI60. The Passing-Bablok regression analysis of absolute count yielded slopes comprised between 0.93 (FlowDiff2 vs DI60) and 1.21 (DI60 vs OM), whereas the intercepts ranged between -0.002 (FlowDiff 1 vs FlowDiff 2) and 0.13 (FlowDiff1 and 2 vs DI60). Bland-Altman plots in absolute values yielded absolute bias comprised between -0.01 × 109 /L (FlowDiff 1 vs FlowDiff 2; DI60 vs OM) and 0.15 × 109 (XN-module vs DI60). CONCLUSION: The results of this analytical evaluation suggest that flow cytometry generates monocyte counts suitable for routine clinical use. OM or DI60 analysis may be useful for identifying morphologic abnormalities, but does not achieve a satisfactory level of accuracy for enumerating blood cells types such as monocytes, which are usually very low in peripheral blood.

Abnormal scattergrams and cell population data generated by fully automated hematological analyzers: New tools for screening malaria infection?

INTRODUCTION: Malaria is a life-threatening infectious disease, which has been for long confined to specific endemic areas. Nevertheless, the recent increase in immigration flows from endemic regions and imported cases has reemphasized many diagnostic challenges in Western countries, thus paving the way to introduce rapid and accurate strategies for screening subjects with suspected Malaria infection. Therefore, the aim of this article was to describe our recent experience with Sysmex XN-module for rapid screening of subjects with suspected Malaria. METHODS: Fourteen patients admitted to the Emergency Department (Papa Giovanni XXIII Hospital Bergamo, Italy) with a clinical suspicion of Malaria infection were evaluated, along with 1047 control samples. The analysis of peripheral blood was performed with XN-module, and results were then compared to optical microscopy. RESULTS: Nine patients were positive to Plasmodim falciparum, 3 to Plasmodim vivax, one to Plasmodim ovale, and one to Plasmodim malarie. Characteristic abnormalities could be observed in both white blood cell differential (WDF) and white cell nucleated (WNR) scattergrams (sensitivity 0.64 and specificity 1.0) in 9 samples with parasites at gametocyte or schizos stage irrespective of Plasmodium species and parasitic index, while characteristic scattergram abnormalities could not be seen in the 5 samples containing only parasites at the trophozoites stage. In these cases, specific variations of some cell population data (CPD) could be recorded (sensitivity 1.00 and specificity 0.91). CONCLUSION: The peculiar abnormalities observed in CPDs, WDF, and WNR-scattergrams may raise a definite suspicion of Malaria infection. Further studies should then be planned for validating these preliminary findings and assessing whether these specific abnormalities may be incorporated in rapid and inexpensive Malaria diagnostic algorithms.

Two-site evaluation of the diagnostic performance of the Sysmex XN Body Fluid (BF) module for cell count and differential in Cerebrospinal Fluid.

INTRODUCTION: Cellular analysis in cerebrospinal fluid (CSF) provides important diagnostic information in many pathological settings. The aim of this two-site study was to evaluate the Sysmex XN Body Fluid mode (XN-BF) for cell analysis of CSF compared to light microscopy (LM). METHODS: Two hundred and seven consecutive CSF samples were analyzed in parallel with XN-BF and LM. The study also included the estimation of the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), carry-over and linearity of XN-BF module. RESULTS: LoQ of white blood cells (WBC) was 3×106  cells/L; linearity was good and carry-over negligible. XN-BF parameters were compared to LM for the following cell classes: total cells, WBC, polymorphonuclear (PMN), and mononuclear (MN) cells. The bias ranged from 1.3 to 15.2×106  cells/L. The receiver operating characteristics curve analysis for WBC showed an area under the curve of 0.98, and the global diagnostic agreement was 95% at a cutoff of 5×106  cells/L. CONCLUSIONS: XN-BF provides rapid and accurate counts in clinically relevant ranges of CSF values, thus providing a valuable alternative to conventional LM analysis. However, microscopic review remains advisable in samples with abnormal cell counts or high fluorescent (HF-BF) cell parameter exceeding 5×106  cells/L.

Performance evaluation of the automated nucleated red blood cell count of five commercial hematological analyzers.

INTRODUCTION: Recent automated hematology analyzers (HAs) can identify and report nucleated red blood cells (NRBC) count as a separate population out of white blood cells (WBC). The aim of this study was to investigate the analytical performances of NRBC enumeration on five top of the range HAs. METHODS: We evaluated the within-run and between-day precision, limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) of XE-2100 and XN-module (Sysmex), ADVIA 2120i (Siemens), BC-6800 (Mindray), and UniCel DxH 800 (Beckman Coulter). Automated NRBC counts were also compared with optical microscopy (OM). RESULTS: The limits of detection for NRBC of the BC-6800, XN-module, XE-2100, UniCel DxH 800, and ADVIA 2120i are 0.035×109 /L, 0.019×109 /L, 0.067×109 /L, 0.038×109 /L, and 0.167×109 /L, respectively. Our data indicated excellent performance in terms of precision. The agreement with OM was excellent for BC-6800, XN-module, and XE-2100 (Bias 0.023, 0.019, and 0.033×109 /L, respectively). ADVIA 2120i displayed a significant constant error and UniCel DxH 800 both proportional and small constant error. CONCLUSION: Regards to NRBC counting, the performances shown by BC-6800, XN-module, and XE-2100 are excellent also a low count, ADVIA 2120i and UniCel DxH 800 need to be improved.

Comparing the performance of three panels rules of blood smear review criteria on an Italian multicenter evaluation.

BACKGROUND: The aims of this study were to compare the diagnostic accuracy of blood smear review criteria, by means of three different panel rules, those proposed by: the International Consensus Group for Hematology [41-ICGH rules], the Italian Survey [IS rules] and the Working Group on Hematology-SIBioC (WGH) consensus rules (WGH rules). METHODS: This study is based on 2707 peripheral blood (PB) samples referred for routine hematological testing to the WGH-associated laboratories displaced all over the Italian territory. The PB samples were processed on seven different hematology analyzers (HAs): Advia 2120i, XE-2100, BC-6800, ABX Pentra, XN-1000, Cell-DYN Sapphire, and DxH800, respectively. All the results provided by the HAs were analyzed through the application of three different blood smear review criteria: that is, the 41-ICGH, IS, and WGH rules. Finally, data were compared with those obtained by optical microscopy (OM), as the current gold standard. RESULTS: The overall the agreement OM classification with ICGH, IS, and WGH panel rules is 0.83, 0.83, and 0.85, respectively. The false negatives are 2.1%, 3.0%, and 2.9%, while false positives are 15.1%, 13.7%, and 11.7%, respectively. All the seven HAs showed variable interinstrument performance, as three different criteria for OM review were adopted on each of them from time to time. CONCLUSION: These results presented show that the customization of validation rules is necessary for enhancing the quality of hematological testing and optimizing workflow.

Reliability of automated synovial fluid cell counting with Mindray BC-6800 body fluid mode.

INTRODUCTION: The enumeration and differentiation of nuclear elements in synovial fluid is a cornerstone for diagnosis and follow-up of many orthopedic and rheumatologic diseases. In this study, we evaluated the analytical performance of Mindray BC-6800 BF mode (BC-6800-BF) for synovial fluid analysis. METHODS: Overall, 78 synovial fluids were collected and analyzed with both BC-6800-BF and light microscopy. The study also entailed the assessment of limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), carryover and linearity. RESULTS: The LoB for the parameters total cells and white blood cells was 6 × 106 cells/L, and the LoD and LoQ were instead 15 and 16 × 106 cells/L, respectively. Linearity was excellent and carryover was negligible. The agreement between BC-6800-BF and light microscopy was satisfactory for all samples pretreated with hyaluronidase, displaying a bias between -5.9% and 8.2%. CONCLUSIONS: The use of BC-6800-BF for synovial fluid analysis enables rapid and accurate assessment, especially for total cell and polymorphonuclear counts. The use of BC-6800-BF may therefore allow the replacement of optical analysis, especially in samples pretreated with hyaluronidase, thus allowing its routine use for the screening of synovial specimens.

Analytical comparison between two hematological analyzer systems: CAL-8000 vs. XN-9000.

INTRODUCTION: This study was aimed to compare the analytical performance of traditional and new parameters and morphological flags of CAL-8000 and XN-9000. The automated differential leukocyte count (DIFF profile) and morphological flags were compared with optical microscopy (OM). METHODS: A total of 1025 peripheral blood samples, collected in K3 EDTA tubes, were analyzed by CAL-8000, by XN-9000, and by OM. Within-run imprecision was performed in low cellularity samples. The comparison was made using Spearman's correlation, Passing-Bablok regression, Bland-Altman bias, and Cohen's K test. RESULTS: Within-run imprecision in low cellularity samples yielded reproducible data between the instruments (imprecision was higher than 10% on samples with platelet count <21 × 109 /L using impedance technology). Passing-Bablok regression (CAL-8000 vs. XN-9000) yielded slopes ranging between 0.2 to 1.16 and intercepts from -6.54 to 21.63. The bias for leukocytes parameters ranged from -1.8% to -82.2%, the red blood cell parameters from -2.9% to 3.1%, platelets parameters from -27.8% to 26%, and reticulocyte parameters from -115.3% to 4.5%. The comparison of morphological flags yielded a K value always <0.55. The DIFF profile vs. OM had a Passing-Bablok regression with slopes ranging between 0.34 to 1.00 and intercepts from -0.01% to 0.11 and bias ranging from -42.9% to 2.6% for XN-9000 parameters and from -2.7% to 35.0% for CAL-8000 parameters. The comparison of morphological flags showed a K value ranging from 0.35 to 0.77 for XN-9000 and from 0.17 to 0.54 for CAL-8000. CONCLUSION: Differences exist between the two analyzers, especially in the generation of morphology flags, thus emphasizing the need of pursuing a major degree of harmonization and/or adopting instrument-specific reference ranges.

Mindray BC-6800 body fluid mode, performance of nucleated cells, and differential count in ascitic and pleural fluids.

INTRODUCTION: An accurate and rapid analysis of cells in body fluids (BFs) is important for diagnosis and follow-up in many pathological conditions. We evaluated the analytical performance of the module BF Mindray BC-6800 (BC-6800-BF) for cytometric analysis of ascitic and pleural fluids. METHODS: A total of 99 ascitic and 45 pleural samples were collected and assessed with BC-6800-BF and optical microscopy. This study also includes the evaluation of limit blank (LoB), limit detection (LoD), limit quantitation, (LoQ), carryover, linearity, and diagnostic concordance between the two methods. RESULTS: For TC-BF, LoB was 1 × 10(6) cells/L, LoD was 3 × 10(6) cells/L, and LoQ was 4 × 10(6) cells/L. Linearity was excellent (r(2) = 0.99) and carryover was negligible. TC-BF performed with the two methods showed Pearson's correlation of 0.99 (P < 0.0001), Passing-Bablok regression y = 1.04x - 1.17, and bias 33.7 cells. In ascitic fluids, polymorphonuclear cells (PMN) showed an area under curve (AUC) of 0.98 (P < 0.0001). In pleural fluids, mononuclear cells (MN) and PMN % displayed an AUC of 0.79 (P < 0.0001) and 0.93 (P < 0.0001), respectively. CONCLUSIONS: BC-6800-BF in ascitic and pleural fluids offers rapid and accurate cell and differential counts in clinically relevant concentration ranges. The use of BC-6800-BF may allow to replace routine optical counting, except for samples displaying abnormal cell counts or abnormal DIFF scattergram.

The impact of perioperative transfusion of blood products on survival after pediatric liver transplantation.

Intraoperative transfusion of red blood cells (RBC) is associated with adverse outcome after LT in adult patients. This relationship in pediatric patients has not been studied in depth, and its analysis is the scope of this study. Forty-one variables associated with outcome, including blood product transfusions, were studied in a cohort of 243 pediatric patients undergoing a cadaveric LT between 2002 and 2009 at the General Hospital of Bergamo. Multivariate stepwise Cox proportional hazards models were adopted with adjustment by propensity scores to minimize factors associated with the use of blood products. Median age at transplant was 1.37 yr. In uni- and multivariate analyses, perioperative transfusion of FFP and RBC was an independent risk factor for predicting one-yr patient and graft survival. The effect on one-yr survival was dose-related with a hazard ratio of 3.15 for three or more units of RBC (p = 0.033) and 3.35 for three or more units of FFP (p = 0.021) when compared with 1 or no units transfused. The negative impact of RBC and FFP transfusion was confirmed by propensity score-adjusted analysis. These findings may have important implications for transfusion practice in the LT pediatric recipients.

Production of epidermal growth factor in human prostatic cells cultured in vitro.

The Epidermal Growth Factor (EGF) plays an important role in the regulation of in vitro growth of prostate cells inducing a strong mitogenic effect. Nevertheless in our previous study we observed that the treatment of human hypertrophic prostate cell line U285 with exogenous EGF produces a restricted effect on the cellular growth rate. This phenomenon could be due to the capacity of the cells to produce EGF. In this study we aimed to verify this hypothesis by evaluating the presence of mRNA of EGF and EGF receptor (EGF-R) and of their translation products in U285 cells, before and after the treatment with suramin and exogenous EGF. Moreover we studied the effects exerted by these substances on the proliferative rate of the cells U285 after different treatment protocols. The presence in the cells of mRNA for EGF and EGF-R and of their translation products was demonstrated by means of reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods respectively. The modification of growth rate induced by these drugs was studied by FRAME Cytotoxicity Test. The operative modalities adopted to carry out these growth assays tended to 1) focus the effects of suramin in relation to in vitro cellular growth phase; 2) verify the reversibility of its effects; 3) ascertain if it was possible to antagonize the action of suramin by adding exogenous EGF. The results obtained from the RT-PCR showed the presence, in the control cells and in the treated ones, of mRNA coding for EGF and EGF-R. The immunocytochemical analysis indicated that 20% of the control cells are EGF positive, and 83% are EGF-R positive, confirming the results obtained with RT-PCR. Moreover, these stainings showed that the treatment with EGF does not significantly modify the percentage of cells marked by the anti-EGF antibody, while treatments with suramin and suramin plus EGF double this percentage. None of the treatments modifies the percentage of EGF-R positive cells. The growth assays showed that the exposition to highest doses of suramin in the first 24 h of cultures causes a decrease (p < 0.05) of the cellular proliferation during the following 48 h and 72 h and that these effects are irreversible. Moreover, a contemporaneous exposition of the cells to EGF and suramin at seeding strengthens the cytotoxic action of the last drug. To sum up, the demonstration of the presence in the U285 cells of mRNA coding for EGF and EGF-R and of the corresponding proteins, confirms the hypothesis that these cells can produce EGF. Moreover, the cytotoxicity experiments allowed a focusing of the role of the endogenous EGF in the regulation of the U285 cells proliferation and confirmed the importance of biological events that take place in U285 cells during the first 24 h of culture.

PCR in meningoencephalitis diagnosis.

Polymerase chain reaction (PCR) detection of a stretch of nucleic acid sequence of microbial origin from a clinical sample is not always diagnostic of disease unless the identified agent is a strict pathogen or its growth is documented. We describe here a case of acute meningoencephalitis in a 21-y-old man, in whom no pathogen was isolated by traditional bacterial or viral culture. Standard DNA PCR performed on the cerebrospinal fluid (CSF) identified the presence of 3 infectious agents: HHV-6, HHV-7 and Mycoplasma pneumoniae. Additional PCRs performed on CSF fractions along with gene transcript analysis proved the bystander role of the 2 herpesviruses and indicated M. pneumoniae as the relevant replicating agent, most likely playing to be a pathogenic role. Until this useful analysis becomes routine, clinicians should deal carefully with DNA PCR results, especially when assessing the aetiological role of agents, such as herpesviruses, which are known to undergo latency.

Changes in HCV viremia following LDL apheresis in a HCV positive patient with familial hypercholesterolemia.

It has been suggested that hepatitis C virus (HCV) can be associated with beta-lipoprotein in human serum. According to this, the LDL receptor could promote endocytosis of such a virus. In the present study, we evaluated the changes in HCV viremia in a HCV positive patient with familial hypercholesterolemia, undergoing both selective (DALI System, Fresenius) and non-selective (plasma exchange) LDL apheresis. HCV-RNA levels did not decrease following selective LDL apheresis, on the contrary showed a random, odd variation pattern (from -35% to +72%). Conversely, plasma exchange steadily induced a drop in HCV viremia (-35/43%), to a lower extent than that of a totally intravascular plasmaprotein, i.e., alpha 2-macroglobulin (-53/54%). These data indicate that beta-lipoprotein may not function as a plasma carrier of HCV, at least in the present case. Moreover, a continuous, quantitatively unforeseeable circulation of HCV virions from the intravascular plasma compartment to other extravascular and intracellular sites, seems to occur during an apheresis session.

Typing of hepatitis C virus by a new method based on restriction fragment length polymorphism.

A new restriction fragment length polymorphism (RFLP) analysis has been developed for hepatitis C virus (HCV) typing in the viral 5' non-coding region and contiguous core region. These genomic sequences were chosen for the relative nucleotide homology among different genotypes and for the presence of polymorphic sites. By employing two endonucleases (AccI and MboI) and, in some instances, a third one (EcoRII), we can unambiguously and reproducibly distinguish between genotypes and subtypes 1a, 1b, 1c, 2a, 2c, 2b, 3a, 3b, 4a, 5a and 6a. The method was applied for diagnosing two Italian groups of HCV-infected individuals reflecting a randomly collected population and a group of intravenous drug users. The accuracy of this method has been validated by comparison with INNOLiPA and by sequencing. Our approach represents an improvement over previous RFLP methods, since typing is accurate and simpler.

Effects of DHT and EGF on human hyperplastic prostate cells cultured in vitro: growth, morphology and phenotype characterisation.

This work studies the effects of dihydrotestosterone (DHT) and epidermal growth factor (EGF) on the growth, morphology and phenotype characterisation of the U285 line obtained from human prostate hyperplastic tissue. Modifications of growth rate induced by these two substances have been evaluated by means of the neutral red assay formulated by Borenfreund and Puerner (1985) as well as by means of Kenacid blue assay described by Knox et al. (1986), culturing cells for 24, 48 and 72 hr with scalar doses of DHT (0.5, 1, 2, 5, 10 microM) and EGF (5, 10, 20, 100 ng/ml). An optical microscope connected to a computer aided system and a scanning electron microscope were used to monitor morphological changes induced by DHT and EGF. The immunophenotype characterisation of the treated and control cells was carried out by using a monoclonal antibody panel. Our results show that the expression of anti-cytokeratin 5+6+18, anti-cytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies varied in relation to the type of treatment undergone by the cells. Moreover, exogenous DHT does provoke a flattening of the U285 cells without modifying their rate of growth, while EGF both shortens the lag phase reactivating the quiescent cells and regulates the subsequent log growth phase, thus causing no cellular overgrowth.

Uptake and intracellular distribution of idarubicin in secondary cultures of normal and neoplastic urothelium.

This study analyzes the uptake and endocellular distribution of idarubicin (IDA) in normal and neoplastic urothelial secondary cultures in relation to the changes in concentration and time of exposure. The urothelial lines were isolated by Freshney's method from biopsy fragments taken from five patients with superficial bladder cancer. Pharmacological experiments were carried out on subcultures previously immunophenotypically characterized and did not exceed ten passages. The uptake and endocellular distribution of IDA was analyzed by densitometric image analysis on cells treated for 10, 20, 30 and 60 min and 2 h with scalar dosages from 10 ng/ml to 2430 ng/ml. Microscopic observations and densitometric analyzes revealed that in the cells treated with IDA, fluorescence was higher in the cytoplasm compared to the nucleus and increased with the change in dosage. Moreover, densitometric data showed that IDA uptake in the first 20 min was higher in the neoplastic cells, but after that period its behavior became heterogeneous at 30 and 60 min, while at 2 h there was an inversion of the trend. These results suggest that the in vitro cytotoxicity should be evaluated in order to verify whether the elevated uptake of IDA in the first 20 min of treatment is really correlated to a more elevated toxicity in the neoplastic cells with respect to the normal cells. This is presently under investigation.

Morphological features and kinetics of primary cultures of BPH tissue supported by TV1 medium.

222 human prostatic biopsies were used to prepare cell cultures by means of a medium--colony formation permissive--containing fetal calf serum, called TV1. After 7, 14 and 21 days, the cultures were examined by optical and scanning electron microscopy. TV1 medium induces the formation and growth of two types of colonies, one mainly composed of epithelioid cells and distinguished by early growth; the second one made up exclusively of fibroblastoid cells which appear later in the culture. Epithelioid colonies, comprising three different cell types, appear to be arranged as a growth halo concentric to the bioptic fragment with a large central area, formed by a monolayer, and a pluristratified edge. Fibroblastoid cells weakly adhere to the substrate and form "satellite growth halos" separated from the primitive bioptic fragment. All the epithelioid cells were positive to cytokeratin LP34 Mab and negative to anti-smooth muscle-actin and anti-proline-4-hydroxylase antibodies. Fibroblastoid cells were only anti-proline-4-hydroxylase positive. The cell kinetics of epithelioid cells were also studied, revealing an extension of the S phase, in contrast to what happened with WAJC 404, and consequently a reduction of the percentage of cells entering mitosis. For this reason, the addition of fetal serum to the culture medium does not allow the use of prostate primary cultures for more than 14 days.

Myofibroblast-derived smooth muscle cells during remodelling of rabbit urinary bladder wall induced by partial outflow obstruction.

BACKGROUND: Fibrosis of serosa, along with smooth muscle (SM) cell hypertrophy, has been shown to occur in the rabbit bladder after partial outflow obstruction. Identification of cells involved in the serosal thickening can be of primary interest to elucidate the functional changes that this organ undergoes. EXPERIMENTAL DESIGN: Cytoskeletal protein composition of cells present in the thickened serosa at different times from the onset of obstruction (7, 15, 30 and 60 days) was evaluated. This was accomplished by means of a panel of monoclonal antibodies specific for a number of differentiation markers of mesenchymal cells (vimentin, desmin, alpha-actin of SM type, nonmuscle (NM) and SM myosins), and by immunocytochemical and immunochemical techniques. RESULTS: The immunocytochemical study revealed that cells in serosal thickening follow a two-step maturation process from pre-existing vimentin-positive cells. In the first time period (7 to 15 days of obstruction), these cells predominantly achieved an immunophenotype corresponding to that of a specific myofibroblast subtype (i.e., containing vimentin, NM myosin, and SM alpha-actin). After 30 days from the onset of obstruction, the cytoskeletal protein content of serosal cells, as also revealed by Western blotting experiments, shifted towards that of fetal-type SM cells (i.e., presence of vimentin, NM myosin, SM alpha-actin, and SM myosin isoforms). Distribution of vimentin, desmin, SM alpha-actin, and SM myosin in tissue culture as well as the ultrastructure in vivo very closely resembled that of SM cells. Bromodeoxyuridine incorporation studies indicated that cells accumulated in the serosa of obstructed bladders did not derive, at least initially, from SM cells of the detrusor muscle. CONCLUSIONS: These findings are consistent with the existence of a differentiation process in which resident mesenchymal cells of bladder serosa may transform to myofibroblasts and, subsequently, in fetal-type SM cells during experimental outflow obstruction.