The role of neutrophil gelatinase-associated lipocalin (NGAL) in cerebrospinal fluids for screening of acute bacterial meningitis.

BACKGROUND: Acute bacterial meningitis is a rare but extremely severe disease. The aim of this study was to investigate whether neutrophil gelatinase-associated lipocalin (NGAL) is present and measurable in cerebrospinal fluid (CSF) and if its assessment may be useful for identifying patients with bacterial meningitis. METHODS: Eligible specimens were all consecutive CSFs of patients with suspect acute bacterial meningitis that were referred from the Unit of Infectious Diseases for routine chemical and morphological analysis over a three months period. CSF measurements consisted in NGAL, glucose, and total protein concentrations, along with cell count and differential. RESULTS: Eighty eight CSFs were received throughout the study period, 58 (66%) with CSF findings compatible with bacterial meningitis. The values of white blood cells (WBC), polymorphonuclear (PMN) and mononuclear (MONO) leukocytes, red blood cells (RBC), total proteins, and NGAL were significantly increased in positive CSFs, whereas that of glucose did not significantly differ. A significant correlation was found between CSF concentration of NGAL and CSF values of PMN, WBC, RBC and total proteins, but not with that of glucose and MONO. The concentration of NGAL in CSF showed an area under the curve (AUC) of 0.94 for identifying positive CSFs, with specificity and sensitivity of 1.00 and 0.741 at a diagnostic threshold of 13 ng/mL. CONCLUSIONS: NGAL is present in CSF of patients with bacterial meningitis and its measurement may be helpful for identifying positive CSFs.

Influence of residual platelet count on routine coagulation, factor VIII, and factor IX testing in postfreeze-thaw samples.

The use of frozen-thawed samples rather than fresh samples for specialized coagulation testing is becoming commonplace, thereby creating novel risks that may jeopardize the quality of hemostasis testing. Residual platelets (PLTs) in frozen plasma are most critical as freezing-induced activation and injury may impair routine and specialized testing after thawing. The aim of this study was to verify the impact of postcentrifugation PLT count in postfreeze-thawed samples on activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, factor VIII (FVIII) activity testing, and factor IX (FIX) activity testing. These parameters were herein assessed in postfreeze-thaw paired plasma samples collected from 15 healthy volunteers and subjected to 4 different centrifugation forces (i.e., 3,000, 1,500, 1,000, and 500g), using data obtained with centrifugation force of 1,500g as the gold standard, in agreement with current recommendations. Compared with reference samples, PLT counts in fresh aliquots were indistinguishable in specimens centrifuged at 1,000g, significantly lower in those centrifuged at 3,000g and significantly higher in those centrifuged at 500g. In all cases except samples centrifuged at 3,000g, the PLT count was significantly decreased in postfreeze-thaw compared with paired fresh specimens. In postfreeze-thaw plasma, APTT was not influenced by residual PLT count. The results of PT and fibrinogen were consistently altered in samples centrifuged at 1,000 and 500g, though the correlation with the reference measures remained clinically acceptable. Data obtained for FVIII and FIX activities revealed a positive bias in all postfreeze-thaw plasmas, achieving statistical significance in samples centrifuged at 3,000g. We conclude that alteration of centrifuge speeds away from the recommended 1,500g may influence the level of residual PLTs in sample centrifuged at lower speeds such as 500g, and therefore may make these specimens unsuitable for hemostasis testing in postfreeze-thawed plasma samples. In addition, although the changes seen in FVIII and FIX in samples centrifuged at 3,000g may reflect non-PLT-related effects, such changes should also be considered in this setting.

Identification of spurious hemolysis in anticoagulated blood with Sysmex XE-2100 and Siemens Advia 2120.

BACKGROUND: Various degrees of hemolysis might occur during collection, processing and storage of blood bags or blood tubes for hematological testing. In both circumstances, the identification of hemolysis is challenging since the centrifugation process is not required. The aim of this study was to identify simple hematological parameters that would help identify the presence of hemolysis in anticoagulated blood. METHODS: Twenty tubes containing K2EDTA anticoagulated blood were randomly selected from outpatient samples and divided in two aliquots. The former was immediately analyzed, whereas the latter was subjected to mechanical hemolysis by aspirating whole blood two times through a very fine needle to generate mechanical hemolysis. Both aliquots were tested on Advia 2120 and Sysmex XE-2100. RESULTS: The double aspiration of the blood through the fine needle caused a remarkable hemolysis with significant decrease of red blood cell (RBC) count (-17 +/- 11%; p < 0.01), hematocrit (-18 +/- 12%; p < 0.01) and reticulocytes (-24 +/- 13%; p < 0.01), but not of hemoglobin, white blood cell or platelet counts. A remarkable increase of immature platelet fraction (IPF) on XE-2100 and RBC ghosts on Advia 2120 was observed in the hemolyzed samples, whereas RBC fragments did not vary significantly. A significant correlation was also observed between hemolysis and reticulocyte count (r = 0.823; p < 0.01), IPF (r = -0.502, p = 0.024) and RBC ghosts (r = -0.711; p < 0.01). Receiver Operating Characteristic (ROC) curve analysis demonstrated a good performance of both IPF and RBC ghosts (i.e., AUCs 0.91 and 0.96, respectively) for distinguishing non-hemolyzed from hemolyzed specimens. CONCLUSIONS: The absolute values of both IPF and RBC ghosts perform efficiently for distinguishing hemolyzed from non-hemolyzed specimens, although neither reached 100% sensitivity and specificity. Nevertheless, the demonstration that both parameters significantly increase after hemolysis can be reliably used to distinguish hemolyzed from non-hemolyzed blood.

Multicentric evaluation of the variability of digital morphology performances also respect to the reference methods by optical microscopy.

INTRODUCTION: Despite the important diagnostic role of peripheral blood morphology, cell classification is subjective. Automated image-processing systems (AIS) provide more accurate and objective morphological evaluation. The aims of this multicenter study were the evaluation of the intra and inter-laboratory variation between different AIS in cell pre-classification and after reclassification, compared with manual optical microscopy, the reference method. METHODS: Six peripheral blood samples were included in this study, for each sample, 70 May-Grunwald and Giemsa stained PB smears were prepared from each specimen and 10 slides were delivered to the seven laboratories involved. Smears were processed by both optical microscopy (OM) and AIS. In addition, the assessment times of both methods were recorded. RESULTS: Within-laboratory Reproducibility ranged between 4.76% and 153.78%; between-laboratory Precision ranged between 2.10% and 82.2%, while Total Imprecision ranged between 5.21% and 20.60%. The relative Bland Altman bias ranged between -0.01% and 20.60%. The mean of assessment times were 326 ± 110 s and 191 ± 68 s for AIS post reclassification and OM, respectively. CONCLUSIONS: AIS can be helpful when the number of cell counted are low and can give advantages in terms of efficiency, objectivity and time saving in the morphological analysis of blood cells. They can also help in the interpretation of some morphological features and can serve as learning and investigation tools.

Multicentric evaluation of analytical performances digital morphology with respect to the reference methods by manual optical microscopy.

AIMS: Optical microscopic (OM) evaluation of peripheral blood (PB) cells is still a crucial step of the laboratory haematological workflow. The morphological cell analysis is time-consuming and expensive and it requires skilled operator. To address these challenges, automated image-processing systems, as digital morphology (DM), were developed in the last few years. The aim of this multicentre study, performed according to international guidelines, is to verify the analytical performance of DM compared with manual OM, the reference method. METHODS: Four hundred and ninety PB samples were evaluated. For each sample, two May Grunwald-stained and Giemsa-stained smears were performed and the morphological evaluation of cells was analysed with both DM and OM. In addition, the assessment times of both methods were recorded. RESULTS: Comparison of DM versus OM methods was assessed with Passing-Bablok and Deming fit regression analysis: slopes ranged between 0.17 for atypical, reactive lymphocytes and plasma cells (LY(AT)) and 1.24 for basophils, and the intercepts ranged between -0.09 for blasts and 0.40 for LY(AT). The Bland-Altman bias ranged between -6.5% for eosinophils and 21.8% for meta-myemielocytes. The diagnostic agreement between the two methods was 0.98. The mean of assessment times were 150 s and 250 s for DM and OM, respectively. CONCLUSION: DM shows excellent performance. Approximately only 1.6% of PB smears need the OM revision, giving advantages in terms of efficiency, standardisation and assessment time of morphological analysis of the cells. The findings of this study may provide useful information regarding the use of DM to improve the haematological workflow.

Multicenter evaluation of analytical performances of platelet counts and platelet parameters: Carryover, precision, and stability.

INTRODUCTION: The correctness of the results of automated platelet analysis is still highly debated. The aim of this multicenter study, conducted according to international guidelines, was to verify the analytical performance of nine different types of hematology analyzers (HAs) in the automated platelet analysis. METHODS: Four hundred eighty-six peripheral blood samples (PB), collected in K3 EDTA tubes, were analyzed by ABX Pentra, ADVIA2120i, BC-6800, BC-6800 Plus, Cell-DYN Sapphire, DxH800, XE-2100, XE-5000, XN-20 with PLT-F App. Within-run imprecision and between-run imprecision were carried out using PB and material control, respectively. The carryover, low limit of quantification (LoQ), and the PB stability were evaluated. RESULTS: The carryover was absent for all HAs. The LoQ of PLT ranged between 2.0 (Cell-Dyn Sapphire) and 25.0 × 109 /L (ADVIA 2120i), while immature platelet fraction (IPF) ranged between 1.0 (XN-20) and 12.0 × 109 /L (XE-5000). The imprecision (%CV) increases as the platelet count decreases. No HAs showed desirable CVAPS for PLT counts less than 50.0 × 109 /L, with the exception of Cell-DYN Sapphire (CV 3.0% with PLT-O mean value of 26.7 × 109 /L), XN-20 (CV 2.4% with PLT-F mean value of 21.5 × 109 /L), and BC-6800 Plus (CV 1.9% with PLT-O mean value of 26.5 × 109 /L). The sample stability ranged between under two hours for MPV by ADVIA2120i and 8 hours for other PLT parameters and HAs. CONCLUSION: The findings of this study may provide useful information regarding carryover, precision, and stability of platelet counts and parameters, especially in thrombocytopenic samples. Moreover, the stability of sample for platelet analysis is conditioned by the HA and by temperature and storage time.

Novel approach for quality assessment and improving diagnostic accuracy in cell-based infection imaging using 99mTc-HMPAO labeled leukocytes.

BACKGROUND AND AIM: Labeled leukocytes with 99mTc-HMPAO are routinely used for infection imaging. Although cell labeling with 99mTc-HMPAO represents an imaging probe to detect infection sites, the diagnostic efficiency of the probe is largely influenced by cell manipulation, multidisciplinary interventions (i.e., biologist, technicians) and available technology (i.e., SPECT, SPECT/CT). The aim of the study was to assess in vitro and in vivo accuracy of a comprehensive approach for quality assessment (QA) of all steps of the procedure. METHODS: Radiochemical purity (RCP), pH, labeling efficiency (LE) were measured in 320 procedures. White Cell Viability Factor (WVF) was determined in consecutive blood samples. Images (490 studies) were scored using a 5-point scale. Training program was evaluated using a Learning Questionnaire and a score system. RESULTS: Pre/post-labelling WVF was 0.99% (max value 1%) in all blood samples. LE (mean value 72%) and RCP (>80% until 55 minutes) yielded considerably high values. The vast majority of images were scored as diagnostic by three independent observer (90% with score ≥4). CONCLUSIONS: This method appears highly reproducible and easy to use in clinical routine for leukocyte labeling, especially when standardized training and total QA system are implemented.

Evaluation of mean platelet volume with four hematological analyzers: harmonization is still an unresolved issue.

The clinical applications of mean platelet volume (MPV) have recently broadened far beyond the differential diagnosis of platelet (PLT) disorders to embrace diagnosis and prognostication of a variety of thrombotic conditions. As the potential usefulness of this simple and inexpensive parameter may be challenged by instrument heterogeneity, we investigated the degree of analytical quality and interinstrument comparability. One hundred consecutive inpatient samples were simultaneously assessed on Abbott Sapphire, Mindray BC6800, Siemens Advia 2120, and Sysmex XE5000. The within-run imprecision of the four hematological analyzers was also assessed according to the Clinical and Laboratory Standards Institute document EP5-A2. The imprecision of PLT count ranged between 1.4 and 4.3%, and hence was always within the desirable quality specifications. The within-run imprecision of MPV ranged between 1.1 and 3.8%, and hence was also within the desirable quality specifications. The optical and impedance measurements displayed excellent correlations. Overall, the PLT count exhibited a modest instrumental variation, with bias always within the desirable quality specifications. A large bias was instead recorded for MPV, with between-instrument variations exceeding the desirable quality specifications in five out of six interinstrumental comparisons. No significant correlation was also observed between PLT count and MPV with any of the instruments tested. These results attest that although there is an optimal degree of analytical quality and comparability for PLT counting among different hemocytometers, the harmonization of MPV is poor, thus making the adoption of universal cutoffs virtually impossible.

What do hemolyzed whole-blood specimens look like? Analysis with a CellaVision DM96 automated image analysis system.

We planned an original study to investigate the morphological changes caused by spurious hemolysis of whole-blood samples, analyzed using an automated image analysis system. Seven whole-blood specimens anticoagulated with EDTA were pooled and divided in two aliquots. The former was left untreated, whereas the latter was subjected to mechanical hemolysis by forced aspiration with an insulin syringe. The complete blood cell count was performed on a Sysmex XE-2100, and the aliquots were then processed with CellaVision DM96. In spuriously hemolyzed samples, the main findings included a rarefaction of erythrocytes, the presence of a remarkable number of cellular debris, a greater degree of microcytosis and anisocytosis, the appearance of band neutrophils, a shift of values between lymphocytes and monocytes, and an increase in smudge cells, artifacts, and large platelets. The results of this study demonstrate for the first time that blood cell morphology may be consistently biased in spuriously hemolyzed whole blood and that the use of an automated image analysis system such as the CellaVision DM96 may be a suitable approach to identify spurious hemolysis in whole-blood specimens.

Evaluation of nucleated red blood cell count by Sysmex XE-2100 in patients with thalassaemia or sickle cell anaemia and in neonates.

BACKGROUND: Current haematology analysers have variable sensitivity and accuracy for counting nucleated red blood cells in samples with low values and in all those conditions characterised by altered sensitivity of red blood cells to the lysing process, such as in beta-thalassaemia or sickle-cell diseases and in neonates. The aim of our study was to evaluate the performance of the automated analyser XE-2100 at counting nucleated red blood cells in the above-mentioned three categories of subjects with potentially altered red blood cell lysis sensitivity and yet a need for accurate nucleated red blood cell counts. MATERIALS AND METHODS: We measured nucleated red blood cell count by XE-2100 in peripheral blood samples of 187 subjects comprising 55 patients with beta-thalassaemia (40 major and 15 traits), 26 sickle-cell patients, 56 neonates and 50 normal subject. Results were compared with those obtained by optical microscopy. Agreement between average values of the two methods was estimated by means of Pearson's correlation and bias analysis, whereas diagnostic accuracy was estimated by analysis of receiver operating characteristic curves. RESULTS: The comparison between the two methods showed a Pearson's correlation of 0.99 (95% CI; 0.98-0.99; p<0.001) and bias of -0.61 (95% CI, -1.5-0.3). The area under the curve of the nucleated red blood cell count in all samples was 0.98 (95% CI, 0.96-1.00; p<0.001). Sub-analysis revealed an area under curve of 0.99 (95% CI, 0.98-1.00; p<0.001) for patients with thalassaemia, 0.94 (95% CI, 0.85-1.00; p<0.001) for patients with sickle cell anaemia, and 1.00 (95% CI, 1.0-1.0) for neonates. DISCUSSION: XE-2100 has excellent performance for nucleated red blood cell counting, especially in critical populations such as patients with haemoglobinopathies and neonates.

Lack of harmonization of red blood cell distribution width (RDW). Evaluation of four hematological analyzers.

OBJECTIVES: To assess analytical imprecision and comparability of red blood cell distribution width (RDW) on Abbott Sapphire, Mindray BC6800, Siemens Advia 2120 and Sysmex XE-5000. DESIGN AND METHODS: Within-run imprecision was assessed on three pools and comparability using 132 inpatient samples. RESULTS: The imprecision of RDW was comprised between 0.3 and 1.2%, but the values exhibited broad variation among different analyzers, with bias exceeding the desirable quality specifications. CONCLUSIONS: Harmonization of RDW is still an unmet need.

Evaluation of white blood cell count in peritoneal fluid with five different hemocytometers.

OBJECTIVES: Evaluation of automated flow cytometric analysis of white blood cell (WBC) count in peritoneal fluids. METHODS: One hundred peritoneal fluids were analyzed with manual microscopy, Sysmex XE-2100 and XE-5000, Siemens Advia 2120, Mindray BC-6800, Abbott Sapphire. RESULTS: High correlations (0.978 to 0.999) and modes biases (-132 to 80 WBC/mm(3)) were found. Agreement at septic peritonitis cutoff ranged between 96% and 99%. CONCLUSIONS: These hemocytometers display acceptable performance for WBC screening in peritoneal fluids.

Evaluation of the fully automated hematological analyzer Sysmex XE-5000 for flow cytometric analysis of peritoneal fluid.

Although microscopy still represents the gold standard for cytometric analysis of peritoneal fluids, automated flow cytometry may improve throughput and accuracy. We evaluated the performance of total nucleated cell (TNC), white blood cell (WBC), polymorphonuclear cell (PMN), and mononuclear cell (MONO) counts of Sysmex XE-5000 on peritoneal fluids. The imprecision was excellent, being always lower than 11%, whereas linearity studies yielded correlation coefficients of 1.00 for all parameters. The carryover was always lower than 0.2%. The comparison between XE-5000 and microscopic analysis of 117 ascitic fluids yielded correlation coefficients always greater than 0.96, with mean biases <11/µL. The diagnostic accuracy versus manual microscopy was greater than that of XE-2100, especially at thresholds for septic ascites (100 versus 98% for ≥500 WBC/µL; 98 versus 93% for ≥250 PMN/µL). The correlation with manual microscopy for macrophages and mesothelial cell count was also higher for XE-5000 than for XE-2100 (0.63 versus 0.55). The results of this evaluation show optimal performance of XE-5000 for routine analysis of ascitic fluids, which are combined with the advantages of automated analysis such as high throughout, shortened turnaround time, no need of sample preparation and trained staff, reduced sample volume, and less likelihood of transcriptional errors.

Assessment of neutrophil gelatinase-associated lipocalin and lactate dehydrogenase in peritoneal fluids for the screening of bacterial peritonitis.

BACKGROUND: Manual microscopy remains the gold standard for enumeration and classification of nucleated cells in peritoneal fluids, especially for diagnosing bacterial peritonitis. However, this approach carries several drawbacks, so that the use of simple and automated tests may be a viable option for initial screening of peritoneal fluids. MATERIALS AND METHODS: Neutrophil gelatinase-associated lipocalin (NGAL), lactate dehydrogenase (LDH), proteins and glucose were assessed in peritoneal fluids from patients with new onset nonmalignant ascites, along with nucleated cell count and differentiation. RESULTS: One hundred and eleven specimens were analyzed, 26 of which (23%) with polymorphonuclear leukocyte (PMN) count≥250/µL, thus compatible with bacterial peritonitis. The median concentration of LDH and NGAL was 3.4 and 3.7-fold higher in samples with ≥250 PMN/µL. The concentration of proteins was also higher in samples with ≥250 PMN/µL, whereas that of glucose was lower. The PMN count significantly correlated with peritoneal fluid values of LDH (r=0.859), NGAL (r=0.774) and proteins (r=0.268), but not with glucose (r=-0.069). The area under ROC curve was 0.88 for LDH, 0.89 for NGAL and 0.94 for their combination (both tests positive), whereas that of proteins and glucose was 0.80 and 0.71, respectively. Sensitivities and specificities were 0.81 and 0.87 for LDH≥227 U/L, 0.96 and 0.75 for NGAL≥120 ng/mL, 0.77 and 0.95 for their combination. The agreement with PMN count was 0.86 for LDH, 0.80 for NGAL, and 0.91 for their combination. CONCLUSIONS: These results suggest that assessment of NGAL in peritoneal fluids, especially in combination with LDH, may be a reliable approach for screening of bacterial peritonitis in patients with new onset nonmalignant ascites.

Influence of temperature and period of freezing on the generation of hemolysate and blood cell lysate.

BACKGROUND: There is little information on temperature and period of freezing for generating hemolysate and blood cell lysate. MATERIALS AND METHODS: Primary tubes containing whole blood were frozen at -20°C and -80°C and then serially thawed to assess cell lysis and clinical chemistry tests in centrifuged plasma. RESULTS AND CONCLUSIONS: Massive amount of blood cells lysis could only be obtained by storing samples 12h at -20°C, or 2h at -80°C.