Analytical performance of free testosterone calculated by direct immunoluminometric method compared with the Vermeulen equation: results from a clinical series.

INTRODUCTION: Testosterone (T) is a hormone that is crucial for primary and secondary sexual development in both males and females. Free testosterone (FT) represents the biologically active form of T, and its measurement is of great importance in clinical practice. While application of either equilibrium dialysis or ultrafiltration is considered to be the gold standard for FT assessment, these methods are expensive and not widely accessible. As an alternative, the Vermeulen formula is a commonly utilized calculated method. METHODS: This clinical study, including 190 consecutive patients, was carried out to compare FT levels obtained through direct immunoluminometric assay and the Vermeulen formula. The comparison was performed using Passing-Bablok and Deming regression as well as the Bland-Altman plot. Sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were assessed. RESULTS: The calculated method employing the Vermeulen formula was considered the gold standard. Passing-Bablok regression indicated a good agreement between the two methods, with slopes close to 1 for the whole series. Although the Bland-Altman plot demonstrated overall agreement, a potential proportional bias was observed in females. Deming regression confirmed excellent agreement and reliable estimates. Sensitivity and specificity analysis revealed that the direct method had a sensitivity of 75.0% and a specificity of 93.4% in all patients. However, sensitivity improved to 81.0% in males and dropped to 18.2% in females likely due to the low number of true positive cases. CONCLUSION: The direct method exhibited comparable performance to the calculated method, but caution should be exercised when interpreting results, particularly in females. Further studies are necessary to validate its sensitivity and specificity in larger series.

Analysis of annual distributions of hemoglobin A2 values as a method to test for HbA2 standardization.

BACKGROUND AND AIMS: The monitoring of yearly distributions of HbA2 measured has been indicated as a reliable indicator of worldwide standardization. MATERIALS AND METHODS: Measurements/year of HbA2 have been collected over three consecutive years in 15 Italian laboratories each using the same analytical method over three years period. HbA2 distributions, cleaned of replicated measurements, were compared by the overlapping area of the raw probability density functions expressed by coefficient eta (η), and by comparing the reference intervals for the central part of each distribution estimated by the indirect method refineR using the R package "refineR". RESULTS: According to the overlapping areas analysis the distributions/year of the data provided by 4 centers able to perform at least 1000 measurements/year were similar in 2 consecutive years. Moreover, the reference intervals provided by 2 centers using the same analytical methods in two separate locations over the three consecutive years, were very similar. The highest overlap (99.7 %) was observed in one center over two consecutive years. The overlapping areas were very high (93.6-95.7%) in 8 out of 9 inter-comparisons. CONCLUSION: Despite the limitations of this study the yearly distribution of the HbA2 measured in various centers appears a reliable tool to test HbA2 standardization over different centers using different analytical methods.

COVID-19: Black Swan or clumsy use?

"Black Swan" is an unexpected and catastrophic event characterized by three conditions: the anomalous and exceptional value, the extreme impact, and predictability a posteriori. On many sides - by politicians, commentators, scientists - there have been attempts to give the COVID-19 pandemic event the meaning of Black Swan. In reality, one should wonder if the Black Swan was not, instead (or even), the way the epidemic was handled. In Italy, an unreasonable use of a two-step protocol - IgM/IgG anti-COVID followed by RT-PCR in the serologically positive only has caused considerable uncertainty, particularly relevant in measures for maintenance or readmission to work of health personnel. This protocol could be affected by serious uncertainties arising from the analytical limitations of the tests used. To quantify this defect, we evaluated the analytical specifications of serological tests under different prevalence conditions. In conclusion, although laboratory diagnostics represent a useful tool, it can only be used for epidemiological purposes and not to provide healthy pass.

Mindray BC-6800 haematological analyser: 3D-DIFF scattergram usefulness in infectious mononucleosis diagnosis.

INTRODUCTION: 3D-DIFF scattergram of the Mindray BC-6800 haematological analyser shows morphological abnormalities and lymphocyte cluster splitting related to the presence of reactive lymphocytes. This study aims to assess whether these cytographic changes are useful in detecting both activated and apoptotic lymphocytes, leading to an improvement in the laboratory diagnostic process of infectious mononucleosis. METHODS: Two hundred three samples with modified shape and doubled lymphocyte cluster of DIFF scattergram (study group) were divided into two different subgroups: with and, respectively, without serological evidence of ongoing IM. Activated and apoptotic cells in peripheral blood were counted by light microscopy or gating in the instrumental dot plots. Values of apoptotic cells counted by microscopy were compared with those resulting from gating. RESULTS: Samples with both shape change and doubled lymphocyte cluster had serological profiles according to the diagnosis of ongoing infectious mononucleosis. Blood smears review was positive for reactive lymphocytes in all 112 samples (100%). An underestimation of apoptotic cell count by light microscopy compared with the gating in the instrumental scatterplot was also observed (96 out of 112, 85.7%). CONCLUSION: The additional lymphocyte cluster was significantly associated with activated and apoptotic lymphocytes in samples with serology suggesting ongoing infectious mononucleosis. Considering the significance of clue for infectious mononucleosis assigned to the apoptotic lymphocytes, a specific flag such as "apoptotic cells?" could be associate with the related cluster. Such a flag could be used for dedicated rules for smears review, thus increasing infectious mononucleosis detection in laboratories that do not usually practise instrumental cytograms observation.

Short preheating at 41°C leads to a red blood cells count comparable to that in RET channel of Sysmex analysers in samples showing cold agglutination.

AIMS: The presence of cold agglutinin in blood samples can cause a spontaneous agglutination of red blood cells (RBCs) when low temperature occurs. This phenomenon causes a spurious lowering of RBC count on the automated haematological analysers that are detected by incongruous values (≥370 g/L) of the mean cellular haemoglobi concentration (MCHC). A preheating at 37°C can remove the RBC agglutination generally resulting in a reliable count. It has been reported that the same result can be reached by using the optical reticulocyte (RET) channel of Sysmex analysers where the RBC count is not influenced by the presence of cold agglutinin. This study aims to evaluate these data in a larger population, with regard to environmental conditions on Sysmex analysers. We have also evaluated the influence of different thermal pretreatments on the RBC count. METHODS: This study was performed on 96 remnants of peripheral blood samples (48 with MCHC in normal range and 48 with MCHC>370 g/L) which have been analysed in different preanalytical conditions on the Sysmex analysers. RESULTS: A preheating of samples at 41°C for 1 min leads to a reversibility of the cold agglutination comparable to the one observed in the RET channel and yields better results compared with 37°C for 2 hours. CONCLUSIONS: None of described procedures assure the complete cold agglutination reversibility in every case. Consequently, since the haematological analysers not yet provide reliable parameters to confirm the complete resolution of agglutination, further verification of RBC count accuracy needs to be performed.

Automatic wedge smears preparation may cause traumatic morphological changes in peripheral blood cells.

In recent years, several automated analysers that prepare and stain blood smears have been introduced in clinical laboratories. Despite the use of instrumental settings based on physical characteristic of individual samples, traumatic injuries of neutrophil and lymphocytes can be observed. Some samples present a very high percentage of damaged cells, allowing the speculation that a cellular susceptibility may enhance mechanical traumatism. These artefacts can puzzle morphological evaluation in both traditional and digitised microscopy; in addition, unskilled operators can be misled.

A prolonged microscopic observation improves detection of underpopulated cells in peripheral blood smears.

We evaluated an extended time in the microscopic review in samples in which the potential clinical information could be increased with respect to those that could be achieved with the usual laboratory methodologies. We used samples containing nucleated red blood cells in a small amount and cytopenic samples. For these purposes for each peripheral blood smear, the timing of eye-count differential was increased up to 20 min, regardless of the final number of cells which could be counted. In addition, an automated system for digital analysis of peripheral blood smears was employed and the number of cells counted was brought up to 1000 leukocytes. In both manual and automatic light microscopy extended observation, we obtained more diagnostic information in respect to those with routine or standard methods. Both automated and manual increase systems of the timing for microscopic review are useful tools to find diagnostic information that otherwise would be lost using normal and standard procedures. So, these methods should be used especially when there is a higher pre-test probability for discovery of pathological cells.

Cytographic changes on BC-6800 Haematological Analyzer related to the presence of Candida albicans in peripheral blood. A new tool to suspect candidemia?

BACKGROUND: We studied the quantitative and cytographic changes that the presence of Candida albicans (C. albicans) in peripheral blood (PB) samples causes on the Mindray BC-6800 Haematological Analyzer. METHODS: A simulated in vitro candidemia was obtained by adding a different amount of C. albicans to discarded remnants of PB samples. Quantitative data and cytographic features were evaluated immediately as well as after 120 and 240 min of the yeast addition. A microscopic slides review was even performed at the same time. RESULTS: After yeasts addition, an increase of total leucocytes, neutrophils and basophils have been observed, but these increases are not certainly descriptive of C. albicans presence.Instead, extracellular blastospores cause a false increase in nucleated red blood cells (nRBCs), which appear as a new population in the specific counting channel for erytroblasts (NRBC channel). Regardless of the numbers, C. albicans form a pseudo-erythroblastic cluster in the NRBC channel whose resulting shape is so different than the 'normal' nRBC that it demands a microscopic review. Even cytographic changes related with the neutrophilic phagocytic activity have been observed on leucocyte's differential count citogram (DIFF) of the BC-6800. CONCLUSIONS: Our observations suggest that the results of the BC-6800, which are due to C. albicans' presence, might be useful to speculate earlier diagnosis of sepsis.