Pseudo-NRBC in the Mindray BC-6800Plus analyzer: A clue for diagnostic anticipation of fungemia. Experimental and preliminary clinical reports.

INTRODUCTION: Candidemia can be a significant cause of death in immunosuppressed or debilitated patients particularly. Abnormalities of the instrumental cytograms of some hematological analyzers, such as Mindray BC-6800Plus, can be related to circulating Candida. We studied the possible diagnostic usefulness of this information. METHODS: A fungal bloodstream infection has been simulated by adding aliquots of Candida albicans, Candida parapsilosis, and Candida glabrata to 75 leftovers and anonymized peripheral blood samples. Cytographic abnormalities like those of experimental samples were used to select patients with possible fungemia. The microscopic review of peripheral blood smears constituted the confirmatory method. RESULTS: In all experimental samples, the various Candida types caused pseudo-NRBC and morphological abnormalities of WNB and DIFF cytograms. Circulating blastospores, free or engulfed by neutrophils, were the microscopic findings in the peripheral blood smears. In the clinical verification, 72 patients were recruited based on the presence of an evocative cluster in the WNB and DIFF cytograms. The microscopic review of 39 out of 72 samples was positive for NRBC. According to blood cultures, light microscopy revealed fungal forms of several Candida or non-Candida types in the remaining 33 samples. Nine of these cases were not yet known to suffer from bloodstream infection. CONCLUSIONS: Although further confirmatory clinical studies are required for these diagnostic abilities, the BC 6800Plus cytographic abnormalities related to fungemia have proven helpful in rapidly monitoring persistent fungemia in already diagnosed patients. In unknown or undiagnosed cases, they could be the trigger point for the subsequent diagnostic-therapeutic pathway.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy.

SARS-CoV-2 is constantly evolving, leading to new variants. We analysed data from 4400 SARS-CoV-2-positive samples in order to pursue epidemiological variant surveillance and to evaluate their impact on public health in Italy in the period of April-December 2021. The main circulating strain (76.2%) was the Delta variant, followed by the Alpha (13.3%), the Omicron (5.3%), and the Gamma variants (2.9%). The B.1.1 lineages, Eta, Beta, Iota, Mu, and Kappa variants, represented around 1% of cases. There were 48.2% of subjects who had not been vaccinated, and they had a lower median age compared to the vaccinated subjects (47 vs. 61 years). An increasing number of infections in the vaccinated subjects were observed over time, with the highest proportion in November (85.2%). The variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed with the Delta variant, while subjects harbouring the Gamma variant showed the highest proportion of asymptomatic infection (21.6%), albeit also deaths (5.4%). The Omicron variant was only found in the vaccinated subjects, of which 47% had been hospitalised. The diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at the national and international levels. Our study provides data on the rapid changes in the epidemiological landscape of the SARS-CoV-2 variants in Italy.

A peptide-based assay discriminates individual antibody response to SARS-CoV-2.

SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. Understanding the antibody response to SARS-CoV-2 is crucial for the development of vaccines, therapeutics and public health interventions. However, lack of consistency in methods used to monitor antibody response to SARS-CoV-2 leaves some uncertainty in our fine understanding of the human antibody response mounted following SARS-CoV-2 infection. We developed a peptide-based enzyme-linked immunosorbent assay (ELISA) by selecting 7 synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of SARS-CoV-2, which effectively detects the antibody response mounted by all COVID-19 convalescent tested. Strikingly, the assay shows a profound difference in antibody response among individual subjects, which may have a significant impact on disease severity. Together, our results define an efficient and specific serological assay to consistently measure the antibody response following SARS-CoV-2 infection, as well as help the design of vaccine and therapeuticals for prevention and treatment of COVID-19.

A Diagnostic Model to Predict SARS-CoV-2 Positivity in Emergency Department Using Routine Admission Hematological Parameters.

Early detection of SARS-CoV-2 in the emergency department (ED) is a crucial necessity, especially in settings of overcrowding: establishing a pre-diagnostic test probability of infection would help to triage patients and reduce diagnostic errors, and it could be useful in resource-limited countries. Here, we established and validated a clinical predictor of infection based on routine admission hematological parameters. The diagnostic model was developed by comparing 85 consecutive patients with symptomatic COVID-19 confirmed by RT-PCR with 85 symptomatic, SARS-CoV-2-negative controls. Abnormal hematological parameters significantly (p < 0.05) associated with SARS-CoV-2 infection were used to derive a "cumulative score" between 0 and 16. The model was validated in an independent cohort of 170 SARS-CoV-2-positive patients. Several routine hematology parameters were significantly (p < 0.05) associated with SARS-CoV-2 infection. A "cumulative score" score ≥7 discriminated COVID-19-postive patients from controls with a sensitivity of 94% and specificity of 100% (p < 0.001). The high sensitivity of the predictive model was confirmed in the prospective validation set, and the cumulative score (i) predicted SARS-CoV-2 positivity even when the first oro-nasopharyngeal swab RT-PCR result was reported as a false negative in both cohorts and (ii) resulted to be independent from disease severity. The cumulative score based on routine blood parameters can be used to predict an early and accurate diagnosis of SARS-CoV-2 infection in symptomatic patients, thereby facilitating triage and optimizing early management and isolation from the COVID-19 free population, particularly useful in overcrowding situations and in resource-poor settings.

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.

The diagnostic use of ADVIA 2120i Siemens and an "APL criteria" can help to reduce the rate of early death in the APL.

INTRODUCTION: Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) with a life-threatening coagulopathy. Once it is suspected, ATRA should be started. Appreciation of APL details is critical, but an experienced hematopathologist may not be available. We developed an algorithm, based on the parameters generated by automated blood cell counter ADVIA 2120i Siemens that can aid the diagnosis of APL. METHODS: All parameters in the algorithm were selected on the bases of the pathophysiology of the APL and the analyzer's technology. We used c1 cutoff: PLT < 150 × 103 ; % Mono<10; % LUC<4; % hyperchromic cells > 2; %saturated cells ≥ 1; % blasts > 4. Satisfying at least five of six cutoffs, we obtained an "APL criteria". It was tested on 247.209 raw-data from routine and emergency samples and on 124 raw-data from APL. We performed a multiparametric analyses and obtained definitive cutoffs (c2). It was validated on a new group of 51.002 raw-data from routine and emergency. Finally, it was tested on AML and ALL, MDS, MPN, oncologic samples, and hemolytic and megaloblastic anemia. RESULTS: The algorithm provided a high sensitivity (86.30%) and high specificity (99.83%) in APL with high or normal number of WBC and satisfactory results in APL with cytopenia (80%). The specificity was very high also in groups of hematological and nonhematological diseases. It can be used as an "APL criteria" to alert pathologists of the possible presence of APL. It together with instrumental and classical morphology may allow to reduce the time of diagnosis with further reduction in early death.

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.

Detection of Apoptotic Lymphocytes Through Sysmex XN-1000 As a Diagnostic Marker for Mononucleosis Syndrome.

BACKGROUND: The infectious mononucleosis (IM) includes clue elements, apoptotic and atypical lymphocytes. In IM, the evaluation of dot plot provided by Sysmex XN-1000 analyzer revealed a stretched lymphocytic cluster, white cell differential channel (WDF), on cytogram. METHODS: In this study, we analyzed 698 samples that include 39 IM, 76 chronic lymphoproliferative disorders, 25 nonclonal lymphocytosis, and 40 healthy donors. Five hundred eighteen samples with other diseases or interference were evaluated. The algorithm was validated on 40,000 files that were received from internal database of Sysmex-Dasit. RESULTS: The analysis of flow cytometry standard (FCS) files in WDF channel and presumed apoptotic lymphocytes counts on side scatter/forward scatter (SSC/FSC) and SSC/SFL (where SFL is side fluorescence) dot plot revealed excellent correlation among apoptotic cells on peripheral blood smear (R(2)  = 0.79 and 0.75). There was a variation of positional parameters in lymphocyte clusters WX, WY, and WZ. If WX-SSC > 500 and WY-SFL > 1,000 and WZ-FSC > 700, specificity equals to 99% and sensitivity equals to 100%. If nucleated red blood cell (NRBC) <0.03 × 10(3) /µl, specificity equals to 100%. In received files, positives were 1% adding the simultaneous presence of a percentage of events in the two gates relating to the apoptotic lymphocytes of 1.2% of WBC. CONCLUSION: On Sysmex XN-1000, dot-plot observation allowed immediate detection of IM. Meanwhile, an algorithm based on the parameters on these plots can be calculated with excellent performance.