Efficiency, efficacy and subjective user satisfaction of alternative laboratory report formats. An investigation on behalf of the Working Group for Postanalytical Phase (WG-POST), of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM).

OBJECTIVES: Although laboratory result presentation may lead to information overload and subsequent missed or delayed diagnosis, little has been done in the past to improve this post-analytical issue. We aimed to investigate the efficiency, efficacy and user satisfaction of alternative report formats. METHODS: We redesigned cumulative (sparkline format) and single reports (improved tabular and z-log format) and tested these on 46 physicians, nurses and medical students in comparison to the classical tabular formats, by asking standardized questions on general items on the reports as well as on suspected diagnosis and follow-up treatment or diagnostics. RESULTS: Efficacy remained at a very high level both in the new formats as well as in the classical formats. We found no significant difference in any of the groups. Efficiency improved in all groups when using the sparkline cumulative format and marginally when showing the improved tabular format. When asking medical questions, efficiency and efficacy remained similar between report formats and groups. All alternative reports were subjectively more attractive to the majority of participants. CONCLUSIONS: Showing cumulative reports as a graphical display led to faster detection of general information on the report with the same level of correctness. Considering the familiarity bias of the classical single report formats, the borderline-significant improvement of the alternative tabular format and the non-inferiority of the z-log format, suggests that single reports might benefit from some improvements derived from basic information design.

The clinically effective use of cardiac markers by restructuring laboratory profiles at Cardiology wards.

Background: Laboratory overutilization is associated with diagnostic error and potential patient risk. We applied a demand management strategy in collaboration with the local Department of Cardiology to reduce the cardiac markers high-sensitive troponin T (hsTropT) and N-terminal pro brain natriuretic peptide (NTproBNP) in laboratory ordering profiles (LOPs). The present study aimed to retrospectively evaluate the implemented strategies. Methods: Strategies included educational measures and evidence-guided, active test de-selection from all cardiology ward LOPs, and/or permanent removal from LOPs. Tests remained available at all times. We evaluated overutilization by reductions in monthly orders, and assessed differences in 30-day all-cause readmission rate and length of patients' hospital stay. Results: Overall, we observed a mean reduction of 66.1% ± 7.6% (n = 277 ± 31) in hsTropT tests. Educational measures effectively reduced NTproBNP orders by 52.8% ± 17.7% (n = 60 ± 20). Permanent removal of tests from LOPs additionally decreased orders to a final extent of 75.8% ± 8.0% (n = 322 ± 31) in NTproBNP tests. The 30-day readmission rate and overall length of hospital stay did not increase. Conclusions: Our results indicate that cardiac markers in routine care are subject to extensive overutilization when used within LOPs. Educational measures are an effective strategy to overcome the overutilization of cardiac markers but may be more effective when combined with the removal of cardiac markers from LOPs.

Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.

BACKGROUND: Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology. METHODS: Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed. RESULTS: Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions. CONCLUSION: All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

Heparin and citrate additive carryover during blood collection.

Background Published evidence on the risk of additive carryover during phlebotomy remains elusive. We aimed to assess potential carryover of citrated and heparinized blood and the relative volume needed to bias clinical chemistry and coagulation tests. Methods We simulated standardized phlebotomies to quantify the risk of carryover of citrate and heparin additives in distilled water, using sodium and lithium as surrogates. We also investigated the effects of contamination of heparinized blood samples with increasing volumes of citrated blood and pure citrate on measurements of sodium, potassium, chloride, magnesium, total and ionized calcium and phosphate. Likewise, we studied the effects of contamination of citrated blood samples with increasing volumes of heparinized blood on heparin (anti-Xa) activity, lithium, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). We interpreted these results based on measurement deviations beyond analytical, biological and clinical significance. Results Standardized phlebotomy simulations revealed no significant differences in concentration of surrogate markers. Clinically significant alterations were observed after contamination of heparinized blood samples with volumes of citrated blood beyond 5-50 µL for ionized calcium and beyond 100-1000 µL for sodium, chloride and total calcium. Investigations of pure citrate carryover revealed similar results at somewhat lower volumes. Heparinized blood carryover showed clinically significant interference of coagulation testing at volumes beyond 5-100 µL. Conclusions Our results suggest that during a standardized phlebotomy, heparin or citrate contamination is highly unlikely. However, smaller volumes are sufficient to severely alter test results when deviating from phlebotomy guidelines.

To report or not to report: a proposal on how to deal with altered test results in hemolytic samples.

Preanalytically altered test results are a challenge every laboratory has to face. The release of such results may be to the harm of the patient by triggering wrong clinical decision making in monitoring or treatment. On the other hand, their deletion also might be to the harm of the patient by delaying the time to decision making as the exact value sometimes is not even necessary but rather an answer to the question "Is it raised or lowered". Based on this dilemma and forced to produce laboratory values without any clinical information on the respective patient, laboratories have developed their own preferred way on how to deal with preanalytically altered test results. Some release the value with a comment, some reject the value with or without a comment and others again provide only general information about the hemolytic sample. To date there is no guideline or standardization to this postanalytical topic. Therefore, with this opinion paper, we want to start the scientific discussion on this important issue by providing one possible method to overcome the lack of clinical information which the laboratory would need to correctly decide whether or not to release an altered test result. We suggest providing the clinician with all the information on the hemolytic sample and its impact on the respective parameter needed to make his/her own decision on the usage of the respective test result. We believe that reporting a preanalytically altered laboratory value including a respective comment is preferable to not reporting it.

The relationship between vacuum and hemolysis during catheter blood collection: a retrospective analysis of six large cohorts.

BACKGROUND: Blood collection through intravenous (IV) catheters is a common practice at emergency departments (EDs). This technique is associated with higher in vitro hemolysis rates and may even be amplified by the use of vacuum collection tubes. Our aim was to investigate the association of five different vacuum tubes with hemolysis rates in comparison to an aspiration system under real-life conditions and to propose an equation to estimate the amount of hemolysis, depending on the vacuum collection tube type. METHODS: We retrospectively evaluated hemolysis data of plasma samples from our ED, where blood is drawn through IV catheters. Over the past 5 years, we compared 19,001 hemolysis index values amongst each other and against the respective vacuum pressure (Pv) of the collection tubes, which were used within the six observational periods. RESULTS: The highest hemolysis rates were associated with full-draw evacuated tubes. Significantly reduced hemolysis was observed for two kinds of partial-draw tubes. The hemolysis rate of one partial-draw blood collection tube was comparable to those of the aspiration system. Regression analysis of Pv and mean free hemoglobin (fHb) values yielded the formula fHb (g/L)=0.0082*Pv2-0.1143*Pv+ 0.5314 with an R2 of 0.99. CONCLUSIONS: If IV catheters are used for blood collection, hemolysis rates directly correlate with the vacuum within the tubes and can be estimated by the proposed formula. By the use of partial-draw vacuum blood collection tubes, hemolysis rates in IV catheter collections can be reduced to levels comparable with collections performed by aspiration systems.

Hemolysis rates in blood samples: differences between blood collected by clinicians and nurses and the effect of phlebotomy training.

BACKGROUND: Hemolytic samples are one of the most challenging preanalytical issues in laboratory medicine. Even causes leading to hemolytic specimen are various, including phlebotomy practices. Respective educational interventions as well as the reduction of the number of people involved in blood collections are claimed to influence the sample quality for the better. In our hospital 70 junior doctors were in charge of routine phlebotomy until 2012, when this task was shifted to 874 nurses, including a preceding training in phlebotomy and preanalytics. Our aim was to evaluate the impact of this training effect and the increase of people involved on sample quality. METHODS: The hemolysis index (HI) of 43,875 samples was measured before (n=21,512) and after (n=22,363) the switch of blood collection responsibilities. Differences in overall hemolysis rates and the amount of plasma samples with a concentration of free hemoglobin (fHb) above 0.5 g/L and 1 g/L were calculated. RESULTS: Overall HI as well as the percentage of samples with an fHb concentration >0.5 g/L decreased after the responsibility for phlebotomy changed. The rate of samples with an fHb concentration >1 g/L remained unchanged. CONCLUSIONS: Hemolysis rates were reduced upon passing phlebotomy tasks from untrained physicians on to a trained nursing staff. We therefore conclude that the number of people performing phlebotomy seems to play a minor role, compared to the effect of a standardized training. However, whether a reduction in the number of people involved in blood collection could lead to further improvement of sample quality, remains to be investigated in future studies.

Relevance of EDTA carryover during blood collection.

BACKGROUND: The order of draw is regarded as a preanalytical issue to prevent carryover of additives during blood collection. Our objective was to prove the theory of ethylenediaminetetraacetic acid (EDTA) carryover for a closed vacuum system and the influence of EDTA on concentrations of selected biomarkers. METHODS: To test the carryover of EDTA, a blood collection with tripotassium EDTA (K3EDTA) and subsequent non-additive tubes was simulated using distilled water as substitute for blood. EDTA concentrations were measured by tandem mass spectrometry. Then we added increasing concentrations of EDTA to heparinized blood and measured routine biomarkers, thereby simulating a carryover of EDTA whole blood and pure EDTA, respectively. Additionally, we tested for EDTA contamination and biomarker alteration in samples collected from 10 healthy volunteers by a syringe with subsequent transfer into sample tubes. RESULTS: No EDTA contamination was detected in samples collected subsequent to a K3EDTA tube when adhering to guidelines of blood sampling. Magnesium, calcium, and potassium levels were altered by artificial K3EDTA whole-blood contamination as well as when adding 1 µL pure K3EDTA. Iron values were altered at EDTA concentrations of 4.4 mmol/L. All other parameters remained unaffected. A slight EDTA carryover was observed in syringe collection and subsequent transfer into EDTA and heparin tubes, however, without any biomarker alteration. CONCLUSIONS: An EDTA carryover during blood collection using a closed vacuum system is highly unlikely. Even if carryover of EDTA whole blood occurs, an absolute volume larger than 10 µL would be necessary to alter test results. However, contamination of samples with preloaded pure K3EDTA solution by severe neglect of current recommendations in blood collection may significantly alter testing results.

Collection efficiency of mononuclear cells in offline extracorporeal photopheresis: can processing time be shortened?

BACKGROUND: Extracorporeal photopheresis (ECP) is a well-established but lengthy and burdensome cell-based therapy for various diseases such as cutaneous T-cell lymphoma, graft-versus-host disease and organ rejection after transplantation. The number of mononuclear cells (MNCs) that needs to be collected to obtain a clinical response to ECP is still under debate. The purpose of this retrospective study was to determine the number of lymphocytes, monocytes and neutrophils in mononuclear cell products (MCP) by flow cytometry and the collection efficiency in the offline ECP setting. MATERIALS AND METHODS: We collected data from 10 different patients undergoing 162 ECP procedures using the Spectra Optia device for MNC collection. White blood cell (WBC) count of MCP was determined using a hematology analyzer. MNCs were analyzed for CD45 and CD14 expression by flow cytometry to exactly determine the collected lymphocyte and monocyte fractions. RESULTS: Collected MCP showed high cell yields with 55.3×106/kg MNCs and 41.1×106/kg lymphocytes. MCP were characterized by high MNC (81.3%) and low neutrophils (18.7%) percentage. Mean collection efficiency for WBCs and for MNCs was 23.9% and 62.0%, respectively. The MNC fraction showed a moderate to high correlation between peripheral blood cell count of patients and MCP count. DISCUSSION: This study is one of a few reports showing the monocyte-to-lymphocyte relation in MCP for ECP determined by flow cytometry. In comparison to historical data from inline ECP, the offline ECP processing one total blood volume results in considerably higher cell yields. For this reason, and to reduce the burden on patients, we propose that the offline ECP processing time can be substantially reduced.

Diagnostic Workup of Microcytic Anemia: An Evaluation of Underuse or Misuse of Laboratory Testing in a Hospital Setting Using the AlinIQ System.

CONTEXT.­: Underuse of laboratory testing has been previously investigated in preselected populations, such as documented malpractice claims. However, these numbers might not reflect real-life situations. OBJECTIVE.­: To evaluate the underuse and misuse of laboratory follow-up testing in a real-life hospital patient population with microcytic anemia, using laboratory results ordered during routine patient care. DESIGN.­: From all patients in whom a microcytic anemia was detected during routine diagnostics in 2018, all available laboratory data were collected and screened for appropriateness of diagnostic workup of iron deficiency and thalassemia. Subgroup analysis was performed for patient groups with mean corpuscular volume values 75 to 79 µm3 (group 1), 65 to 74 µm3 (group 2), and <65 µm3 (group 3). RESULTS.­: A total of 2244 patients with microcytic anemia were identified. Follow-up testing for iron deficiency was not performed in 761 cases (34%). For inconclusive ferritin levels due to elevated C-reactive protein results (n = 336), reticulocyte hemoglobin content or soluble transferrin receptor levels were missing in 86 cases (26%). In patients with suspected thalassemia (n = 127), follow-up testing for hemoglobin variants was not performed in 70 cases (55%). Subgroup analysis showed that the frequency of underuse of iron status as well as thalassemia/hemoglobinopathy testing decreased from group 1 to group 3. When considering relevant preexisting anemia diagnoses, laboratory tests were underused in 904 cases (40.3%). CONCLUSIONS.­: Because 40% (n = 904) of the patients with microcytic anemia were potentially not followed up correctly, laboratory specialists are advised to act by implementing demand management strategies in collaboration with clinicians to overcome underuse of laboratory tests and to improve patient safety.

Long-term immunological consequences of anti-CD20 therapies on humoral responses to COVID-19 vaccines in multiple sclerosis: an observational study.

Background: Anti-CD20 therapies induce pronounced B-cell depletion and blunt humoral responses to vaccines. Recovery kinetics of anti-CD20 therapy-mediated cellular and humoral effects in people with multiple sclerosis (pwMS) are poorly defined. Objective: To investigate the duration of the anti-CD20 treatment-induced effects on humoral responses to COVID-19 vaccines. Methods: This retrospective observational study included pwMS who had discontinued anti-CD20 therapy for ⩾12 months and remained without immunomodulation. We retrieved demographics and laboratory parameters including B-cell counts and immunoglobulin (IgG, IgM, IgA) levels prior to anti-CD20 commencement (baseline) and longitudinally after anti-CD20 treatment discontinuation from electronic medical records. Humoral responses to SARS-CoV-2 vaccines were compared with a population of 11 pwMS with ongoing anti-CD20 medication (control cohort). Results: A total of 24 pwMS had discontinued anti-CD20 therapy for a median of 34 months (range: 16-38 months). Antibody responses to COVID-19 vaccines were available in 17 (71%). Most individuals (n = 15, 88%) elicited a measurable antibody response [mean: 774 BAU/ml (±SD 1283 BAU/ml)] to SARS-CoV-2 immunization on average 22 months (range: 10-30 months) from the last anti-CD20 infusion, which was higher compared with the population with ongoing anti-CD20 therapy (n = 11, mean: 12.36 ± SD 11.94 BAU/ml; p < 0.00001). Significantly increased antibody levels compared with the control cohort were found among pwMS who were vaccinated >18 months after treatment discontinuation (19-24 months: n = 2, p = 0.013; 25-36 months: n = 9; p < 0.001). The interindividual kinetics for B-cell reconstitution were heterogeneous and mean B-cell counts approached normal ranges 18 months after treatment discontinuation. There was no correlation of B-cell repopulation and vaccine responses. Mean total IgG, IgM, and IgA levels remained within the reference range. Conclusion: Anti-CD20-induced inhibition of humoral responses to COVID-19 vaccines is transient and antibody production was more pronounced >18 months after anti-CD20 treatment discontinuation. The immunological effect on B-cell counts appears to wane by the same time.

Acidification of 24-hour urine in urolithiasis risk testing: An obsolete relic?

BACKGROUND: Recommendations on the optimal preservation of 24 h urine for the metabolic work-up in urolithiasis patients are very heterogeneous. In case two such tests with different storage condition recommendations are being analysed, multiple collections would be needed, challenging especially elderly and very young patients. We therefore aimed to evaluate the stability of urine constituents under different storage conditions. MATERIAL AND METHODS: We collected urine samples from ten healthy volunteers and prepared aliquots to be stored either at room temperature or 4 °C. Some aliquots were preserved using hydrochloric acid prior to storage, some thereafter, some using the BD Urine preservation tube and some were not preserved at all. Storage duration was 0, 24, 48 or 72 h. In all samples calcium, magnesium, phosphorus, creatinine, oxalate, citrate and uric acid were measured and compared to the according reference sample. RESULTS: We could not find any significant deviation for any of the analytes and preanalytical treatment conditions compared to the associated reference sample. CONCLUSION: Preservation of 24 h urine for the metabolic evaluation in stone formers might not be necessary for sample storage up to 72 h.

Biopsy procedures for molecular tissue diagnosis of invasive fungal infections.

The incidence of invasive fungal infections (IFI) has increased substantially and the epidemiology has changed dramatically in recent years. Candida albicans is still most important, but non-albicans species, Aspergillus species, Glomeromycota (formerly Zygomycetes) and Fusarium species are an increasing cause of IFIs. Due to this growing diversity, the identification of the causative organism to genus and species level is important to perform best and adequate treatment. The early, sensitive and specific detection of IFIs remains challenging and current conventional methods are limited. The golden standard for the definite diagnosis of proven pulmonary infection remains either histopathologic, cytopathologic or direct tissue examination. Invasive procedures are necessary to obtain reliable specimens and biopsies may be taken percutaneously, bronchoscopically, via open surgery or via video-assisted thorascopic surgery. Molecular methods, like PCR or in situ hybridization, are a promising diagnostic tool for rapid and reliable species identification and should be performed in addition to microscopic examination and culture to increase the sensitivity for the diagnosis of IFI. Combining culture, microscopy, serology, and PCR in lung tissues and/or bronchial samples will increase the diagnostic yield by 99%. Here, we give an overview of biopsy procedures for molecular tissue diagnosis of IFI.

Laboratory Demand Management Strategies-An Overview.

Inappropriate laboratory test selection in the form of overutilization as well as underutilization frequently occurs despite available guidelines. There is broad approval among laboratory specialists as well as clinicians that demand management strategies are useful tools to avoid this issue. Most of these tools are based on automated algorithms or other types of machine learning. This review summarizes the available demand management strategies that may be adopted to local settings. We believe that artificial intelligence may help to further improve these available tools.

In Vitro Susceptibility Testing in Fungi: What is its Role in Clinical Practice?

An increasing number of patients are undergoing transplantation procedures or receiving aggressive immunosuppression and chemotherapy. The growing population of immunocompromised hosts has led to a rise in the prevalence of invasive fungal infections due to yeasts and molds. The introduction of new antifungal agents and recent reports of resistance emerging during treatment of fungal infections have highlighted the need for in vitro susceptibility testing. Various testing procedures have been proposed, including macrodilution and microdilution, agar diffusion, disk diffusion, and Etest (AB Biodisk, Solna, Sweden). Establishing clinical correlation with antifungal susceptibility testing, however, is a huge challenge because susceptibility techniques do not take into account the dynamic and complex biology of fungi exposed to an antifungal in vivo. This paper reviews the available methods for antifungal susceptibility testing of yeasts and filamentous fungi and the data regarding the clinical implications of in vitro testing.

Errors within the total laboratory testing process, from test selection to medical decision-making - A review of causes, consequences, surveillance and solutions.

Laboratory analyses are crucial for diagnosis, follow-up and treatment decisions. Since mistakes in every step of the total testing process may potentially affect patient safety, a broad knowledge and systematic assessment of laboratory errors is essential for future improvement. In this review, we aim to discuss the types and frequencies of potential errors in the total testing process, quality management options, as well as tentative solutions for improvement. Unlike most currently available reviews on this topic, we also include errors in test-selection, reporting and interpretation/action of test results. We believe that laboratory specialists will need to refocus on many process steps belonging to the extra-analytical phases, intensifying collaborations with clinicians and supporting test selection and interpretation. This would hopefully lead to substantial improvements in these activities, but may also bring more value to the role of laboratory specialists within the health care setting.