Point-of-care testing: state-of-the art and perspectives.

Point-of-care testing (POCT) is becoming an increasingly popular way to perform laboratory tests closer to the patient. This option has several recognized advantages, such as accessibility, portability, speed, convenience, ease of use, ever-growing test panels, lower cumulative healthcare costs when used within appropriate clinical pathways, better patient empowerment and engagement, and reduction of certain pre-analytical errors, especially those related to specimen transportation. On the other hand, POCT also poses some limitations and risks, namely the risk of lower accuracy and reliability compared to traditional laboratory tests, quality control and connectivity issues, high dependence on operators (with varying levels of expertise or training), challenges related to patient data management, higher costs per individual test, regulatory and compliance issues such as the need for appropriate validation prior to clinical use (especially for rapid diagnostic tests; RDTs), as well as additional preanalytical sources of error that may remain undetected in this type of testing, which is usually based on whole blood samples (i.e., presence of interfering substances, clotting, hemolysis, etc.). There is no doubt that POCT is a breakthrough innovation in laboratory medicine, but the discussion on its appropriate use requires further debate and initiatives. This collective opinion paper, composed of abstracts of the lectures presented at the two-day expert meeting "Point-Of-Care-Testing: State of the Art and Perspective" (Venice, April 4-5, 2024), aims to provide a thoughtful overview of the state-of-the-art in POCT, its current applications, advantages and potential limitations, as well as some interesting reflections on the future perspectives of this particular field of laboratory medicine.

Point-of-care testing performed by healthcare professionals outside the hospital setting: consensus based recommendations from the IFCC Committee on Point-of-Care Testing (IFCC C-POCT).

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Point-of-Care Testing (C-POCT) supports the use of point-of-care testing (POCT) outside of the hospital setting performed by healthcare professionals without formal laboratory education because of its numerous benefits. However, these benefits are associated with risks that must be managed, to ensure the provision of reliable test results and minimize harm to the patient. Healthcare professionals, local regulatory bodies, accredited laboratories as well as manufacturers should actively be engaged in education, oversight and advice to ensure that the healthcare professional selects the appropriate equipment and is able to analyze, troubleshoot and correctly interpret the point-of-care (POC) test results.

Artificial Intelligence in Point-of-Care Testing.

With the projected increase in the global population, current healthcare delivery models will face severe challenges. Rural and remote areas, whether in developed or developing countries, are characterized by the same challenges: the unavailability of hospitals, lack of trained and skilled staff performing tests, and poor compliance with quality assurance protocols. Point-of-care testing using artificial intelligence (AI) is poised to be able to address these challenges. In this review, we highlight some key areas of application of AI in point-of-care testing, including lateral flow immunoassays, bright-field microscopy, and hematology, demonstrating this rapidly expanding field of laboratory medicine.

Comparison of BNT162b2-, mRNA-1273- and Ad26.COV2.S-Elicited IgG and Neutralizing Titers against SARS-CoV-2 and Its Variants.

Because the vaccine-elicited antibody and neutralizing activity against spike protein of SARS-CoV-2 are associated with protection from COVID-19, it is important to determine the levels of specific IgG and neutralization titers against SARS-CoV-2 elicited by the vaccines. While three widely used vaccine brands (Pfizer-BNT162b2, Moderna-mRNA-1273 and Johnson-Ad26.COV2.S) are effective in preventing SARS-CoV-2 infection and alleviating COVID-19 illness, they have different efficacy against COVID-19. It is unclear whether the differences are due to varying ability of the vaccines to elicit a specific IgG antibody response and neutralization activity against spike protein of the virus. In this study, we compared the plasma IgG and neutralization titers against spike proteins of wild-type SARS-CoV-2 and eight variants in healthy subjects who received the mRNA-1273, BNT162b2 or Ad26.COV2.S vaccine. We demonstrated that subjects vaccinated with Ad26.COV2.S vaccine had significantly lower levels of IgG and neutralizing titers as compared to those who received the mRNA vaccines. While the linear regression analysis showed a positive correlation between IgG levels and neutralizing activities against SARS-CoV-2 WT and the variants, there was an overall reduction in neutralizing titers against the variants in subjects across the three groups. These findings suggest that people who received one dose of Ad26.COV2.S vaccine have a more limited IgG response and lower neutralization activity against SARS-CoV-2 WT and its variants than recipients of the mRNA vaccines. Thus, monitoring the plasma or serum levels of anti-SARS-CoV-2 spike IgG titer and neutralization activity is necessary for the selection of suitable vaccines, vaccine dosage and regimens.

Epigallocatechin gallate from green tea effectively blocks infection of SARS-CoV-2 and new variants by inhibiting spike binding to ACE2 receptor.

BACKGROUND: As the COVID-19 pandemic rages on, the new SARS-CoV-2 variants have emerged in the different regions of the world. These newly emerged variants have mutations in their spike (S) protein that may confer resistance to vaccine-elicited immunity and existing neutralizing antibody therapeutics. Therefore, there is still an urgent need of safe, effective, and affordable agents for prevention/treatment of SARS-CoV-2 and its variant infection. RESULTS: We demonstrated that green tea beverage (GTB) or its major ingredient, epigallocatechin gallate (EGCG), were highly effective in inhibiting infection of live SARS-CoV-2 and human coronavirus (HCoV OC43). In addition, infection of the pseudoviruses with spikes of the new variants (UK-B.1.1.7, SA-B.1.351, and CA-B.1.429) was efficiently blocked by GTB or EGCG. Among the 4 active green tea catechins at noncytotoxic doses, EGCG was the most potent in the action against the viruses. The highest inhibitory activity was observed when the viruses or the cells were pre-incubated with EGCG prior to the infection. Mechanistic studies revealed that EGCG blocked infection at the entry step through interfering with the engagement of the receptor binding domain (RBD) of the viral spikes to angiotensin-converting enzyme 2 (ACE2) receptor of the host cells. CONCLUSIONS: These data support further clinical evaluation and development of EGCG as a novel, safe, and cost-effective natural product for prevention/treatment of SARS-CoV-2 transmission and infection.

Best Laboratory Practices Regarding POCT in Different Settings (Hospital and Outside the Hospital).

Point-of-care testing is proliferating at an alarming rate as technological improvements in miniaturization coupled with the need for rapid diagnostics drive the market globally. This review highlights best laboratory practices that must be communicated to the diverse group of people employing POC testing in their respective settings both inside and outside the hospital setting so that reliable results can be obtained.

Catering for the Point-of-Care Testing Explosion.

The ease of performing a laboratory test near to the patient, at the point-of-care, has resulted in the integration of point-of-care tests into healthcare treatment algorithms. However, their importance in patient care necessitates regular oversight and enforcement of best laboratory practices. This review discusses why this oversight is needed, it's importance in ensuring quality results and processes that can be placed to ensure point-of-care tests are chosen carefully so that both oversight can be maintained and patient care is improved. Furthermore, it highlights the importance of delivering focused webinars and continuing education in a variety of formats.

Exosomes Transport Anti-Human Immunodeficiency Virus Factors from Human Cervical Epithelial Cells to Macrophages.

The female reproductive tract (FRT) is a major site of HIV sexual transmission. As the outermost layer of cells in the FRT, the human cervical epithelial cells (HCEs) have direct contact with HIV or infected cells. Our early work showed that supernatant (SN) from TLR3-activated HCEs contain the antiviral factors that could potently inhibit HIV replication in macrophages. However, it remains to be determined how HCEs transport the anti-HIV factors to macrophages. This follow-up study examined the role of exosomes in HCE-mediated anti-HIV activity. We found that TLR3 activation of HCEs resulted in the release of exosomes that contained multiple IFN-stimulated genes (ISGs: ISG56, OAS1, MxA, and Mx2) and the HIV restriction microRNAs (miR-28, miR-29 family members, miR-125b, miR-150, miR-382, miR-223, miR-20a, and miR-198). The depletion of exosomes from SN of TLR3-activated HCEs diminished HCE-mediated anti-HIV activity in macrophages, indicating that HCE-derived exosomes are responsible for transporting the antiviral molecules to macrophages. These in vitro findings suggest a novel antiviral mechanism by which HCEs participate in the FRT innate immunity against HIV infection. Further in vivo studies are necessary in order to develop an exosome-based delivery system for prevention and treatment of HIV infection through sexual transmission.

Multiple Myeloma: The Case of the Disappearing Band.

This case study presents a patient with multiple myeloma whose serum specimen exhibits 2 distinct bands in serum protein electrophoresis but only one band in immunofixation electrophoresis. This latter, single band corresponds to the M-spike. An investigation is presented to determine the identity of this disappearing or phantom band. Furthermore, this case is used as a teaching point to explain the criteria used for staging multiple myeloma, how a cell can become a myeloma propagating cell, methods that can be used to identify unexpected bands in serum protein electrophoresis, possible explanations for bands in the beta region, the usual treatment regimens in multiple myeloma and finally specimen collecting and handling procedures for serum protein electrophoresis.

False positive acetaminophen concentrations in patients with liver injury.

BACKGROUND: Acetaminophen toxicity is the most common form of acute liver failure in the U.S. After acetaminophen overdoses, quantitation of plasma acetaminophen can aid in predicting severity of injury. However, recent case reports have suggested that acetaminophen concentrations may be falsely increased in the presence of hyperbilirubinemia. METHODS: We tested sera obtained from 43 patients with acute liver failure, mostly unrelated to acetaminophen, utilizing 6 different acetaminophen quantitation systems to determine the significance of this effect. In 36 of the 43 samples with bilirubin concentrations ranging from 1.0-61.5 mg/dl no acetaminophen was detectable by gas chromatography-mass spectroscopy. These 36 samples were then utilized to test the performance characteristics of 2 immunoassay and 4 enzymatic-colorimetric methods. RESULTS: Three of four colorimetric methods demonstrated 'detectable' values for acetaminophen in from 4 to 27 of the 36 negative samples, low concentration positive values being observed when serum bilirubin concentrations exceeded 10 mg/dl. By contrast, the 2 immunoassay methods (EMIT, FPIA) were virtually unaffected. The false positive values obtained were, in general, proportional to the quantity of bilirubin in the sample. However, prepared samples of normal human serum with added bilirubin showed a dose-response curve for only one of the 4 colorimetric assays. CONCLUSIONS: False positive acetaminophen tests may result when enzymatic-colorimetric assays are used, most commonly with bilirubin concentrations >10 mg/dl, leading to potential clinical errors in this setting. Bilirubin (or possibly other substances in acute liver failure sera) appears to affect the reliable measurement of acetaminophen, particularly with enzymatic-colorimetric assays.

The variability of results between point-of-care testing glucose meters and the central laboratory analyzer.

CONTEXT: Point-of-care testing glucose meters are strongly recommended in the management of diabetes and are increasingly being used for making therapeutically important decisions. Thus, it is essential that their results correlate well with those of laboratory analyzers. OBJECTIVES: To test the reliability of point-of-care testing glucose meters. DESIGN: Two studies were performed: (1), an in-house study comparing accuracy of point-of-care testing glucose meters with a reference analyzer using fresh whole blood specimens (2), a real-time comparison of (a) 2 successive glucose meter readings and (b) glucose meter reading to central laboratory analyzer reading. SETTING: (1), Seven glucose meters from 4 manufacturers were compared with the Yellow Springs YSI 2300 blood glucose analyzer using whole blood without preservative. (2), (a) Whole blood samples were read within 5 minutes of each other using Accu-Chek meters and (b) between a glucose meter and a Hitachi laboratory analyzer. RESULTS: (1) Within the Accu-Chek group of glucose meters, fresh, preservative-free whole blood samples showed the lowest bias. (2) At the hypoglycemic level, successive glucose meter readings agreed well, but there was considerable disagreement between glucose meter and central laboratory values. Because laboratory analyzers are of proven accuracy, they are used as the reference. In the glucose meter-central laboratory analyzer correlation, for both hypoglycemic and hyperglycemic values, readings in which the differences were greater than 10% occurred more than 61% of the time. In the hypoglycemic range, differences greater than 20% occurred 57% of the time. CONCLUSIONS: One should scrutinize point-of-care testing glucose meter readings at the hypoglycemic and hyperglycemic levels and whenever possible to corroborate these clinical results with central laboratory analyzers.

Modified plasma-based ecarin clotting time assay for monitoring of recombinant hirudin during cardiac surgery.

Recombinant hirudin (r-hirudin) is being used increasingly for therapeutic anticoagulation in patients with heparin-induced thrombocytopenia undergoing cardiovascular surgery. Although multiple laboratory methods are available for measuring r-hirudin, the ecarin clotting time (ECT) is the most commonly used for this purpose. Ecarin (extracted from snake venom) converts prothrombin to meizothrombin, which promotes clot formation. Direct thrombin inhibitors, like r-hirudin, bind meizothrombin and yield a linear, dose-dependent prolongation of ECT. Low levels of prothrombin and fibrinogen in plasma samples can lead to higher ECT; suggesting falsely elevated r-hirudin levels. A modified ECT assay with prothrombin and fibrinogen in excess was optimized using an orthogonal array method to eliminate the variations in patients' plasma prothrombin and/or fibrinogen levels for accurate determinations of plasma r-hirudin levels. By using the modified ECT assay, falsely elevated r-hirudin levels can be avoided in patients undergoing cardiopulmonary bypass, thus providing reliable and accurate r-hirudin monitoring in this clinical setting.

Lipopolysaccharide: a p38 MAPK-dependent disrupter of neutrophil chemotaxis.

In sepsis, and in models of sepsis including endotoxemia, impaired neutrophil recruitment and chemotaxis have been reported. The inability of the endotoxemic neutrophil to chemotax could be attributed to the fact that intracellular signaling via LPS overrides signals from endogenous chemokines or, alternatively, that sequestration of neutrophils into lungs prevents access to peripheral tissues. Using both in vitro and in vivo chemotaxis assays the authors established that neutrophils from healthy mice chemotaxed in vivo toward MIP-2, whereas endotoxemic neutrophils did not. Since LPS activates leukocytes via the p38 MAPK pathway, SKF86002, a p38 MAPK inhibitor, was given to endotoxemic animals. SKF86002 significantly reversed the LPS-induced impairment in emigration of endotoxic neutrophils in response to MIP-2. Neutrophil chemotaxis in vitro was also impaired by LPS, via a p38 MAPK-dependent pathway, and this impairment could be reversed via p38 MAPK inhibition. Although neutrophil numbers dropped in the circulation and trapped in lungs during endotoxemia, SKF86002 did not reverse these parameters, demonstrating that p38 MAPK inhibition did not release trapped neutrophils from the lungs. In conclusion, the data suggest that the impaired emigration and chemotaxis of neutrophils at peripheral sites during endotoxemia may be partially due to a p38 MAPK-mediated inhibition of neutrophil responses to endogenous chemokines.

Role of CD44 and hyaluronan in neutrophil recruitment.

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44(-/-) and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44(-/-) mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44(-/-) mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44(-/-) mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44(-/-) mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.

L-Selectin ligands in lymphoid tissues and models of inflammation.

Both lymphocyte recirculation through the lymphoid tissues and leukocyte recruitment to sites of inflammation are essential components of immune surveillance, and are necessary for sustained protection against pathogens. This process is mediated by the leukocyte-endothelial adhesion cascade of which the interaction of leukocyte L-Selectin with its endothelial ligand initiates the first critical tethering and rolling step. As well as discussing the constitutive L-Selectin ligands in lymphoid tissues, this review examines the literature regarding their induction in inflammation, and draws attention to recent findings regarding soluble L-Selectin ligands that suggest an emerging multifunctional role in leukocyte recirculation and inflammation.

Leukocyte migration is regulated by L-selectin endoproteolytic release.

L-selectin mediates lymphocyte migration to peripheral lymph nodes and leukocyte rolling on vascular endothelium during inflammation. One unique feature that distinguishes L-selectin from other adhesion molecules is that it is rapidly cleaved from the cell surface after cellular activation. The biological significance of L-selectin endoproteolytic release was determined by generating gene-targeted mice expressing a modified receptor that was not cleaved from the cell surface. Blocking L-selectin cleavage on antigen-stimulated lymphocytes allowed their continued migration to peripheral lymph nodes and inhibited their short-term redirection to the spleen. Blocking homeostatic L-selectin cleavage also resulted in a constitutive 2-fold increase in overall L-selectin expression by leukocytes. As a result, neutrophils entered the inflamed peritoneum in greater numbers or for a longer duration. Thus, endoproteolytic cleavage regulates both homeostatic and activation-induced changes in cell surface L-selectin density, which directs the migration patterns of activated lymphocytes and neutrophils in vivo.

L-selectin: an emerging player in chemokine function.

The emigration of leukocytes across the blood-endothelium barrier and their subsequent transmigration through the interstitium is a complex process that is vital for maintaining the efficiency of the body's innate and adaptive immunity. The chemokines, a family of low-molecular-weight chemoattractant cytokines, are well recognized to be key players in this process. However, recent investigations have highlighted an important role played by the selectin family of adhesion molecules in enhancing chemokine functions. This review summarizes the in vitro and in vivo studies that support this growing notion. It discusses chemotaxis in the context of the phosphoinositide 3-kinase and p38 mitogen-activated protein kinase pathways, and their relation to several chemoattractants (i.e., interleukin-8, leukotriene-B(4), formyl-methionyl-leucyl-phenylalanine, keratinocyte-derived cytokine, and macrophage inflammatory protein-2), the possible role played by L-selectin, and finally how chemotaxis can be altered in different inflammatory settings, such as lipopolysaccharide-mediated endotoxemia or chronic vasculitis.