D-dimer is a soluble fibrin degradation product deriving from the plasmin-mediated degradation of cross-linked fibrin. D-dimer can hence be considered a biomarker of activation of coagulation and fibrinolysis, and it is routinely used for ruling out venous thromboembolism (VTE). D-dimer is increasingly used to assess the risk of VTE recurrence and to help define the optimal duration of anticoagulation treatment in patients with VTE, for diagnosing disseminated intravascular coagulation, and for screening medical patients at increased risk of VTE. This review is aimed at (1) revising the definition of D-dimer; (2) discussing preanalytical variables affecting the measurement of D-dimer; (3) reviewing and comparing assay performance and some postanalytical variables (e.g. different units and age-adjusted cutoffs); and (4) discussing the use of D-dimer measurement across different clinical settings.

D-dimer: Preanalytical, analytical, postanalytical variables, and clinical applications

Convalescent plasma (CP) recurs as a frontline treatment in epidemics because it is available as soon as there are survivors. The COVID-19 pandemic represented the first large-scale opportunity to shed light on the mechanisms of action, safety, and efficacy of CP using modern evidence-based medicine approaches. Studies ranging from observational case series to randomized controlled trials (RCTs) have reported highly variable efficacy results for COVID-19 CP (CCP), resulting in uncertainty. SUMMARY Convalescent plasma (CP) recurs as a frontline treatment in epidemics because it is available as soon as there are survivors. The COVID-19 pandemic represented the first large-scale opportunity to shed light on the mechanisms of action, safety, and efficacy of CP using modern evidence-based medicine approaches. Studies ranging from observational case series to randomized controlled trials (RCTs) have reported highly variable efficacy results for COVID-19 CP (CCP), resulting in uncertainty. We analyzed variables associated with efficacy, such as clinical settings, disease severity, CCP SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antibody levels and function, dose, timing of administration (variously defined as time from onset of symptoms, molecular diagnosis, diagnosis of pneumonia, or hospitalization, or by serostatus), outcomes (defined as hospitalization, requirement for ventilation, clinical improvement, or mortality), CCP provenance and time for collection, and criteria for efficacy. The conflicting trial results, along with both recent WHO guidelines discouraging CCP usage and the recent expansion of the FDA emergency use authorization (EUA) to include outpatient use of CCP, create confusion for both clinicians and patients about the appropriate use of CCP. A review of 30 available RCTs demonstrated that signals of efficacy (including reductions in mortality) were more likely if the CCP neutralizing titer was >160 and the time to randomization was less than 9 days. The emergence of the Omicron variant also reminds us of the benefits of polyclonal antibody therapies, especially as a bridge to the development and availability of more specific therapies.

COVID-19 Convalescent Plasma and Clinical Trials: Understanding Conflicting Outcomes

Many preanalytical variables may affect the results of routine coagulation assays While advances in laboratory instrumentation have partially addressed the laboratory's ability to recognize some of these variables, there remains an increased reliance on laboratory personnel to recognize the three potential areas where coagulation testing preanalytical issues may arise: (1) specimen collection (including patient selection), (2) specimen transportation and stability, and (3) specimen processing and storage The purpose of this article is to identify the preanalytical variables associated with coagulation-related testing and provide laboratory practice recommendations in an effort to improve the quality of coagulation testing and accuracy of result reporting

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Preanalytical Variables in Coagulation Testing: Setting the Stage for Accurate Results.

Hemolysis is conventionally defined as membrane disruption of red blood cells and other blood cells that is accompanied by subsequent release of intracellular components into the serum or plasma. It accounts for over 60% of blood sample rejections in the laboratory and is the most common preanalytical error in laboratory medicine. Hemolysis can occur both in vivo and in vitro. Intravascular hemolysis (in vivo) is always associated with an underlying pathological condition or disease, and thus careful steps should always be taken by the laboratory to exclude in vivo hemolysis with confidence. In vitro hemolysis, on the other hand, is highly preventable. It may occur at all stages of the preanalytical phase (i.e. sample collection, transport, handling and storage), and may lead to clinically relevant, yet spurious, changes in patient results by interfering with laboratory measurements. Hemolysis interference is exerted through several mechanisms: (1) spectrophotometric interference, (2) release of intracellular components, (3) sample dilution and (4) chemical interference. The degree of interference observed depends on the level of hemolysis and also on the assay methodology. Recent evidence shows that preanalytical practices related to detection and management of hemolyzed samples are highly heterogeneous and need to be standardized. The Working Group for Preanalytical Phase (WG-PRE) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has published many recommendations for facilitating standardization and improvement of this important preanalytical issue. Some key EFLM WG-PRE publications related to hemolysis involve: (i) a call for more transparency and some practical recommendations for improving the harmonization of the automatic assessment of serum indices and their clinical usefulness, specifically the hemolysis index (H-index), (ii) recommendations on how to manage local quality assurance of serum or plasma hemolysis/icterus/lipemia-indices (HIL-indices) and (iii) recommendations on how to detect and manage hemolyzed samples in clinical chemistry testing. In this review we provide a comprehensive overview of hemolysis, including its causes and effects on clinical laboratory assays. Furthermore, we list and discuss the most recent recommendations aimed at managing hemolyzed samples in everyday practice. Given the high prevalence of hemolyzed blood samples, the associated costs, the great heterogeneity in how hemolysis is handled across healthcare settings, countries and continents, and increasing patient cross-border mobility, standardization and quality improvement processes aimed at combatting this important preanalytical problem are clearly warranted.

Managing hemolyzed samples in clinical laboratories.

SummaryIntroduction
Haemolysis, icterus and lipaemia (HIL) may affect haemostasis test results. This may be influenced by the level of interfering substance and the reagents and end-point detection system used.

Methods
We assessed the influence of HIL on prothrombin time, activated partial thromboplastin time and fibrinogen assay using a viscosity-based detection analyser.

Results
Spontaneous haemolysis that occurred during sample collection and processing had no effect on PT with either a rabbit tissue factor extract or recombinant human tissue factor reagents. In contrast, addition of mechanically haemolysed cells impacted on PT for the highest haemoglobin concentration.

For APTTs determined with STA®-Cephascreen® reagent, there was no significant difference between results in haemolysed and nonhaemolysed samples. For the other two reagents studied, APTTs were statistically significantly shorter in haemolysed samples compared with nonhaemolysed samples. This bias was clinically significant only for STA®-PTT Automate. For all three APTT reagents, the impact of haemolysis was sufficient to impact patient management decisions, and in some samples, the effects of lipaemia and icterus were not clinically significant.

Conclusion
Overall, our results confirm that PT and fibrinogen were not clinically significantly affected by HIL. The APTTs of some haemolysed samples were falsely normal. Haemolysed samples for APTT determination should be rejected.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/ijlh.12498

Effects of haemolysis, icterus and lipaemia on coagulation tests as performed on Stago STA-Compact-Max analyser

Key Points Question What patient characteristics are associated with benefit from treatment with COVID-19 convalescent plasma (CCP)? Findings This prognostic study of 2287 patients hospitalized with COVID-19 identified a combination of baseline characteristics that predict a gradation of benefit from CCP compared with treatment without CCP. Preexisting health conditions (diabetes, cardiovascular and pulmonary diseases), blood type A or AB, and earlier stage of COVID-19 were associated with a larger treatment benefit. Meaning These findings suggest that simple patient information collected at hospitalization can be used to guide CCP treatment decisions for patients with COVID-19.

Development and Validation of a Treatment Benefit Index to Identify Hospitalized Patients With COVID-19 Who May Benefit From Convalescent Plasma

Summary
Introduction

Our study was designed to evaluate, on healthy subjects and patients on oral anticoagulant therapy vitamin K antagonist (OAT-vka), the possible interference caused by hemolysis on the main coagulation tests.



Methods

To obtain hemolyzed samples, two methods were used: heat shock and mechanical system. The coagulation tests on hemolyzed samples were performed employing optical automated analyser BCSxp (Siemens Healthcare®). Moreover, the prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests were also carried out manually using an electromechanical device (KC4 – Amelung).



Results

The PT test, on healthy subjects, in case of moderate hemolysis can be performed without significant interference on automatic instrument. On manual instrument, the PT test can be performed even in case of marked hemolysis. For patients on OAT-vka, the PT test in case of marked hemolysis can be performed both on automatic and manual instrument. For the aPTT test, it can be carried out manually, because also in case of marked hemolysis a statistically significant difference was not observed. For the fibrinogen test, a dramatic concentration decrease was already clear for weak hemolysis. A decreased function on antithrombin test was statistically significant for mild-moderate hemolysis. The D-dimer test showed increased values for mild hemolysis.



Conclusions

The rejection of hemolyzed sample and/or the request of a second sample are not always the proper attitudes to take for performing clotting tests. The rational management of the hemolyzed samples decreases the employment of both nursing and technical staff significantly, the turnaround time and, consequently, does not lead to additional costs for each patient involved.

Evaluation of the main coagulation tests in the presence of hemolysis in healthy subjects and patients on oral anticoagulant therapy.

The efficacy of COVID‐19 convalescent plasma (CCP) as a treatment for hospitalized patients with COVID‐19 remains somewhat controversial; however, many studies have not evaluated CCP documented to have high neutralizing antibody titer by a highly accurate assay. To evaluate the correlation of the administration of CCP with titer determined by a live viral neutralization assay with 7‐ and 28‐day death rates during hospitalization, a total of 23 118 patients receiving a single unit of CCP were stratified into two groups: those receiving high titer CCP (>250 50% inhibitory dilution, ID50; n = 13 636) or low titer CCP (≤250 ID50; n = 9482). Multivariable Cox regression was performed to assess risk factors. Non‐intubated patients who were transfused with high titer CCP showed 1.1% and 1.7% absolute reductions in overall 7‐ and 28‐day death rates, respectively, compared to those non‐intubated patients receiving low titer CCP. No benefit of CCP was observed in intubated patients. The relative benefit of high titer CCP was confirmed in multivariable Cox regression. Administration of CCP with high titer antibody content determined by live viral neutralization assay to non‐intubated patients is associated with modest clinical efficacy. Although shown to be only of modest clinical benefit, CCP may play a role in the future should viral variants develop that are not neutralized by other available therapeutics.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ajh.26531

Early administration of COVID‐19 convalescent plasma with high titer antibody content by live viral neutralization assay is associated with modest clinical efficacy

Background Although frequently used in the early pandemic, data on the effectiveness of COVID-19 convalescent plasma (CCP) remain mixed. We investigated the effectiveness and safety of CCP in hospitalized COVID-19 patients in real-world practices during the first two waves of the pandemic in a multi-hospital healthcare system in Texas. Methods and findings Among 11,322 hospitalized patients with confirmed COVID-19 infection from July 1, 2020 to April 15, 2021, we included patients who received CCP and matched them with those who did not receive CCP within ±2 days of the transfusion date across sites within strata of sex, age groups, days and use of dexamethasone from hospital admission to the match date, and oxygen requirements 4–12 hours prior to the match date. Cox proportional hazards model estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for effectiveness outcomes in a propensity score 1:1 matched cohort. Pre-defined safety outcomes were described. We included 1,245 patients each in the CCP treated and untreated groups. Oxygen support was required by 93% of patients at the baseline. The pre-defined primary effectiveness outcome of 28-day in-hospital all-cause mortality (HR = 0.85; 95%CI: 0.66,1.10) were similar between treatment groups. Sensitivity and stratified analyses found similar null results. CCP-treated patients were less likely to be discharged alive (HR = 0.82; 95%CI: 0.74, 0.91), and more likely to receive mechanical ventilation (HR = 1.48; 95%CI: 1.12, 1.96). Safety outcomes were rare and similar between treatment groups. Conclusion The findings in this large, matched cohort of patients hospitalized with COVID-19 and mostly requiring oxygen support at the time of treatment, do not support a clinical benefit in 28-day in-hospital all-cause mortality for CCP. Future studies should assess the potential benefits with specifically high-titer units in perhaps certain subgroups of patients (e.g. those with early disease or immunocompromised).

/pdf/multi-center-matched-cohort-study-of-convalescent-plasma-for-3s4sewwe.pdf

C. Villa

Papers

Development and Validation of a Treatment Benefit Index to Identify Hospitalized Patients With COVID-19 Who May Benefit From Convalescent Plasma

Evaluation of the main coagulation tests in the presence of hemolysis in healthy subjects and patients on oral anticoagulant therapy.

Early administration of COVID‐19 convalescent plasma with high titer antibody content by live viral neutralization assay is associated with modest clinical efficacy

Multi-center matched cohort study of convalescent plasma for hospitalized patients with COVID-19