Spotlights on the trends in performance assessment of qualitative in vitro diagnostic medical devices in transfusion medicine.

For reliable clinical decisions in transfusion medicine, assessing the performance of qualitative tests performed in medical laboratories is critical. When false results are reported, these can lead to an adverse reaction to blood components. Good performance assessment practices are essential for this kind of scenario, and they still remain as one of the many unmet high-priority challenges in this area. This paper aims to provide an overview of the current trends in this field. A review of the IFCC-IUPC. qualitative vocabulary was carried out, and a particular focus was given to the evaluation protocols CLSI EP12-A3 and Eurachem AQA, such as the European Union Regulation for class D in vitro diagnostic medical devices. There is a consistency between the current protocols and recognized performance assessment principles, which are mandatory in transfusion service labs. We believe that a revised imprecision interval approach and models based on emerging qualitative test types may prove beneficial in the long run. It is also important to emphasize the uncertainty of proportions to mitigate the risk of misclassification.

Assessment of extracellular vesicles using IFC for application in transfusion medicine.

Extracellular vesicles (EVs) have been shown to be involved in various physiological and pathophysiological processes. With respect to Transfusion Medicine, the accumulation of EVs in blood products during hypothermic storage is an indicator of the storage lesion and reportedly correlates with adverse effects after transfusion, including but not limited to immunomodulation, activation of coagulation, endothelial activation, and others. To optimally reduce such an impact on blood product quality degradation and improve post-transfusion outcomes, better methods for detection, enumeration, characterisation by size and phenotype, and functional involvement of EVs in different pathophysiological and physiological processes are required. Currently, Imaging Flow Cytometry (IFC) technology provides the most comprehensive assessment of EV subsets in different body fluids. The unique ability of IFC to detect EVs of 20 nm size by registration of a single pixel of fluorescence signal makes this approach highly promising for comprehensive studies of EVs. In this review, we will focus on the recent breakthrough and advantages of using the ImageStreamX MKII IFC platform for the detection and characterisation of EVs and its future prospects for routine application of IFC in Transfusion Medicine.

Poor economics - Transforming challenges in transfusion medicine and science into opportunities.

The prospect of cryopreservation of cellular components in the low and medium income (poor economics) part of the world absolutely needs a solid and sustainable infrastructure to build on in line with science, technology and globalization, based on rational thinking, standardization and harmonization of future advances we are currently witnessing in limited parts of the world. With the stepwise development of the healthcare stimulated by the 2012 UN Universal Health Coverage (UHC) program and supported by WHO Model List of Essential Medicines (EM) and Essential in vitro Diagnostics (ED), a slowly growing number of countries will reach a point where quality cryopreservation of cellular components becomes feasible as an advance for implementing specific health care visions, policies and strategies in line with the Sustainable Development Goals 2016-2030.

Donor health assessment - When is blood donation safe?

Blood donation is a highly regulated practice in the world, ensuring the safety and efficacy of collected blood and its components whether used as irreplaceable parts of modern transfusion medicine, as a therapeutic modality or additional support to other clinical therapies. In Norway blood donation is regulated by governmental regulations ("Blodforskriften") and further instructed by national guidelines, "Veileder for transfusjonstjenesten" [1], providing an aid for assessment of donor health. This concise review touches upon: definitions of donor health and disease; some important pitfalls; and the handling of some common and less common pathophysiological conditions; with an example from the Blood center of Oslo University Hospital, Norway's largest blood center. I also comment on some medications used by a number of blood donors, although wounds, ulcers and surgery are not included. Considering the panorama of conditions blood donors can suffer from, blood donation can never be completely safe for everybody, as zero risk does not exist, but it is our task through donor evaluation to identify and reduce risk as much as possible.

Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40sec; heterozygous patients have clotting times of about 40-60sec and normal individuals yield clotting times >70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin therapies, and has no interference with lupus anticoagulant (LA). This new assay for Factor V-Leiden can be easily used in any coagulation laboratory, is performed as a single test, and is quantitative. This assay has a high robustness, is accurate and presents a good intra- (<3%) and inter-assay (<5%) variability. It contributes solving most of the laboratory issues faced when testing factor V-Leiden. Quantitation of Factor V-L could contribute to a better assessment of thrombotic risk in affected patients, as this complication is first associated to and caused by the presence of a defined amount of FVa.

The state of the art of removal of prion proteins in SD-FFP, by specific prion affinity chromatography and its impact on the hemostatic characteristics of the product.

In recent Coimbra' Conference, on the pre-launch of pathogen reduced-FFP for the local clinical use, the question was raised, by the moderator, on the efficacy of the current methodology used for prion removal processes and its influence on the overall quality and safety of the final product. This brief paper put together by speaker of this session and the moderator, as a consensus of opinions, which was largely discussed during Q&A session, to make it available to a large group of readers of transfusion apheresis science, who might be interested to this topic. In short the capacity of the current process of Octaplas to remove prion is in order of 5.6 log10/ID50 reduction based on several animal studies. Moreover the changes in coagulation and inhibitors are within acceptable range and bioequivalent to untreated FFP with no sign of inferiority. This paper describes in brief a technology update on solvent/detergent treated plasma, an alternative to FFP but with increased pathogen safety. The biochemical profile of the final product is comparable with FFP and contains all clinically relevant plasma proteins. Furthermore, Octaplas is a product that, in long term, reduces health care costs.

Multilayer-strategy to enhance optimal safety of the blood supply: The role of pathogen inactivation for optimizing recipient safety and helping health care cost containment: Moderator views.

This brief paper is based on the Coimbra 'conference presentation by the moderator', prior to the two main lectures on pathogen reduction treatment [PRT] of plasma. Being an educationist and teacher in core and having a great interest to simplify the message convey to conference' participants and readers I decided to maintain the slide format of the presentation. To highlight most effectively the role played by pathogen reduction to supplement the multilayer-strategy already in place, emphasizes were placed by going back to basic focusing on: where we were, where we are now and where we are going!. The unresolved problems of viral safety of blood components and criteria of universal acceptability of PRT are highlighted so is the need for further DDR strategies both in incremental and innovative ways. Finally the issue of who would benefit from implementation of PRT is described based on published data and also providing some visionary foresights for the long term benefits of PRT in both optimizing the safety of blood supply and helping at least in health care containment. I hope this new approach will be useful to readers, providing at least some conceptual and technical supports in understanding the role of PRT in optimizing the safety of blood supply.

Highlights of PBTI Coimbra Conference on PRT of Plasma & Current Opinions on Pathogen Reduction Treatment of Blood Components.

Two experts from Octapharma and from Cerus addressed, in very concise ways, the concerns about non-viral inactivated FFP and how they managed to obtain highest standard of safety margin for pathogen reduction treatment [PRT] of plasma. The session was moderated by Portuguese Institute of Blood and Transplantation (PIBT) consultant advisor [Jerard Seghatchian] with long standing familiarity and international recognition in PR technologies for plasma, platelets and WB/red cells. The focus of conference was mainly on the criteria of acceptability of PRT-FFP; added values of having diversity in choice without fears of liability, as both of PRT technologies provide an excellent safeguard margins, for more than a decade of usage. In most European countries, it is believed that patients' safety come first followed by the safe usage initiatives, in particular using locally available products. Portugal is finally going forward with the implementation PRT plasma using its own FFP for their clinical use. The round table Q&A session focused on the impacts of the additional processing, which is still continuously improving, on the residual/emerging pathogen infectivity; eliminating the clinical impacts of donors viable leukocytes; the degree of altered product potency in particular cold activation of FVII; and loss of endothelial permeability factors during fluid storage of plasma. Both speakers highlighted their product safety and clinical efficacy using both routine in vitro, including the modern proteomic tests to establish the relevant changes in various parameters and in the overall clinical outcomes. The advancements in pharmacovigilance and hemovigilance, regulatory aspects and cost effectiveness were also highlighted. A local speaker [from the PIBT] described the state of the art of local processing issues and overall required standards used both during validation and the intercept process scale up, which is going ahead smoothly to providing the highest safety standards PRT-intercept plasma locally, in production now. Overall this was an excellent conference, open to transfusion medicine specialists and other health care professionals, for feedback and quality awareness, of providing diversity in choice, to local clinicians, who demand the best for the ultimate patient requirements. This is achieved in a period where both cost effectiveness and affordability matter, so is the clinical outcome, which ultimately counts. There was an atmosphere of non-competitive collaboration and sharing knowledge and working togetherness, even between two manufacturer representatives, which made the conference most joyful event. This was the opportunity to the scientific update and sharing "the once upon a time" impossible task of going for PRT-plasmas in Portugal, making it a reality in their local setting, in real time using Portuguese plasmas. This is in fact the timely news in the period of austerity, to provide a pre launching session for reporting the state of the arts of Portugal achievements to staff, academician, laboratory experts and clinician alike.

Quality management in European screening laboratories in blood establishments: A view of current approaches and trends.

The screening laboratory has a critical role in the post-transfusion safety. The success of its targets and efficiency depends on the management system used. Even though the European Union directive 2002/98/EC requires a quality management system in blood establishments, its requirements for screening laboratories are generic. Complementary approaches are needed to implement a quality management system focused on screening laboratories. This article briefly discusses the current good manufacturing practices and good laboratory practices, as well as the trends in quality management system standards. ISO 9001 is widely accepted in some European Union blood establishments as the quality management standard, however this is not synonymous of its successful application. The ISO "risk-based thinking" is interrelated with the quality risk-management process of the EuBIS "Standards and criteria for the inspection of blood establishments". ISO 15189 should be the next step on the quality assurance of a screening laboratory, since it is focused on medical laboratory. To standardize the quality management systems in blood establishments' screening laboratories, new national and European claims focused on technical requirements following ISO 15189 is needed.

"Go no Go" in plasma fractionation in the world's emerging economies: still a question asked 70 years after the COHN process was developed!

In the late 1980s, following the human immunodeficiency virus (HIV) epidemic and transfusion-transmitted infections from plasma-derived coagulation factor concentrates to hemophiliacs, many "advanced thinkers" claimed that plasma-derived products would be completely replaced by the year 2000 by safe recombinant products in most developed countries. However, things have not turned out that way, due to both the continual progress witnessed in plasma fractionation and viral-reduction technologies and technical difficulties still being encountered in developing more cost-effective non-immunogenic, fully active recombinant therapeutic proteins. Accordingly, plasma fractionation remains a reasonably healthy industry worldwide, with an ever-increasing volume of plasma fractionated each year to meet the demands for safe and effective plasma-derived medicines at the global level. While high-income countries currently have generally good access to a panel of plasma-derived and recombinant products, desperate shortages of fractionated plasma products remain in developing economies,and patients still have to be treated inadequately. The steady development of the collection of whole blood in developing economies, to gradually cover the recognized needs for red blood cell concentrates, generates an increasing volume of recovered plasma that is currently wasted. Incentives are therefore high for those countries to consider fractionating such plasma as a means of enhancing their supply of products to treat patients, thereby also decreasing the level of dependence on imported products. Challenges of local plasma fractionation in developing economies are high, in a context where the technological and regulatory sophistication of the plasma fractionation industry is often underestimated, and the blood supply may be exposed to emerging infectious agents. In parallel, plasma product quality requirements and drivers are evolving in developed economies as is the awareness of clinicians to newer uses of products such as intravenous immunoglobulins, somewhat deviating from what currently remain the basic needs of developing countries in terms of affordable safe plasma products. Global market trends for plasma-derived products, through plasma fractionation, are still increasing, despite increasing use of recombinant products, and attention is being focused on the five Ws of the fractionation field: which products; where; when; what and how much; and who will be the main suppliers?

Analytical model for calculating indeterminate results interval of screening tests, the effect on seroconversion window period: a brief evaluation of the impact of uncertain results on the blood establishment budget.

The evaluation of measurement uncertainty is not required by the European Union regulation or blood establishments' laboratory tests. However, it is required for tests accredited by ISO 15189. Also, the forthcoming ISO 9001 edition requires "risk based thinking" with risk described as "the effect of uncertainty on an expected result". ISO recommends GUM models for determination of measurement uncertainty, but their application is not intended for ordinal value measurements, such as what happens with screening test binary results. This article reviews, discusses and proposes concepts intended for measurement uncertainty of screening test results. The precision model focuses on cutoff level allowing the evaluation of the indeterminate interval using analytical sources of variance. The intervalis considered in the estimation of the seroconversion window period. The delta-value of patients and healthy subjects' samples allows ranking two tests according to the probability of the two classes of indeterminate results: chance of false negative results and chance of false positive results (waste on budget).

A national quality assessment scheme for counting residual leucocytes in unfixed leucodepleted products: the effect of standardisation and 48 hour storage.

BACKGROUND: WBC counting, an essential part of quality monitoring of WBC-reduced blood components, is carried out logistically within 48-72 h of collection. The between-laboratory variability and effects of 24-48 h storage were investigated using three major counting technologies. STUDY DESIGN AND METHODS: Samples of RBC and platelets with WBC in the range 0-50/microl were transported by courier. WBC counting was performed on days 1 and 2, by IMAGN 2000, flow cytometry and Nageotte, initially using local protocols and then using a national flow protocol. Up to 15 laboratories participated in each exercise. RESULTS: For "real failed leucodepleted" red cell products, higher levels of variability were observed for flow and Nageotte, as compared to IMAGN. For spiked RBC samples at critical decision making point (3-20 WBC/microl), between-laboratory the coefficients of variation (CVs) were low for IMAGN and were the highest for Nageotte. Flow cytometry CVs were generally high but improved subsequent to standardisation of sampling and the gating strategy. A similar pattern in the variability of results was observed for platelet concentrates. Sign tests using all samples (carried out for each method in each exercise; 25 in total) demonstrated no overall tendency for larger WBC counts to be recorded on day 1 when compared to day 2, although this difference was significant (p < 0.001) in certain cases depending on the nature of the spiked product. CONCLUSIONS: We conclude that while a good performance is achieved using validated automated technologies for low residual leucocyte counting, the unification of reagents and standardisation of sampling and gating strategies are essential in obtaining interchangeable results. Unfixed RBC and platelet samples can generally be stored for 48 h before WBC counting.

A platelet quality assessment scheme for comparing the performance of quality monitoring laboratories in the UK National Blood Service.

This exercise focused on performance of NBS quality monitoring establishments with respect to enumeration of low leucocyte and other quality indexes of platelet concentration. Paired identical leucodepleted platelet samples, spiked with WBC (20 cells/microl) in 'vacuette' or 'pouch' were assessed by participants (n = 20) on days 1, 2 and 5. For low WBC counting, all laboratories gave estimates within acceptable range (+/-25%) and good agreement between storage and assay methods was observed on days 1 and 2. Day 5 results showed greater variability. Under improved performance criteria (+/-15%), only one laboratory under-estimated at days 1 and 2. Similarly, other parameters demonstrated good agreement between storage methods on days 1 and 2. At day 5, mean results were often significantly different to previous days. Improved performance target (+/-15%) will allow identification of non-conformers.