Laboratory testing for platelet antibodies

Laboratory testing for immune‐mediated thrombocytopenias involves identification and classification of antibodies present in patient sera or attached to patient platelets. This article summarizes the available types of platelet antibody testing and applications in disorders such as neonatal alloimmune thrombocytopenia, post‐transfusion purpura, multiple platelet transfusion refractoriness, immune thrombocytopenia, and drug‐induced thrombocytopenia. Am. J. Hematol. 88:818–821, 2013. © 2013 Wiley Periodicals, Inc.     

Laboratory testing for cryoglobulins

Cryoglobulins are immunoglobulins that precipitate below 37°C and can cause multiorgan damage. There are three types of cryoglobulins: Type I (also called simple), which is mostly associated with monoclonal gammopathy and/or other hematologic disorders and Type II and Type III (known as mixed cryoglobulins), which are associated with infectious and systemic diseases. Testing for cryoglobulins is complicated by lack of reference range, standards, and stringency in maintaining testing temperature conditions. Identification of cryoprecipitate can be critical for patient care; therefore, correct testing conditions are crucial for reliable cryoglobulin testing. The patient's blood sample should be kept at 37°C initially to avoid premature precipitation of cryoglobulins and thereby decreasing the yield for subsequent identification. This could cause a false negative result. After warm centrifugation or warm cell precipitation, the clear serum is observed at 4°C for formation of cryoprecipitate. The cryoprecipitate is then washed in cold buffer, and the resulting precipitate is warmed to 37°C and subjected to further analysis by immunodiffusion and immunofixation. In addition to Meltzer's triad of purpura, weakness and arthralgias, cryoglobulinemias have protean manifestations involving skin, joints, kidney, nervous system, as well as the hematopoietic system. The management of cryoglobulinemia especially in patients with organ damage remains difficult. Treatment of cryoglobulinemia focuses on management of the underlying lymphoproliferative disorder or infectious or systemic causes. Medical management may also include corticosteroids and other immunosuppressive agents and plasmapheresis. Rituximab therapy seems to abrogate the aberrant B cell response.     

Epidemiology of VITT

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a life-threatening syndrome of aggressive thrombosis, often profound thrombocytopenia, and frequently overt disseminated intravascular coagulation. It has been associated with 2 adenovirus vector COVID-19 vaccines: ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen). Unlike the myriad of other conditions that cause thrombosis and thrombocytopenia, VITT has an important distinguishing feature: affected individuals have platelet activating anti-PF4 antibodies that appear in a predictable time frame following vaccination. The reported incidence of VITT differs between jurisdictions; it is dependent on accurate ascertainment of cases and accurate estimates of the size of the vaccinated population. The incidence ranges from 1 case per 26,500 to 127,3000 first doses of ChAdOx1 nCoV-19 administered. It is estimated at 1 case per 518,181 second doses of ChAdOx1 nCoV-19 administered, and 1 case per 263,000 Ad26.COV2.S doses administered. There are no clear risk factors for VITT, including sex, age, or comorbidities. VITT is a rare event, but its considerable morbidity and mortality merit ongoing pharmacovigilance, and accurate case ascertainment.     

Laboratory testing for fibrinogen abnormalities

Fibrinogen is essential for the formation of a fibrin clot. Acquired and congenital disorders of fibrinogen may result in decreased concentration or altered function of fibrinogen, often leading to an increased risk of bleeding. Routine coagulation testing and specialized laboratory investigations can guide diagnosis in patients suspected of having a fibrinogen abnormality. This article summarizes the types of laboratory assays that are used to assess fibrinogen disorders, and key abnormalities found in different types of fibrinogen disorders.     

Longitudinal Aspects of VITT

In hundreds of patients worldwide, vaccination against COVID-19 with adenovirus vector vaccines (ChAdOx1 nCoV-19; Ad26.COV2.S) triggered platelet-activating anti-platelet factor 4 (PF4) antibodies inducing vaccine-induced immune thrombotic thrombocytopenia (VITT). In most VITT patients, platelet-activating anti-PF4-antibodies are transient and the disorder is discrete and non-recurring. However, in some patients platelet-activating antibodies persist, associated with recurrent thrombocytopenia and sometimes with relapse of thrombosis despite therapeutic-dose anticoagulation. Anti-PF4 IgG antibodies measured by enzyme-immunoassay (EIA) are usually detectable for longer than platelet-activating antibodies in functional assays, but duration of detectability is highly assay-dependent. As more than 1 vaccination dose against COVID-19 is required to achieve sufficient protection, at least 69 VITT patients have undergone subsequent vaccination with an mRNA vaccine, with no relevant subsequent increase in anti-PF4 antibody titers, thrombocytopenia, or thrombotic complications. Also, re-exposure to adenoviral vector-based vaccines in 5 VITT patients was not associated with adverse reactions. Although data are limited, vaccination against influenza also appears to be safe. SARS-CoV-2 infection reported in 1 patient with preceding VITT did not influence anti-PF4 antibody levels. We discuss how these temporal characteristics of VITT provide insights into pathogenesis.     

Clinical picture of VITT

This chapter explores the clinical features of vaccine-induced immune thrombotic thrombocytopenia, also called vaccine-induced immune thrombocytopenia and thrombosis (VITT). Whilst the etiology is distinct from other causes of thrombotic thrombocytopenia syndrome (TTS), presentation may be similar and hence the need for strict diagnostic criteria to ensure accurate and prompt diagnosis and early treatment. Studies have identified prognostic markers of the disease, directing therapy and management pathways, and mortality and morbidity from this rare but life-threatening and potentially disabling consequence of the ChAdOx1 nCov-19 vaccine has declined.     

Laboratory testing in hyperthyroidism

The clinical diagnosis of hypo- or hyperthyroidism is difficult (full text available online: http://education.amjmed.com/pp1/272). Clinical symptoms and signs are often non-specific, and there is incomplete correlation between structural and functional thyroid gland changes. Laboratory testing is therefore indispensible in establishing the diagnosis of thyrotoxicosis. Similar considerations apply to treatment monitoring. Laboratory testing also plays a crucial role in establishing the most likely cause for a patient's hyperthyroidism. Finally, during pregnancy, when isotopic scanning is relatively contraindicated and ultrasound is more difficult to interpret, laboratory testing becomes even more important.     

Laboratory testing for cobalamin deficiency in megaloblastic anemia

Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations. Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalamin and folate status. Current total serum cobalamin measurements are performed in the clinical laboratory with competitive binding luminescence assays, whose results may not always accurately reflect actual cobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocysteine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by themselves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, has been proposed as a replacement for current total cobalamin assays. Although holotranscobalamin measurements appear to have slighter better sensitivity, the specificity of this assay remains to be determined. The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patients with suspected megaloblastic anemia are discussed. Am. J. Hematol. 88:522–526, 2013. © 2013 Wiley Periodicals, Inc.     

Interpretation and recommended testing for antiphospholipid antibodies

The antiphospholipid syndrome (APS) is defined by the association of arterial and/or venous thrombosis and/or pregnancy complications with the presence of at least one of the main laboratory-detected antiphospholipid antibodies (aPL) (i.e., lupus anticoagulants [LA], IgG and/or IgM anticardiolipin antibodies [aCL], and IgG and/or IgM anti-β2-glycoprotein I antibodies [aβ2GPI]). During the last decade efforts have been made to improve the harmonization and reproducibility of laboratory detection of aPL and guidelines have been published. The prognostic significance of aPL is being clarified through the fine elucidation of their antigenic targets and pathogenic mechanisms. Several clinical studies have consistently reported that LA is a stronger risk factor for both arterial and venous thrombosis compared with aCL and aβ2GPI. In particular, LA activity dependent on the first domain of β2-glycoprotein I and triple aPL positivity are prognosticators of the thrombotic and obstetric risks. Hopefully, this increasing knowledge will help improve diagnostic and treatment strategies for APS.     

Laboratory testing: current recommendations for older adults

Efficient use of laboratory testing is essential in the care of the elderly, both for making accurate diagnoses and keeping costs in line. Further, primary care physicians treating the elderly need to have an understanding of the effect of age on laboratory values. Clinically significant change occurs with age in some values but not in others. This review focuses first on the effect of aging on different laboratory values and then discusses current recommendations for the most commonly used laboratory tests.     

Laboratory testing for prothrombotic states: clinical utility

It is possible to detect a genetic contribution to venous thrombosis in a significant proportion of patients. This has led to a huge expenditure in clinical time and health resource. However, the gains to be made from the uncritical investigation of the causes of venous thromboembolism are limited and the approach raises significant issues in relation to the appropriateness of this form of genetic testing. In contrast, there are some acquired prothrombotic states that should be identified because the risk of further thrombosis may be sufficient to influence therapy. These states include antiphospholipid syndrome, myeloproliferative disorders, and cancer.     

The deglycosylated form of 1E12 inhibits platelet activation and prothrombotic effects induced by VITT antibodies

In order to improve the safety of COVID-19 vaccines, there is an urgent need to unravel the pathogenesis of vaccine-induced immune thrombotic thrombocytopenia (VITT), a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, and likely due to anti-platelet factor 4 (PF4) IgG antibodies. In this study, we demonstrated that 1E12, a chimeric anti-PF4 antibody with a human Fc fragment, fully mimics the effects of human VITT antibodies, as it activates platelets to a similar level in the presence of platelet factor 4 (PF4). Incubated with neutrophils, platelets and PF4, 1E12 also strongly induces NETosis, and in a microfluidic model of whole blood thrombosis, it triggers the formation of large platelet/leukocyte thrombi containing fibrin(ogen). In addition, a deglycosylated form of 1E12 (DG-1E12), which still binds PF4 but no longer interacts with Fcγ receptors, inhibits platelet, granulocyte and clotting activation induced by human anti-PF4 VITT antibodies. This strongly supports that 1E12 and VITT antibodies recognize overlapping epitopes on PF4. In conclusion, 1E12 is a potentially important tool to study the pathophysiology of VITT, and for establishing mouse models. On the other hand, DG-1E12 may help the development of a new drug that specifically neutralizes the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT.     

Laboratory testing in autoimmune rheumatic diseases

There are a number of pathological conditions in which tissue damage occurs in association with immune activation directed against components of normal tissue. The initial damaging events usually involve cells of the immune system, the T-cells, but the cell damage releases antigens that become targets for an antibody response. The detection and quantification of autoantibodies has become an important component in the diagnosis and management of autoimmune rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, the systemic vasculitides and systemic sclerosis. Each of these diseases is associated with a particular autoantibody or group of autoantibodies. They are usually detected by their reaction against tissue components using subjective methods such as indirect immunofluorescence. Any positive samples are further analysed using more specific and quantitative methods for the 'quantification' of the specific autoantibody concentration. It is important that these autoantibodies are not considered to be 'gold standard' tests: they are no more than markers of the disease with significant limitations. They are best used as part of a diagnostic panel rather than as a marker indicating one particular disease. Techniques are gradually improving, giving numerical results rather than titres, but a lack of standardization makes these results extremely variable. Many of the markers show no correlation with disease activity. Their use should be restricted to the initial investigation and not repeated every time the patient is followed up. Other markers do, however, correlate with disease activity and can be used to monitor disease. When investigating patients who have symptoms associated with autoimmune rheumatic diseases, analytes such as immunoglobulins, complement components and C-reactive protein may all be measured.     

Laboratory testing in disseminated intravascular coagulation.

The lack of improvement in the clinical prognosis of disseminated intravascular coagulation (DIC) poses searching questions on the value of current laboratory testing in this area. There is little immediate chance of advancement unless new tests or new modes of analyzing currently available tests place an emphasis on identifying the early phase of DIC. An equal emphasis is also required on practicality to ensure that recommendations are applicable to the routine clinical setting. These two priorities need to be fulfilled to facilitate earlier therapeutic intervention in the clinical trials required for formal assessment of promising and emerging new molecules from successful animal studies. This article therefore discusses the advantages and disadvantages of available assays in DIC with these perspectives in mind. It also proposes a format to bring together the necessary elements required to make an impact on ultimately delivering clinical improvement in this critical area.