IVIG

Intravenous immunoglobulin G (IVIG) is a biological product obtained by fractionation of human plasma. It is a replacement therapy in humoral deficiency, and is also used for immunomodulatory therapy for a variety of autoimmune disorders and transplantation. As it is a biological product, it contains inherent risk of non-infectious contaminants or transmission of pathogens resulting from donors or the manufacturing processes.  Safety testing for pathogens and other contaminating proteins, as part of standard quality testing, decreases the risk of adverse effects (AEs).

Thrombosis is severe AEs associated with IVIG administration. IVIG-induced thrombosis is reported both as venous events such as thrombosis stroke, pulmonary embolism (PE), deep venous thrombosis (DVT), and arterial ischemia events such as myocardial infarct and stroke.  Incidence of IVIG-induced thrombosis is reported to range from 3 to 13%.  Risk factors for thrombotic events are linked to underlying risk factors in the recipient, but are also associated with high-dose and high-speed IVIG infusions. Although the mechanisms driving IVIG-associated thrombosis are still not clear; three mechanisms have been proposed.

Three IVIG Thrombosis Mechanisms:

1. Increased blood viscosity, causing a hypercoagulable state
2. Anticardiolipin antibodies passively transferred through IVIG
3. Highly active coagulation factors, Factor XIa or PKA, passively transferred via IgG concentrates

Avoiding activated coagulation factors, like Factor XIa or PKA, in IgG concentrates starts with appropriate anticoagulation of donated sample and continues over the whole fractionation or manufacturing process. Quality control and/or safety testing for activated Factor XIa or Prekallikrein (PKA) through sensitive and specific chromogenic testing assays is an additional measure put in place when manufacturing and releasing each batch of IVIG products.

Factor XIa Testing Products

Plasma fractionation procedures may sometimes be associated with inadvertent activation of Factor XIa (FXIa). Routine tests, such as the NAPTT, may not be sufficiently sensitive to detect relevant low levels of FXIa. Rox Factor XIa offers a highly sensitive chromogenic method for determination of sub-picomolar levels of FXIa, therewith allowing high sample dilutions and minimizing interference from the sample matrix and from contaminating proteins such as kallikrein, zymogen FXI and FIXa.

PKA Testing Products

Fractionation of Human plasma to produce Human Albumin and Immunoglobulin preparations can also result in concentration of PKA. At high levels, PKA can release kallikrein and patients may go into shock. PKA is therefore removed by post fractionation chromatography. Blood product licensing authorities require manufacturers to validate each batch of final product before it can be released. PKA content has been limited to levels of less than 35 IU/ml as outlined in European Pharmacopoeia guidelines.

PKA assays are in general performed using in-house methods with an externally sourced chromogenic substrate (Chromogenix S-2302™) and standards (Human Prekallikrein Pool, PW30304). An in-house method can be a difficult and laborious process, so a ready-to-use commercially available PKA assay kits can streamline the release of human Immunoglobulin and Albumin preparations.

The ready-to-use commercial kits tests measures PKA present in the blood product which will activate a known concentration of Prekallikrein to release the enzyme Kallikrein. The kallikrein formed releases chromogene p-nitroaniline (pNA) from the chromogenic kallikrein substrate. The rate at which pNA is released is measured photometrically in a microtiter plate reader. The amount of pNA released is proportional to the amount of PKA present in the preparation.  The assay can be performed as rate method or by end point.

TGA Testing Products

Thrombin Generation Assay (TGA) is a more global downstream read out for the kinetics of thrombin generation during initiation, amplification and down regulation of thrombin formation from activation of proteins in the coagulation cascade, like PKA and FXIa. Technothrombin® TGA is a complete fluorogenic tool for a broad range of applications and is therefore a universal assay kit for analyzing and researching the function of the hemostatic system.