TransFix^®

Immunophenotyping by flow cytometry is an established diagnostic procedure used to analyse lymphocytes in blood, CSF and other body fluids. Situations often arise where samples cannot be analysed immediately follwowing collection. This often occurs when samples are taken during evenings and weekends or where samples must be transported to the laboratory. TransFix^® will preserve and stabilise samples for up to 10 days until flow cytometry can be performed enabling treated samples to be stored or transported safely.

TransFix^® can be used to extend the viability of blood samples for up to 10 days when stored at 4°C. Research has also demonstrated (Bergeron et al) that blood samples treated with TransFix^® are also stable when stored between 20°C to 24°C for up to 7 days and at 37°C for up to 3 days. Transportation of samples between clinical sites often raises concerns about sample integrity. TransFix^® can be used in resource poor areas of the world to extend sample viability without refrigeration and enable samples to be batched and transported for examination. (Jani et al, 2001).

Fresh Blood Samples
Day 0 (No TransFix^®)

TransFix^® Blood Sample
Day 10

Flow Cytometry comparison of fresh whole blood with a TransFix^®sup>® stabilised sample at Day10 demonstrates that TransFix^® stabilised whole blood has the same flow cytometry staining pattern as seen in fresh whole blood. Staining examples with CD45-PE-Cy5 and CD3 FITC antibodies are shown above.

Immune Monitoring for Management of HIV/AIDS

Monitoring T-cell subsets of human immunodeciency virus (HIV)-infected individuals is performed routinely by flow cytometry. It is the most commonly used method for monitoring the immune status of HIV-infected individuals. Using traditional or homogeneous gating strategies for T-cell subset enumeration, specimens must be processed within 24 h to preserve their integrity and obtain a reliable absolute CD4+ T-cell value. Samples treated with TransFix^® remain viable for up to 10 days. Samples treated with TransFix^® retain morphology and cell surface expression over time. Primary CD4+ gating can be performed on blood samples treated with TransFix^® with great precision.

TransFix^® is mixed with whole blood at a ratio of 1:5 offering the benefit of minimal sample dilution. Absolute counts from blood samples treated with TransFix^® must be adjusted for the dilution factor by dividing by 0.8.

Transportation of samples between clinical sites often raises concerns about sample integrity. TransFix treated samples are stable at ambient temperatures between 4⁰C and 37⁰C for a minimum of 4 days, enabling them to be transported safely between clinical sites.

Shipping and handling of HIV+ samples involves inherent elevated costs and a risk of exposure to HIV. The addition of TransFix^® results in a significant reduction in HIV replication as measured by HIV p24 antigen production. (Kim et al, 2001). However, samples must still be handled with universal precaution protocols.

Fresh Blood Samples
Day 0 (No TransFix^®)

TransFix^® Blood Sample
Day 10

Flow Cytometry comparison of fresh whole blood with a TransFix^® stabilised sample at Day10 demonstrates that TransFix^® stabilised whole blood has the same flow cytometry staining pattern as seen in fresh whole blood. Staining examples with (CD3 FITC and CD4 APC) and (CD3 FITC and CD8 PE) antibodies are shown above.

Immunophenotyping

Subsets of leucocytes can be distinguished on the basis of cell surface antigens using flow cytometry. Quantitative and qualitative changes in leucocyte sub sets are used to identify and monitor haematologic diseases. TransFix^® has been shown to preserve a wide range of cellular markers (Barnett et al, 1999) for up to 10 days.

A full haematological profile can also be obtained up to 7 days after the addition of TransFix^®.

The wide range of clones and conjugates available make it essential that all conjugates are tested in conjunction with TransFix^® to ensure there is no interference with antibody binding. Care should also be taken with regards to intracellular antigens as TransFix^® causes a progressive permeabilisation of the cellular membrane. As a direct result intracellular markers will leach out over time. As a general guide intracellular antigen measurement on TransFix^® treated samples is only possible up to 3 days after the addition of TransFix^®.

Other Species

TransFix^® has been tested on mouse, rat, guinea pig, sheep, horse, pig and green turtle blood samples. The amount of TransFix^® required to stabilise blood varies considerably in different species. In some species the volume required also varies by breed and type.

The user must determine the optimal dilution required. The optimal dilution is the concentration where the same flow cytometry light scatter result remains unchanged after the treated sample has been stored for 10 days at 4°C.The protocol for determining the optimal concentration needed can be downloaded here.

Functional Assays

Samples treated with TransFix^® cannot be used in functional assays.

Storage

TransFix^® is stored at room temperature (18 - 24°C). In countries where the ambient temperature is greater than 24⁰C TransFix^® can be stored at 4°C. However, it must be allowed to warm up to room temperature before use. TransFix^® must NEVER be frozen.

Wherever possible TransFix^® treated samples should be refrigerated at 4°C. Treated samples are also stable at temperatures up to 25°C for 7 days and at temperatures between 26°C and 37°C for 4 days.

History

TransFix^® was developed by medical scientists from UK NEQAS, which is part of Sheffield Teaching Hospitals NHS Foundation Trust. TransFix^® was developed out of the requirement to produce stabilised blood samples that could be used in External Quality Assurance programs for flow cytometry. The first publication on TransFix^® as a cellular antigen stabilizing reagent was made in 1999 by D. Barnett et al.

TransFix^® has been in circulation in the research world from 1999 until the present day, generating a number of scientific publications. In 2005 the Sheffield Teaching Hospitals NHS Foundation Trust licensed TransFix^® to Cytomark in order to make it more generally available to the clinical and scientific world.