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Stabilise antigens for up to 14 days

FAQ

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TransFix®

General Information

1. Where did TransFix® originate?

TransFix® was developed by scientists working for UK NEQAS. This organisation is the External Quality Assurance (EQA) assessor for all pathological diagnostic laboratories in the UK. UK NEQAS also supports many other laboratories in Europe and beyond.

Whole blood is very unstable due to proteolytic degradation. The result is that blood samples older than 48 hours are no longer usable for flow cytometric examination. For the same reason unstabilised whole blood is unsuitable for use as an EQA sample.

TransFix® was developed by UK NEQAS as a solution to this problem. When added to whole human blood it stabilises the blood cell profile and inhibits proteolytic deterioration.

TransFix: A clinical sample stabilising fluid for use in clinical haematology and immunology.
D. Barnett, V.Granger, A.G. Pockley, J.M.saxton, I storie, L.Whitby & J.T.Reilly
Cytometry (1999) 38:88 (abstract)

2. Can TransFix® be used for Immunophenotyping?

Yes. For human whole blood samples, we have demonstrated that the immunophenotypic profile is maintained for 14 days for cells with markers: CD3, CD4, CD8, CD16, CD19, CD45, and CD56. The assessment of these cell subsets after stabilisation form a part of the quality control testing procedure for TransFix.

3. Will TransFix® preserve intracellular antigens?

Yes, but it increases the permeability of the cellular membrane. Therefore, it is advisable to analyse intracellular antigens within 3 days of TransFix® treatment. We recommend the use of the An der Grub Fix and Perm kit (product code: GAS-002) for flow cytometric examination of intracellular blood cell markers after TransFix® treatment.

4. Are TransFix products in vitro Diagnostic Medical Devices (IVDs)?

For a list of countries where TransFix products are licenced as IVDs, please click here.

Product and Sample Storage

5. How should TransFix® be stored before it is used for sample stabilisation?

All TransFix® products (TransFix®, TransFix® Sample Storage Tubes, TransFix/EDTA CSF Sample Storage Tubes and TransFix/EDTA Vacuum Blood Collection Tubes) can be stored at 2-8°C and must not be frozen.

TransFix® bottles can be stored at 2-25°C although it is advisable to store them at 2-8°C to reduce the risk of evaporation. It has a shelf life of up to 2 years or until the expiration date on the label.

TransFix® Sample Storage Tubes can be stored at 2-25°C although it is advisable to store them at 2-8°C to reduce the risk of evaporation. They have a shelf life of up to 1 year from the date of manufacture or until the expiration date on the label.

TransFix/EDTA Vacuum Blood Collection Tubes are supplied in a sealed foil pouch. Tubes in an unopened pouch can be stored at 2-25°C for 18 months from the date of manufacture or until the expiration date on the label. TransFix/EDTA Vacuum Blood Collection Tubes from opened packages must be stored at 2-8°C and have a shelf life of 6 months from the date that the pouch is opened.

TransFix/EDTA CSF Sample Storage Tubes must be stored at 2-8°Cand have a shelf life of 1 year from the date of manufacture or until the expiration date on the label.

6. How should TransFix® treated samples be stored?

The immunophenotypic profile is maintained in human blood samples stabilised using TransFix®, TransFix® Sample Storage Tubes and TransFix/EDTA Vacuum Blood Collection Tubes after storage at 2-8°C for 14 days.

CSF samples administered into TransFix/EDTA CSF Sample Storage Tubes must be stored at 2-8°C for up to 10 days.

Studies have demonstrated that it is possible to analyse TransFix® stabilised blood cells that have been stored at temperatures of up to 37°C after 4 days (refer to references below), but these conditions are not validated at Cytomark.

Barbara Canonico et al, Clinical Cytometry, Vol. 78, 2010, 267-278, Flow Cytometric Profiles, Biomolecular and Morphological Aspects of Transfixed leucocytes and Red Cells.

M. Bergeron et al, Clinical Cytometry, Vol. 50, 2002, 86-91, Evaluation of stabilised blood cell products as candidate preparations for quality assessment programs for CD4 T-cell counting.

7. How can I tell if TransFix® has deteriorated?

The normal appearance of TransFix® is that of a clear green liquid. It should not be used if this is different. TransFix® should not be used beyond the expiry date on the package, vial or bottle.

8. Why is there moisture in the foil pouch?

The foil outer packaging for TransFix/EDTA Vacuum Blood Collection Tubes contains moisture to form a protective humid environment for the tubes. This helps to prevent TransFix evaporating and does not mean the tubes have leaked.

Once the foil pouch has been opened, this protective atmosphere is lost, and the shelf-life of the tubes is decreased to 6 months from the date that the pouch is opened. Tubes in an unopened pouch are stable at 2 - 8°C for 18 months from date of manufacture or until the expiration date on the label.

9. It appears that blood stabilised using TransFix/EDTA Vacuum Blood Collection Tubes has coagulated in storage. Is this an issue?

Blood taken into our TVTs are immediately stabilised and anti-coagulated post collection as long as the tubes are inverted sufficiently (x10) to mix the blood with the TransFix/EDTA solution. There is a sufficient concentration of K3EDTA within the TransFix solution (1.5 mg/ml final concentration) to anti-coagulate the blood sample and prevent clotting.

What is observed within blood filled TVTs during the storage period is a precipitation of the cells, whereby heavier cells and blood components sediment, forming two distinct layers. This is perfectly normal and will not affect analysis so long as the sample is mixed sufficiently before testing. This is a reversible process and is not a sign that the blood has clotted.

To homogenise TransFix/EDTA-treated blood, incubate the TVT at room temperature (18 - 25°C) for 15 minutes prior to use. Then mix the TransFix/EDTA-treated blood by rolling the TVT between the hands 10 times and by inverting. Repeat the mixing process if necessary until there is no cell sediment remaining.

How to Use TransFix®

10. How much TransFix® should I use to stabilise my sample?

For stabilising human whole blood samples the amount of TransFix® to use is 0.2ml per 1ml blood. A 1:5 ratio of TransFix® to human whole blood has been validated by Cytomark for use in a routine clinical setting. TransFix® Sample Storage Tubes contain 0.2ml TransFix® for the stabilisation of 1ml of blood. TransFix/EDTA Vacuum Blood Collection Tubes contain the correct amount of TransFix/EDTA to stabilise the volume of blood which the tube is designed to fill to.

For stabilising cerebrospinal fluid (CSF), 1-5ml can be added to TransFix/EDTA CSF Sample Storage Tubes that contain 0.2ml TransFix/EDTA.

Lower concentrations of TransFix® can be used for research purposes. In these circumstances we recommend that the user determine the optimal dilution required by setting up a titration experiment, starting with 0.2ml per 1ml of sample and reducing the volume of TransFix® in 0.05ml steps.

N.B. Early publications mention that a concentration of 0.1ml TransFix® per 1ml of blood is ideal,but the formulation of TransFix® has since changed so it is no longer applicable.

11. When should I add TransFix® to the sample?

For the best results, the sample should be treated with TransFix® as soon as possible. TransFix® instantly preserves the cell profile of whole blood and CSF samples at the time it is applied. If a 24 hour old sample is treated with TransFix® then the profile preserved will be for a 24 hour old sample. Therefore, TransFix/EDTA Vacuum Blood Collection Tubes are beneficial for immediate anticoagulation and stabilisation of blood cells.

If instant blood stabilisation is not possible then ensure that samples are TransFix® treated within 6 hours of venepuncture. Do not refrigerate the sample before treatment with TransFix.

If instant CSF stabilisation is not possible then ensure that samples are transferred into TransFix/EDTA CSF Sample Storage Tubes when they are less than 2 hours old. Do not refrigerate the sample before treatment with TransFix/EDTA.

12. Which antibody conjugates can be used with TransFix?

This link provides details of the antibody conjugates that have been validated by Cytomark for use with TransFix.

CD138 is known to be an unstable marker, and it may not be possible to stabilise with TransFix.

N.B. Cross linking of proteins may occur when using TransFix and we recommend that all antibody conjugates are validated in association with TransFix prior to use.

13. Does the type of anticoagulant used interfere with TransFix?

We recommend the use of K3 EDTA over other anticoagulants. However, there are publications that demonstrate the use of TransFix® with other commonly used anticoagulants.

14. What lysis buffer should be used in combination with TransFix® stabilised samples?

TransFix® is compatible with Quicklysis (product code: CYT-QL-1), an erythrocyte lysing solution that does not contain fixatives as well as BD FACS-Lyse. We recommend that the lysing buffer is validated when used in combination with TransFix® for research purposes.

15. Is it necessary to perform wash steps before staining TransFix® stabilised samples with antibodies?

It is not necessary to wash stabilised peripheral blood samples prior to staining and analysis; Cytomark use a lyse/no wash method when performing Quality Control assays for TransFix. For other sample types, it may be necessary to isolate cells by washing prior to TransFix® treatment. Cytomark recommends that all antibodies should be validated prior to routine use and a list of antibodies validated by Cytomark as suitable for use with TransFix® can be found here.

Stabilisation of Alternative Sample Types

16. Will TransFix® preserve cell lines?

Yes, but TransFix® is a fixative so the functionality of the cells will be lost. The user must define the optimal concentration of TransFix® to use in their particular application. Please refer to question 9.

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix® is used in applications other than those validated by Cytomark.

17. Has TransFix® been tested for other species?

Yes, it has been tested on mouse, rat, guinea pig, sheep, horse, pig and green turtle. The volume of TransFix® required to effectively stabilise 1ml of blood in different species varies considerably. In some species the volume required also varies by breed and type.

Research has been undertaken and publications made indicating the use of TransFix® in stabilising animal blood samples:

A rapid high-precision flow cytometry based technique for total white blood cell counting in chickens. C.Seliger,et al. Veterinary Immunology and Immunopathology (2012) Volume 145, 86–99.

Effects of Cyclosporin A induced T-lymphocyte depletion on the course of avian Metapneumovirus (aMPV) infection in turkeys. D. Rubbenstroth, et al. Developmental & Comparative Immunology (2010) 34, Issue 5, 518-529.

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix® is used in applications other than those validated by Cytomark.

18. Can TransFix® stabilise tissue biopsy samples?

The creators, UK NEQAS, attempted to stabilise fine needle aspirates with TransFix. Cytomark are able to provide the protocol for this experiment, but we have not independently verified these findings.

Cytomark does not take liability if TransFix® is used in applications other than those validated by Cytomark.

19. Is TransFix suitable for stabilising platelets for flow cytometric analysis?

There are limited settings in which TransFix may be useful for platelet flow cytometry analysis, and any assay would require the development and validation of a customised process. Possibilities for using TransFix include measurement of platelet antigen levels; measurement of platelet receptor occupancy by drug; and measurement of platelet functional responses in minimally processed samples. Note that unless the assay is EDTA-compatible and requires no sample processing, TransFix Sample Storage Tubes (rather than TransFix/EDTA Vacuum Blood Collection Tubes) will be required.

20. Can TransFix stabilise erythrocytes?

Yes. TransFix has been used to stabilise erythrocytes.

Canonico et al. analysed DNA cell content and membrane structure of erythrocytes, and found that Transfixed red cell protein profile as well as their morphological features appears almost unaltered.

Canonico, B., et al. (2010) Flow Cytometric Profiles, Biomolecular and Morphological Aspects of Transfixed Leukocytes and Red Cells. Cytometry Part B 78B: 267–278.

TransFix has also been used to create reagent cells for the identification of erythrocyte antibodies in the serum./ González et al. found that the addition of TransFix to erythrocytes expressing low-incidence blood group antigens (Dia and Lua) allowed cell stabilisation and proper antigen detection for at least 120 days after freeze-drying, which facilitating their use as reagent cells.

González, C., et al. (2016) Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent Cells. PLoS ONE 11(9): e0161968.doi:10.1371/journal.pone.0161968

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix is used in applications other than those validated by Cytomark.

21. Will TransFix stabilise trophozoite/schizont infected erythrocytes?

Yes. TransFix has been used to stabilise variant surface antigens (VSA) present on Trophozoite/schizont infected erythrocytes (isolated using gel floatation), for analysis via flow cytometry.

Mohamed, S. N. et al. (2016) The Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response to P. falciparum Infected Erythrocytes among Asymptomatic Children in Sudan. Published online 2016 Jun 28. doi: 10.1155/2016/7987686

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix is used in applications other than those validated by Cytomark.

22. Has TransFix been used to stabilise circulating endothelial cells?

Yes. TransFix has been used to stabilise circulating endothelial cells (CECs) in the context of analysing the correlation between cardiovascular risk factors and body adiposity index.

The following antibodies were used to stain CECs for flow cytometric analysis: CD31 (BD Biosciences FITC 560984), CD45 BD (Biosciences PerCP 347464), CD45 (BD Bioscience APC 340910), CD133 (Mylteny Biotec APC 130090826), CD105 (BD Bioscience PE 562380), CD106 (Southern Biotech PE 9510-09), KDR/flk1 (BD Biosciences Alexa 647 560495), CD146 (Chemicon International FITC P4321), CD34 (BD Biosciences PE 348057), CXCR4 (BD Biosciences PerCP 560670), CD117(c-kit) (BD Biosciences PE 555714).

Dias, I.B.F. et al. (2013) Relationships between emerging cardiovascular risk factors, z-BMI, waist circumference and body adiposity index (BAI) on adolescents. Clinical Endocrinology (2013) 79, 667–674

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix is used in applications other than those validated by Cytomark.

Further Applications of TransFix Stabilised Samples

23. Does TransFix® affect HIV Replication?

Published data indicates that HIV+ blood samples show a 1-log reduction in HIV replication as measured by HIV p24 antigen production. The results show that TransFix® has the ability to cause significant reduction in HIV replication.

Evaluation of TransFix®, a commercial whole blood stabilizing reagent. This product reduces HIV replication. Kim et al: Cytometry, Clinical Cytometry (2002) 50: 281

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix® is used in applications other than those validated by Cytomark.

24. Can cells preserved with TransFix® be used for PCR?

Yes, but the user must define the optimal concentration of TransFix® to use in their particular application. Please refer to question 9.

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix® is used in applications other than those validated by Cytomark.

25. Can cells preserved with TransFix® be used in Functional Assays?

No, TransFix® is a preservative that fixates the cells. As a result they cannot be used in functional studies.

26. How can I analyse TransFix-stabilised circulating tumour cells?

We have validated our Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes to stabilise CTCs in Human blood for 5 days at ambient temperature. Cells were detectable by flow cytometry, based on the epCAM marker.

Other published downstream applications include:

  • Staining with anti-acH4 and DAPI, and analysed by imaging flow cytometer (1).
  • Staining with anti-EpCAM, cytokeratin, survivin and CD45 expression and DAPI analysed by imaging flow cytometer (2).
  • CTC isolation via size-exclusion filtration (Screen Cell Cyto Tubes), then analysed by Facs, DepArray, DNA amplification, and Sanger sequencing (3).
  • CTC isolation via size-exclusion filtration (Hamilton STARlet™ robot) and analysed by multiplex immunoassay (4).

Cytomark has not independently verified these findings. Cytomark does not take liability if TransFix is used in applications other than those validated by Cytomark.

27. Can I isolate peripheral blood mononuclear cells (PBMCs) via Ficoll separation on a TransFix® treated blood sample?

It is possible to isolate PBMCs via Ficoll separation on a TransFix® treated sample, but for best results we recommend that the user isolates the PBMCs from blood prior to TransFix® treatment.

28. Can TransFix® treated plasma be used for clinical chemistry analysis?

Cytomark have not evaluated TransFix® in this application. However, plasma from TransFix® treated blood samples will contain paraformaldehyde, buffering solution and anti-proteolytic reagents. All of these components have the potential to interfere with clinical chemistry analysis. On this basis we recommend that if you want to perform clinical chemistry analysis you take a second tube of blood which is not TransFix® stabilised.

29. Can TransFix® stabilised cells be used in combination with cell viability stains?

We do not recommend that TransFix® stabilised cells are analysed in this way as the reagent contains a low concentration of paraformaldehyde that fixes the cells as soon as they come into contact with TransFix®, therefore they are no longer viable.

30. Can Blood Controls for immunophenotyping be prepared using TransFix?

Yes, blood controls for immunophenotyping can be prepared using TransFix, and can help towards meeting the ISO15189:2012 standard.

The ISO standard entitled 'ISO 15189: Medical laboratories - Particular requirements for quality and competence', refers to the 'use of quality control materials that react to the examining system in a manner as close as possible to patient samples' (section 5.6.2.2), and that 'the use of independent third party control materials should be considered, either instead of, or in addition to, any control materials supplied by the reagent or instrument manufacturer' (section 5.6.2.2, Note 2).

It is common practice in immunophenotyping and immune monitoring laboratories to use a patient sample as a daily or even weekly control for flow cytometry. In addition, when a rare immunophenotype is detected there is an opportunity to preserve it for teaching or comparison purposes.

All samples left untreated are subject to apoptosis of cells and proteolytic degradation leading to immediate changes in the phenotypic profile. This poses a challenge, not only with analysing the sample in a short time scale but when preserving samples of interest for re-testing.

Simple addition of TransFix to a blood sample enables stabilisation of the immunophenotype for up to 14 days. Subject to validation at a clinical flow facility, this could provide a solution when such reference material is not available and would address the clauses referenced above.

Therefore, using TransFix, it is possible to stabilise blood samples (diseased and healthy) for use as a reference tool during flow cytometry analysis.


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