[Histonet] Validation of parriffin

[Tony Henwood]

Hi John,

Following is the process we used when a change in wax was needed (different supplier, change in formulation etc).

Validation of tissue processing waxes
Changing processing and embedding waxes could improve or badly affect your histology quality. Your lab has years of experience with a particular wax and have developed and optimised stains (including immunohistochemistry) on tissues embedded in this wax. To change requires re-validation of all these stains.

  1.  Compare data sheet with old wax datasheet (melting point, additives).
  2.  Wax Purity Test

If components of the wax are not soluble in the clearing agent, then tissue samples will not be effectively infiltrated during processing nor will they dewax adequately for staining methods. The following test procedure has been recommended:

  1.  Take 1 gram of wax and mix in 20ml of xylene
  2.  Record time for full dissolution
  3.  Check for any undissolved residues and contaminants

  1.  Occurrence of nuclear birefringence

Incomplete wax removal can cause several problems including birefringence of cell nuclei (Vlachos 1968, Stain Technol. 43: 89–95), so-called pink disease in which the distribution of stains is patchy and distinct nuclear margins are lost, and weak to false negative immunohistochemical staining (Henwood 2012, Biotechnic & Histochemistry, 87(1), 46-50). Although there may be several causes of pink disease, one common cause is the incomplete removal of wax. It is commonly accepted that birefringent cell nuclei are due to incomplete wax removal by xylene (Vlachos 1968). Vlachos (1968) also noted that nuclear staining was weaker in areas that showed increased birefringence.
Using routine dewaxing and rehydration methods followed by observation using polarisation microscopy can indicate differences in wax properties that might require changes in dewaxing procedures prior to routine staining.

  1.  Tissue Wax Test

It is important to ensure that use of the wax results in well-processed blocks that are easy to section. Whenever a new wax is being assessed it is important that several tissues are used. I would recommend:

  *   Brain – a tissue rich in lipids and delicate neural fibres, can 6-7μm sections be easily sectioned?
  *   Liver – regular polyhedral cells bound in cords, that will show any variation in infiltration and possible shrinkage across the tissue
  *   Skin – a mix of keratinised squamous cells, elastic and connective tissue
  *   Tonsil or lymph node – how do the lymphoid cells appear especially when thin 2μm sections are cut?
  *   Appendix – a mix of delicate epithelial cells, collagen and muscle. Do stained sections exhibit folding of the epithelial cells due to excessive shrinkage?
  *   Kidney – what is the quality of 2μm sections?

  1.  It is important to compare the new wax with the old (ie run the tests in parallel).




Regards,



Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)

Principal Scientist, the Children’s Hospital at Westmead (Retired)

Adjunct Fellow, School of Medicine, University of Western Sydney.

________________________________
From: John O’Brien via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 11 March 2025 07:41
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Validation of parriffin

Histonetters
  ?When a new paraffin is introduced to a Tissue processor is validation required?
If so what is protocol?
Thank you
IMEB Inc

_______________________________________________
Histonet mailing list
Histonet at lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet