Molecular and Histological Evaluation of Histofix SAN a New, Economic, Non Carcinogenic, Non Formalin Tissue Preservative
Dr. Zahir Hilmi
Zahir A. Hilmi 1,2 , Umer A. Hamd 2, Mai Aldaw M . Yousif 2, Korolin M. A. Sanhory 5, Al Gaili M. Algaili 3, Adel Merghani Babiker 4 ,Imad M. Fadl Almola 6, Nasr al deen M. A. El Wali 4 and Mohamed A. Sanhory 5.
1- College of Medical laboratory Science, Sudan University For Science and Technology
2- Center For Bioscience and Biotechnology, University Geazira
3- Faculty of medical laboratory Science, University Geazira
4- Institute of Nuclear Medicine and Oncology, University of Gezira
5- Sanhorial Factory for Medicines and Cosmetics, Khartoum , Sudan
6- Faculty of medical laboratory Science, Al Neleen University.
Introduction and objectives:
Formalin is commonly used for tissue preservation, but degraded DNA. This study aimed to evaluate Histofix SAN (5% and I %) for preservation of human blood molecular biology. Also it aimed to evaluate the effects of different tissues preservation methods, including the new fixative Histofix SAN, on the fixation and staining quality of sheep different tissues for different durations of time.
Materials and Methods:
Human blood specimens (300µl each) were preserved in ; formalin 10%, buffered formalin, Histofix SAN (5% and 1%), ethanol, 70% at 4ºC and freezing at – 20ºC and -60ºC. A Promega DNA extraction kits were used to extract the DNA. Three preservation periods were applied (3 days, 15 days and 30 days). The DNA concentration (in ng/µl) and purity were measured. The DNA samples were loaded in 8% acrylamide gel electrophoresis. PCR for interferon-γ receptor was performed.
Sheep tissues (Kidney, liver, small intestine and brain) were fixed in Formalin 10% and Histofix SAN (5% and 1%). The tissues were fixed for five preservation period (One, two, three, seven and thirty days). The effects of fixatives on fixation quality, tissues features, and morphology were considered. The staining quality (H & E) and ranking were assessed on the basis of clear contrast between the nucleus and cytoplasm.
Histofix SAN 5% obtained the highest DNA concentrations (159, 133 and 108) for the three durations. In contrast, formalin 10% and buffered formalin obtained the lowest DNA concentrations (46, 40, and 1.0) (42, 21 and 0.6) respectively. The DNA concentration by Histofix SAN was found to be more than hundred times that of formalin and buffered formalin after thirty days. The PCR reaction gave results only the DNA extracted from Histofix SAN.
The fixation quality was good in all tissue types for both preservatives. Similar excellent and very good staining qualities were obtained by both Histofix SAN 1% and Formalin 10% for three days according to tissue types. After fixation for seven days, the staining quality decreased to good in Formalin 10%, while Histofix SAN 1% mostly gave excellent to very good staining, not only for seven days but up to thirty days. Histofix SAN 5% obtained excellent staining quality for short periods (one to two days), decreased gradually to reach poor staining quality thirty days later.
In contrast to formalin, Histofix SAN 5% yielded higher DNA concentration and purity. It did not degrade DNA; also it stopped all endonucleases activities that may decrease the concentration and purity.
Histofix SAN 1% good fixation quality and excellent to very good staining quality could be explained as follows. Histofix SAN 1% did not interact or form complexes with tissues macromolecules. It has antimicrobial activities and surrounded the tissues with thin protectivelayer that prevents water loss or shrinkage. It preserved the tissues as similar fresh tissues in colour with soft texture. Histofix SAN has a nice smell not as formalin.
The present research recommended Histofix SAN as a new, economic, and non carcinogenic, alternative for formalin 10% for histopathology and molecular biology. Further research on its effects on RNA, proteins and immunohistochemical applications are highly needed.