The influence of different fixatives and preparation methods on morphology, immunohistochemistry and molecular analyses

Lone Bojesen1, Dorte Skriver-Jensen1, Wojciech Skovrider-Ruminski1, Nadja Beekhuijen1, Reema Butt1, Anne-Mette Wenning1, Birgitte Bols1, Estrid Høgdall1 and Hanne Bjørn1.

1. Department of Pathology, Herlev Hospital, University of Copenhagen, Denmark

• Formaldehyde is the most commonly used fixative in the area of pathology. Unfortunately it is carcinogenic and allergenic.

• Alcohol based fixative used for molecular analysis may result in similar morphology, more intense background staining in the H&E stain but also diverging immunohistochemical staining results (1,2).

• DNA and RNA yielded longer fragments (1,2).

INTRODUCTION• MF is not optimal for substitution of Formaldehyde as a routine fixative for

traditional diagnosis• MF were superior for molecular diagnostic. • Further investigations of reasons which may explain the differences by

preparation method or ingredient in the solutions used are warranted.

CONCLUSION

REFERENCES1: Cox et al: Assessment of fixatives, fixation and tisssue processing on morphology and RNA integrity. Experimental and Moleculat Pathology. 2006; 80: 183-1912: Vladimir Vincek et al: A Tissue Fixative that Protects Macromolecules (DNA, RNA and Protein) and Histomorphology in Clinical Samples. Laboratory Investigation. 2003; 83 (10): 1427-1435

RESULTS STAINS

MATERIALS AND METHODS

RECOMMENDATIONS

Samples: large intestine cut into small biopsies (approx. 2x2x4 mm) fixed for 4 and 6 hours in either 4% neutrally buffered formaldehyde (NBF) or Tissue Tek® Xpress® Molecular Fixative (MF) processed on both Tissue Tek® VIP® 5 and Tissue Tek® Xpress®.

Stains: H&E and Alcian PikroSirius (APS). Immunhistochemical stains: CD117, Ki67, Actin SMM-1, CK20 and PMS2. Morphology, histochemical and immunohistochemical stains were evaluated with a score from 0-3, where 3 is optimal and 0 is poor.

DNA quality and quantity: Fragment analysis and RealTime PCR.

Morphology, histochemical and immunohistochemical results are shown I table 1. We found that Molecular Fixative causes shrinking in varying degree (Picture 1-2). This is most likely do to the coagulant nature of MF, which precipitates proteins. The background staining with Eosin in the HE staining is intensified and the competitive staining of muscle and connective tissue in APS is not optimal (Picture 3-4), since there are more reactive amino groups available in the MF fixed tissue, and we did not optimize the stain for the new fixative. We found no discernable difference between preparation method or fixation time.

Picture 1: 4 hours NBF, Xpress HE Picture 2: 4 hours MF, Xpress HE

Picture 3: 4 hours NBF, Xpress APS Picture 4: 4 hours MF, Xpress APS

The molecular analysis performed showed a tendency toward a better quality and quantity using MF (Figure 1-2), since MF is not a crosslinking fixative. Furthermore it is observed that method of tissue preparation may also influence the molecular results (Figure 3-4), depending on the analysis performed. There is some speculation on whether this might be causes by the microwaves in the Xpress.

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Figure 1: 400bp quantity from NBF and MF fixed tissue

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Figure 2: 600bp quantity from NBF and MF fixed tissue

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Figure 3: 400bp quantity from VIP and Xpress processed tissue

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Figure 4: 600bp quantity from VIP and Xpress processed tissue

• Encourage optimal handling and verification of fresh tissue for molecular analyses with registration in a biobanking facility.

• Process 1 block of NBF tissue on the VIP (overnight) for molecular analyses if fresh tissue is not possible.

AIMTo compare the influence of different fixatives and different preparation methods on molecular pathology results

Average score NBF 2,94 3,00 2,92 2,91 2,42 2,92 2,94 2,83Average score MF 2,00 3,00 2,14 2,36 2,89 3,00 2,78 2,08

Average NBF+ VIP 2,94 3,00 3,00 2,83 2,67 2,94 3,00 2,89

Average NBF + Xpress 2,94 3,00 2,83 3,00 2,17 2,89 2,89 2,78

Average MF + VIP 2,00 3,00 2,00 2,11 3,00 3,00 2,89 2,06

Average MF + Xpress 2,00 3,00 2,29 2,61 2,78 3,00 2,67 2,11

Table 1: Average score for morphology, histochemical and immunohistochemical stains

Immunohistochemical staining varies and is largely dependent on the specific antibody. CK20 and PMS2 (Picuture 5-6) shows a decline in stainability in tissue fixed in MF, whereas Ki67, Actin SMM-1 and CD117 showed no difference between the different variables. MF is a coagulant fixative which opens the proteins tertiary structure, which means that some epitopes are more easily accessible and others are hidden.

Picture 5: 4 hours NBF, Xpress PMS2

Picture 5: 4 hours MF, Xpress PMS2

RESULTS STAINSCONTINUED

RESULTS MOLECULAR ANALYSES

Morphology HE APS CK20Actin SMM-1 Ki67 CD117 PMS2