[1920] DOE Methodological Evaluation of Efficacy of Residual Paraffin Removal during IHC Process Development.

Sean Liu, Gaylee Nuttman, Julie Erickson, Grant Toland, Therese Phillips, Scott Webster, Rosanne Welcher. Dako North America, Carpinteria, CA

Background: Non-toxic and biodegradable Histoclear^TM is widely used as a xylene substitute for immunohistochemical (IHC) deparaffinization and post-IHC clearing during permanent coverslipping. However, Histoclear has been reported to be a less efficient clearing agent and can fail to completely remove paraffin from the specimens. We have observed that residual paraffin can be trapped in cell structures, especially in the nuclei of some formalin-fixed, paraffin-embedded (FFPE) cell pellets when the sections are subjected to heat pretreatment. This results in an artifact which is manifested as an apparent patchy distribution of IHC staining and a loss of clearly distinguishable cell margins. Since both heat pretreatment and the uses of Histoclear are common practices in IHC, we investigate which combination of these factors will reduce the incidence of paraffin artifacts.
Design: This study employs design of experiments (DOE) methodology to compare the aforementioned procedures for the treatment of FFPE cell pellet sections before and after IHC staining. Three factors, deparaffinization, heat, and post-IHC clearing were chosen for the 2^k factorial experimental design. The contribution of any one factor and the interactive contribution of any two factors of the procedure were evaluated monitoring the level of residual paraffin. Data was analyzed by Minitab® software.
Results: The results confirmed that 1) Heat pretreatment cause a major increase in residual paraffin staining artifact, 2) Use of Histoclear in deparaffinization procedures was not as effective for removal of paraffin as xylene when heat pretreatment was subsequently performed and 3) Post-IHC clearing with Histoclear prior to permanent mounting of coverslips was essential to achieve a satisfactory solution of quality staining with minimal residual paraffin.

Factors and Levels

Factor	Description	Low level of factors (-)	High level of factors (+)
A	Deparaffinization	Histoclear	Xylene
B	Heat	No	Yes
C	Post-IHC clearing with Histoclear	No	Yes

Two-level 3 factor full factorial DOE results

Run	Combination	Factor A	Factor B	Factor C	Score
1	-1	-	-	-	1.5
2	a	+	-	-	1
3	b	-	+	-	70.5
4	ab	+	+	-	12.5
5	c	-	-	+	0
6	ac	+	-	+	0.5
7	bc	-	+	+	0
8 = 2x2x2	abc	+	+	+	0

Conclusions: DOE technique is a powerful tool for the systematic evaluation of several concurrent response variables and the efficient improvement of process development. This study recommends a higher-quality and eco-friendly procedure using the combination of Histoclear deparaffinization and post-IHC clearing when residual paraffin is observed within cell structures.
Category: Techniques

Monday, February 28, 2011 1:00 PM

Poster Session II # 274, Monday Afternoon