Leica PELORIS DUAL User Manual

High-Speed Processing of Large Specimens on the PelorisTM Dual Retort Tissue Processor

Geoffrey Rolls

Leica Microsystems, Biosystems Division, Melbourne, Australia

High-Speed Processing of Large Specimens on the

PelorisTM Dual Retort Tissue Processor

Geoffrey Rolls

Leica Microsystems, Biosystems Division, Melbourne, Australia

This paper presents the results of comparative evaluations carried out during field trials in two large public hospitals, that set out to examine whether the design features of the Peloris dual retort tissue processor led to reduced processing times for large specimens without compromising quality. In the context of a busy histopathology laboratory, we demonstrate that the introduction of Peloris processing would allow the processing of large, dense specimens in 6 hours leading to the completion of more runs in a working day and the reduction of turnaround times.

Ten sequential six hour and nine hour processing runs were undertaken on a Peloris processor at either 45 °C or 55 °C and the resultant blocks and sections were evaluated for quality using a comprehensive scoring system. Peloris results were compared to those achieved on a matched panel of specimens processed using a processor that represents the industry standard (the Tissue-Tek® VIP™). The results show that for large, dense specimens Peloris can produce results of an equivalent standard to an “overnight” schedule (13 hours) run on a VIP using much shorter schedules. Results also indicate that there is some advantage in processing at higher temperature (55 °C) for both the six and nine hour schedules.

Introduction

A fundamental requirement in the histopathology laboratory is to safely and effectively process specimens. Sectioning of paraffin blocks should be straightforward with the resultant sections being of a high standard demonstrating excellent morphological detail. This applies to the complete range of specimen types and sizes, ranging from tiny, delicate fragments to sizeable wedges of dense tissue. In busy laboratories these requirements must be balanced against increasing demands to reduce turnaround times, to process greater numbers of specimens and to complete more runs in the working day.

The traditional view embraces a conservative approach to processing of all specimen types. A survey of standard histology texts published in the last twenty years reveals an average processing time of 15.7 hours for “routine overnight processing” using xylene as a clearing agent. A survey of local and international laboratories shows that the average duration of a “routine overnight” schedule on an enclosed processor using xylene and excluding any additional fixation steps is 10.3 hours (see Table 1 for details). Of course these laboratories do use shorter schedules for special purposes, where particular types of specimens are to be

processed (eg. endoscopic biopsies), or where specimens falling within strict size limits are included.

These field trials were conducted by Vision BioSystems (VBS). Vision Biosystems has since formed part of the Biosystems Division of Leica Microsystems. What is needed then, is a processor which can effectively process a full load of cassettes, say 200 – 300 per retort, containing the complete range of tissues normally included in a “routine overnight run”, but doing so in a much shorter time, preferably within the six hour threshold that would allow them to be completed within one working day. The Peloris dual retort tissue processor has been designed with these requirements in mind. Compared to processors representing the current industry standard it has a much faster and more even heating response in the retorts, fast fill and drain actions, a basket design allowing better fluid exchange with reduced reagent carryover, and more effective agitation. These features are designed to reduce processing times. The purpose of this paper is to present the results of comparative evaluations carried out during field trials that set out to show that the design features of Peloris lead to reduced processing times without compromising quality.

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Number of Labs	Types of Processor		Schedule Description	Average Number	Average Number	Average Total Time	Average Total Time
	(number)			of Steps	of Steps Excluding	Excluding Fixation	(minutes)
					Fixation	(minutes)
25	Leica TP1050	(20)	Routine overnight	13	12	620	694
	VIP	(4)
	Shandon	(1)

Table 1. Routine overnight processing schedules

Independent field trials were carried out in two large public teaching hospitals in Melbourne, Australia, during 2003 and 2004. They were conducted in the Anatomical Pathology laboratories of the Royal Melbourne Hospital (RMH) (1200 beds) and at Austin Health (AH) (840 beds). The evaluations relating to processing speed were conducted in a similar fashion during both field trials. They involved senior histology scientists from both external laboratories together with scientists from Vision BioSystems, in the assessment of processed blocks and stained sections from a number of processing runs. Using duplicate sets of matched specimens of a variety of dimensions and types, multiple processing runs were carried out using Peloris and an “industry standard” processor in each laboratory (in both cases a Tissue-Tek VIP). Results from Peloris rapid schedules run at 45 °C and 55 °C were compared with VIP standard “routine overnight schedules” currently used in each laboratory which served as a normal control.

Processing at elevated temperatures is widely accepted as a means of accelerating processing by increasing diffusion rates in specimens (1, 2) although some authors believe that this can cause additional shrinkage and staining problems (2, 3). We have certainly not found this to be the case during extensive testing of Peloris. These trials provided an opportunity to compare results of rapid processing at 45 °C and 55 °C and to confirm that there were no adverse effects from using the higher temperatures.

The assessments were carried out independently with staff at the external sites using slightly different scoring methods, with the assessors being unaware of the schedules used to process the specimens. The scoring system used by VBS staff has been extensively used throughout the development and testing of Peloris to evaluate the quality of tissue processing and as a mechanism for optimizing standard processing protocols. A score is calculated by assessing 23 parameters and is expressed as a percentage. The complete details are provided elsewhere(4).

Method

Testing throughout the RMH and AH trials was conducted such that all processing results were compared directly to those of their existing tissue processors (RMH - Tissue-Tek VIP 4, AH Tissue-Tek VIP 5). As far as possible specimens for assessment were kept identical in terms of size, fixation and source on both instruments for each processing run. All specimens were thoroughly fixed. Not every specimen in each run was evaluated. Any specimens that were not for evaluation but loaded into retorts on Peloris to provide a representative case load comparable to the VIP, consisted of pig tissue supplied by VBS. Typical specimens used in assessment of processing and their approximate dimensions are shown in Table 2.

At each laboratory for each of 10 sequential working days, three processing runs were carried out. Two rapid schedules were run on Peloris using the two retorts. Retort A was used for the 6 hour schedule and Retort B for the 9 hour. Runs were carried out daily at either 45 °C or 55 °C. For each 6 and 9 hour run on Peloris a routine overnight run was completed on a VIP containing a normal diagnostic specimen load together with a set of the duplicate test specimens (200 – 250 cassettes). These served as our normal control group. Fresh reagents were provided for run 1 and not changed for the 10 runs on both Peloris and the respective VIP. For each Peloris run, in addition to the test specimens, cassettes containing various pig tissues were included to take the specimen number to 228, which provided an equivalent specimen load to that in the respective VIP processor (75% capacity of each retort). The processing schedules used are shown in Tables 3 and 4. Figure 1 illustrates the difference in step times between the various schedules used. Note that the total pump and drip times in the Peloris schedules are considerably shorter than those of the VIP.

Royal Melbourne Hospital (RMH)		Austin Hospital (AH)
Tissue	Dimensions (mm)	Tissue	Dimensions (mm)
Intestine	15 x 10 x 5	Intestine	30 x 8 x 5
Liver (pig)	25 x 15 x 5	Intestine	20 x 15 x 5
Spleen	15 x 10 x 5	Liver (pig)	20 x 20 x 5
Lung	25 x 15 x 5	Liver	30 x 25 x 5
Kidney (pig)	15 x 10 x 5	Lung	20 x 20 x 5
Heart (pig)	20 x 15 x 5	Lung	20 x 15 x 5
Thyroid	20 x 10 x 5	Kidney (pig)	20 x 15 x 5
Skin	30 x 25 x 5	Kidney	15 x 15 x 5
Breast	30 x 25 x 5	Heart (pig)	20 x 20 x 5
Prostate	Chips	Heart	20 x 15 x 5

Table 2. Typical specimens used in assessment of processing

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