PATHOS Histoprocessor: FAQ

General

What is the benefit of using the PATHOS?

Routine same-day turn-around time for diagnosis on tissue samples received during the working day. Time savings of at least 24 hours.

What is the throughput capacity of the PATHOS?

Up to 210 universal cassettes per processing run, consisting of three separable layers of 70 cassettes. Processing takes as little as 60 minutes for small biopsies, and up to 3 hours for larger blocks.

Isn't the 210-cassette throughput capacity of the PATHOS a limitation compared to conventional overnight processing of up to 300 cassettes?

No. Conventional overnight processing of 300 cassettes varies from 12–14 hours. The PATHOS offers the ability to process 210 universal cassettes every 60, 90, 120, or 180 minutes, depending on the laboratory needs.

Is the PATHOS based on new technology?

Yes. The PATHOS is based on the innovative technology developed for our RHS-1 histoprocessor, covered by European Patent no. 0822403. We have further developed the system to automate histoprocessing for unattended operation.

What are the steps involved in PATHOS histoprocessing?

1. Simultaneous (synergistic) extraction of tissue-bound water and lipids, exploiting the combined properties of an ethanol/isopropanol and long chain hydrocarbon patented solution at preset pressure/temperature conditions.
2. Partial extraction of superficial bound JFC residues on tissue by lowering the vaporization temperature of solution with the use of vacuum (partial drying step).
3. Final extraction (vaporization) of tissue-bound JFC residues, carried out in molten wax under vacuum.

Can I perform microwave fixation of samples with the PATHOS?

Yes. Tissue samples are first allowed to presoak in formalin (up to 30 minutes for small biopsies and up to 1 hour for the larger blocks). Microwaving is then performed at 5 to 10 minutes for each millimeter of tissue thickness. For example, a 3mm-thick block will take 15 to 30 minutes, at a temperature of 50°C. The unit has pre-set fixation programs to correspond to each of the tissue processing programs available.

CAP Requirements

How do Milestone microwave units comply with College of American Pathologists (CAP) requirements for the use of microwave units in laboratories?

A. ANP.27720 - Phase1

Are microwave devices monitored at least annually to ensure that there is less than 5mW/cm2 leakage at a distance of 5cm from the surface?

Each Milestone microwave instrument is tested for radiation leakage by the manufacture. Documentation of this testing is reported in the instrument Quality Control Report. A report of leakage detection testing is included in the Purchased Service Contract yearly preventive maintenance.

B. ANP.28290 - Phase 1

Are microwave devices periodically monitored for temperature reproducibility?

Microwave Wattage Output Testing procedures are integrated into the software of Milestone microwave instruments and can be easily performed by the customer. Written documentation of each run temperature can be produced using the Saved Session procedure.

C. ANP.28860 - Phase 1

Are all containers used in microwave devices made from microwave-transparent material? Note: Examples of microwave-transparent materials include ceramics, sodium borosilicate glass, unleaded quartz, fluoropolymers, and nonpolar plastics such as polypropylene, polyethylene and PTFE compounds.

All containers and accessories supplied by Milestone S.r.l. are made from microwave-transparent material.

D. ANP.29430 - Phase 1

Are microwave devices properly ventilated?

Note: Microwave devices should be placed in an appropriate ventilation hood to contain airborne chemical contaminants and potentially infectious agents. Microwave devices used outside a fume hood should have an integral fume extractor that is certified by the manufacturer for use in a clinical laboratory.

This checklist question does not apply if only non-hazardous reagents are used in the device (e.g., water, certain biological stains).

• Milestone Rapid Microwave Processors are designed with an integral fume extractor.
• An exhaust outlet is located in the back of the units.
• Exhaust hoses are supplied by Milestone with recommendation that the hose be placed inside a chemical fume hood or to an outside venting system. Pathos units have a built-in charcoal/dust filter for stand alone operation.
• The work area is totally enclosed during operation.

Reference:
College of American Pathologists (CAP)
Commission On Laboratory Accreditation
Laboratory Accreditation Program
Anatomic Pathology Checklist, ANP.27720, ANP.28290, ANP.28860, ANP.29430, Revised 06 October 2005

Clinical and Laboratory Standards Institute. Microwave Device Use in the Histology Laboratory; Approved Guideline. CLSI document GP28-A [ISBN 1-56238-563-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.

Operation

How do I determine which processing time to use when I have biopsy samples of varying dimensions?

You can mix small biopsies with larger samples, and the processing time is determined by the dimensions of the largest sample present.

Is there a risk of over-processing small biopsies if using the longer processing programs?

No, if using the PATHOS system.

Does the operator of the PATHOS unit need to be present at all times whlie the unit is operating?

No. The operator is only required to load the cassette rack, then unload it at the end of the processing cycle. The unit is fully automated, and therefore requires no additional time spent attending to it.

Are there any changes required in the retrieval technique fpr Immunohistochemsitry (IHC)?

There will be reduced need for an aggressive and lengthy retrieval process. Why? If using microwave fixation and histoprocessing, you have reduced exposure times to the adverse effects of antigen masking/preservation associated with reagents in conventional histoprocessing. If tissues are formalin fixed with longer conventional times, followed by microwave processing, there will be little or no change to your existing retrieval process and antibody dilutions.

Is any pretreatment of biopsy cassettes required prior to histoprocessing with the PATHOS?

All tissue cassettes are briefly rinsed in a sequence of two reagent-grade alcohols within the PATHOS unit, to eliminate surface residues of water and formalin. This step, like all steps in the PATHOS procedure, is automated, with no need for operator involvement.

Is fixation of tissue samples required prior to PATHOS histoprocessing?

Yes, as is the case for conventional histoprocessing. Tissue samples that are not fixed (e.g., with formalin) prior to histoprocessing will exhibit features of alcoholic fixation, due to the ethanol and isopropanol components of the JFC solution. This is similar to the consequences in conventional processing.

What is the reagent usage rate with the RHS system, and therefore the cost for each paraffin block?

For 210 cassettes, 5,000 ml is equal to:

• 24 ml average per cassette for the first run
• 12 average per cassette if re-used once
• 8 ml average per cassette if re-used twice

Are there variations in processing times for some tissue types?

Yes, when dealing with bone and brain tissue. In the case of bone, the high density of the decalcified bone requires an increase in wax impregnation times, by as much as 50%, to compensate for the increased resistance to wax penetration. In the case of brain tissue, the high density of the lipid content requires extended extraction time for clearing of these lipids, by as much as 50%. The need for an increase in time also applies in the case of conventional processing where longer processing times are usually required, especially for the larger blocks. In both brain and bone, the need for longer times will depend on the size of the samples.

Results

Is PATHOS processing detrimental to preservation?

No. In fact, it can produce better quality of antigen preservation if we combine microwave fixation and microwave histoprocessing. You will also see a number of situations where the antibody dilutions need to be increased due to improved sensitivity. This is a result of reduced exposure to the adverse antigen effects of prolonged exposure to formalin, and the dehydrating clearing process associated with the longer conventional method of histoprocessing.

Does the rapid processing procedure produce difficult-to-cut blocks?

No. On the contrary, the process produces blocks that section as well as, or better than, conventionally processed material. Furthermore, larger blocks become easier to cut, due to the softening/lubricating effect of the rapid processing.

What benefits will lab staff notice when cutting various tissue types processed with the PATHOS?

Toenail sections can be cut very easily (softer), without the risk of sections falling off the slide, when compared to conventional processing.

Decalcified biopsies are easier to cut, not having the hardness seen with conventional processing.

POC (products of conception) which are very hemorhagic do not fragment (pulverize), producing uniformly cut sections with improved nuclear/cytomorphology when compared to conventional processing.

Overall sectioning properties enable sections to be cut easily, enabling a ribbon of sections to be cut.

Paraffin tissue blocks require little or no soaking in water to soften blocks prior to sectioning.

Technical

Hardware

Can I use metal cassette lids inside the unit?

No. No metal parts in the microwave cavity. Cassettes must be made of microwave-transparent material.

What type of biopsy filters can I use with the PATHOS?

Cassettes with mesh lining, biopsy mesh bags and mesh capsules, translucent paper (e.g., tea bags). The use of rubber foam pads as filters is restricted to longer processing times (i.e., ≥ 1¼ hours), as it is a barrier to shorter processing (restricts reagent flow).

Reagents & Wax

What is the composition of the one-step dehydrating/clearing solution (JFC)?

JFC solution is a patented product consisting of a mixture of ethanol, isopropanol, and a long chain hydrocarbon.

Can I use other reagents instead of JFC for the histoprocessing?

Yes. The PATHOS is not a closed system for reagents. It was developed to allow the user to choose the proprietary JFC solution, or ethanol-isopropanol, or a combined use of ethanol-JFC, for use as follows:

Ethanol-Isopropanol-Wax
Ethanol-isopropanol protocol is recommended as the standard method for histoprocessing small to medium-large biopsies, provided that the fat content of the tissue blocks is kept to a minimum. It is a very cost-effective protocol for general histoprocessing. This is the recommended method for processing of renal biopsies.

Ethanol-JFC-Wax
Ethanol-JFC is for use with tissues where overnight fixation of tissue is uncertain, and for partially fixed tissues. In this instance, the ethanol acts as a post-fixative step, since JFC is not a fixative. This protocol is especially suited for routine use and for very fatty tissues, such as breast. The ethanol step will extend the reuse life of the JFC.

JFC-Wax
JFC solution is strongly recommended for tissues known to be well fixed, generally at least overnight, and is of major benefit when dealing with tissues that contain high lipid content, such as larger pieces of breast and brain.

Xylene is not recommended for use, as we have developed the system with safety in mind, to avoid the use of this highly toxic reagent—and the hardware seals reflect this consideration. The seals within the system are not designed to handle xylene, and will risk damage if it is used.

How many times can I re-use the JFC solution?

It can be used at least twice, and possibly further, depending on the quantity and size of samples processed, and whether a preceding microwave step in ethanol is used. It is essential, if you intend to reuse again, to make sure that the container is covered to prevent loss of reagent vapors, and to prevent absorption of atmospheric moisture.

Can I dispose of JFC solution down the drain?

No. Due to the properties of the hydrocarbon (not miscible with water in pure form), it may accumulate and therefore increase risks of flammability in the drainage system. Dispose of JFC solution in accordance with disposal instructions from your local environmental protection agency.

Can I recycle the JFC solution?

No, as the ratio of the individual components is not disclosed. However, if the recycling unit is of a high standard, the individual components may be used for other applications (e.g., ethanol use in staining machines).

What is the temperature of the JFC solution used?

68°C.

How many times can I reuse ethanol or isopropanol?

Provided the cassettes are briefly washed in reagent-grade alcohol steps (included within the PATHOS) to remove superficial water and formalin, it can be used at least twice and possibly more, depending on the quantity and size of samples processed.

What is the temperature of the ethanol and isopropanol used?

65°C for ethanol and 68°C for isopropanol.

Am I restricted in the type of paraffin wax used?

No. However, it is recommended to use histology-grade wax.

What is the wax temperature used in the PATHOS process?

65–70°C.

Do I need to dispose of wax after each processing cycle?

No. Due to vaporization "removal" of the solutions, the wax can be used repeatedly, requiring only an occasional "top up" to replace wax absorbed by tissue—or alternatively at infrequent intervals (e.g., weekly or monthly) to be transferred to the embedding center.

How is the reagent system organized in the PATHOS?

The system is divided into two sections:

1. Fixed storage tanks. These supply the reagents to the processing retort during processing. They are located at the back of the pull-out drawer, at the base of the unit, consisting of six storage tanks.
2. Movable replacement tanks. Located in the front of the pull-out drawer, it is the area for unloading the used reagents from the fixed tanks and then reversing the process by reloading with fresh reagents into the rear fixed tanks. This area holds six unloading/loading containers.

How is the sequence of reagent exchange performed?

As an overview, the system is based on an automatic exchange of reagents, following a set sequence as we perform the reagent exchange via the processing retort (transit station).

Can I do a manual change of reagents?

Yes. The PATHOS is set up to allow the operator to select the fixed storage tank and the destination tank to discard to and reload in the reverse mode.

How is the paraffin wax discarded, when required?

First, the wax can be used repeatedly for periods of up to several months at least, not requiring a regular change, other than the occasional top-up for wax absorbed by the tissue samples. The wax retort is fixed and has a concealed drainage outlet tap attached at the front. The drainage tap is accessed by opening the unit's front panel cover (single access key movement). A bayonet connector with a long drainage tube is attached to the tap, allowing the wax to be drained by gravity feed into a container. Replacement wax is added directly into the wax retort.

How much wax do I need to add to the wax retort?

Approximately 5 liters of molten wax is required, determined by a maximum height liquid sensor.