mitigating contamination in the h&e process

Mitigating contamination in the H&E process


In a recent study of histology lab stainer baths, cross-contamination to blank test slides occurred at a rate of up to 25%. Study details outlined on Slide 12

During this session, we will explore three possible sources

contamination during the H&E process.

1.  Block (grossing, embedding)

2.  Waterbath

3.  Stainer

At the end of this session, you should be able to…

1. Articulate 3 sources of contamination in the H&E process

2. Explain the implications of contamination on H&E slides

3. Implement these mitigation practices in your laboratory


•  Contamination sources in the Histology lab

•  Review of current published literature

•  Procedural detail of a recent contamination study

•  Overview of individual & compiled result data

•  Potential mitigation steps

•  Block contamination –  Usually occurs during grossing or embedding –  Extraneous tissue is typically found at the periphery of primary specimen –  Will typically be present in multiple levels from the tissue block –  When extraneous tissue and primary specimen are same tissue type,

diagnostic difficulty can occur


•  Block contamination –  Potential mitigations:

•  Using new grossing tongs for each case •  Placing tongs in Bunsen burner flame between cases •  Thorough cleaning of grossing or embedding areas between cases


•  Water bath contamination –  Tissue fragments separate from paraffinized sections while floating in water

bath •  Fragments can float on to other sections

–  Often believed to be the primary source of contamination in histology


•  Water bath contamination –  Potential mitigations:

•  Sweep of water surface between cases with a low lint cleaning tissue •  Visual inspection of water bath surface •  Frequent changing of water bath liquid


8

•  Stainer bath contamination –  Tissue fragments separate from paraffinized sections while being agitated in

stainer baths •  Fragments can float on to other sections = possibility of a false positive

diagnosis •  Instances when a floater of “like” tissue migrates onto another slide can

= particularly difficult diagnosis •  Fragments lost in the baths prevent review of all diagnostic material =

possibility of a false negative diagnosis


•  Stainer bath contamination –  Potential mitigations:

•  Frequent changing or filtering of reagents

–  Challenges to mitigation Ø  Labor cost Ø  Reagent cost Ø  Time required Ø  Increased exposure to

chemicals


11


Extraneous Tissue in Surgical Pathology A College of American Pathologists Q-probes Study of 275 Laboratories

Gordon N. Gephardt, MD, Richard J. Zarbo, MD, DMD

(Arch Pathol Lab Med. 1996; Vol. 120)

•  Study of extraneous tissue found in surgical pathology microscopic 275 labs –  Of the slides reviewed retrospectively, 1,653 were found to contain extraneous tissue

•  Degree of diagnostic difficulty caused by the extraneous tissue was evaluated -  Severe in 57 of slides reviewed retrospectively


Tissue Floaters and Contaminants in the Histology Laboratory Eric Platt, BS; Paul Sommer; Linda McDonald, MT, ASCP; Ana Bennett, MD; Jennifer Hunt, MD

(Arch Pathol Lab Med. 2009;133:973–978)

•  Recent increased attention to patient safety –  Decreasing cross-contamination is an example of a patient identity issue for which process

improvement exists

•  Study Goal –  To assess for contaminants in water baths at cutting stations and in linear stainer stain

baths –  To assess tissue discohesion and carryover onto blank slides sent through the stainer

•  Water Bath Contamination –  In the 13 water baths examined, only 1 fragment of tissue was identified.

•  Conclusions –  Cross-contamination to blank slides at a rate of up to 25% in the late afternoon. –  Cross-contamination does occur onto blank slides in the experimental setting.


Risk of Misdiagnosis Due to Tissue Contamination May be Higher for Certain Specimen Types Changes to laboratory staining techniques offer opportunity to reduce contamination events

John B. Carpenter, MD (DARK Daily Laboratory and Pathology News @ darkdaily.com, 21Mar2011)

§  Explores the challenges certain tissue types present as contaminants

§  Highlights 3 tissue types that can present diagnostic challenge due to their fragmented nature

•  Esophageal biopsies •  Curettage specimens •  Lymph node biopsy for metastatic malignancy

§  Reviews elevated risk of specialty pathology

practices-“like” tissue

§  Increased reagent changes and individual slide staining technology should be considered to mitigate contamination

Fig. 3: Biopsy from the lower esophagus showing a fragment of gastric cardia-type mucosa and several detached fragments of columnar mucosa with goblet cells. Adjacent tissue levels did not show the fragments of intestinal-type epithelium. A diagnosis of Barrett’s esophagus was initially rendered on the basis of this field, but later reconsidered given the possibility of slide contamination. The patient will be undergoing increased surveillance due to the appearance of this slide.

Procedural detail of a recent contamination study •  Step 1

–  Intersperse 100 blank slides with the final 100 patient slides and process using the lab’s standard protocol

•  Step 2 (completed immediately after Step 1) –  Collect all fluid from first 1 or 2 (500 ml maximum dependent on stainer brand/model)

xylene (or xylene substitute) baths, 100% alcohol and 95% alcohol

•  Step 3 –  Return items to Ventana Medical Systems, Inc. (Ventana) to a qualified screener

•  marks any potential foreign tissue fragments on the patient simulator slides •  creates spin downs of the reagent bath contents

•  Step 4 –  Both sets of slides are sent to a 3rd party, board certified pathologist for review

•  characterizes each tissue type seen that qualifies as a “floater” per study criteria •  counts the fragments identified


Results from an ongoing Ventana study-

•  “12% of “blank” patient simulator slides contained floaters

•  Reagent bath fluids contained 590 fragments

•  Potential patient slide contamination rate of 3500 and 9905 per year (p=.05), based on 50K SPY

•  Customer has since acquired an H&E system that stains slide individually

Example, 95% Alcohol, invasive poorly differentiated carcinoma, 200x


•  Additional results from the study

–  Potential patient slide contamination rate of 1,723 – 8, 940 per year (p=.05), based on 80K SPY

–  In reference to the study results at this facility, a site pathologist commented that discrete staining is the solution to this problem of cross contamination in the stainer baths and that the site will be working toward the goal of shifting to the staining technology as soon as possible.

Figure 1. Inflammatory Stroma fragment, 200x



valu

e of

the

mea

sure

men

t

0

1000

2000

3000

Average turnover time (Change per week)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Scatter plot of number of tissue fragments and slides*1000 per yearversus. average turnover time

type Number of tissue fregments Slides*1000 per year

The Stainer Bath Contamination Challenge results to date show that the frequency of reagent bath changes does not appear to reduce the # of floaters present.

One mitigation strategy used to limit stainer bath contamination is changing or filtering the reagents regularly.

Potential Mitigation Steps

@ Grossing/Embedding •  Using new grossing tongs for each case •  Placing tongs in Bunsen burner flame between cases •  Proper cleaning of grossing or embedding areas between cases

@ the Waterbath •  Low lint cleaning tissue sweep of water surface after each case •  Visual inspection of water bath surface •  Frequent change of water bath liquid

@ the Stainer •  Frequent filtering or reagent changes (recent study data does not support) •  Adopt individual slide staining technology •  Using charged or coated slides? (recent study data being analyzed currently)


•  Laboratory staff can mitigate contamination in many steps within the H&E process with clean techniques

•  Stainer Bath Contamination is happening in every one of the 72+ linear stainers in accounts that have participated in the study

•  Beyond floaters identified on the patient simulator slides, floaters in the baths represent a potential risk, from both a false positive and false negative standpoint

•  Frequency of reagent bath changes does not appear to decrease the amount of fragments identified in the staining baths

•  Individual slide staining technology protects against cross contamination during staining

Mitigating contamination in the H&E process Conclusions

Mitigating Contamination in the H&E Process

Appendix

24

•  Upon request, the site will receive a VENTANA Stainer Bath Contamination Challenge Kit. Everything needed to conduct the challenge will be included in this kit. The kit includes:

•  Hazardous materials grade box with: –  Individual spaces for 4 reagent containers –  Absorbent-lined (yellow) plastic bags to hold reagent containers (brown plastic) –  Ties for closing the plastic bags –  Red and Clear sealing tape for closure of box prior to shipment

•  2 Boxes of SUPERFROST Plus slides •  100 serialized slide labels •  2 Blue slide boxes, with labels for the name of lab where the study is

being completed.

•  Qty 3 1000 ml brown NALGENE bottles with labels containing the specific reagent to be captured and a space to write the name of the lab where the study is being conducted.

•  Plastic funnel •  Rubber gloves

Mitigating contamination in the H&E process Procedure

Procedure

•  Step 1 –  Process the final 100 patient slides and 100 blank slides using the lab’s standard protocol. When

finished with coverslipping, apply sequentially numbered labels to the slides –  in the order in which the slides leave the coverslipper, load the slides into the blue slide box

provided.

•  Step 2 (To be completed immediately after Step 1) –  Pour all fluid from first 1 or 2 (dependant on stainer brand/model) xylene (or xylene substitute)

baths into 1000ml container labeled “Xylene”. (Maximum 500 ml) –  Pour all fluid from the first 1 or 2 100% alcohol baths into 1000ml container labeled “100%

alcohol”. (Maximum 500 ml) –  Pour all fluid from the first 1 or 2 95% alcohol baths into 1000ml container labeled “95% alcohol”.

(Maximum 500 ml)

•  Step 3 –  All items are returned to Ventana Medical Systems, Inc. (Ventana) where are qualified

screener marks any potential foreign tissue fragments on the patient simulator slides and creates spin downs of the reagent bath contents

•  Step 4 –  Both sets of slides are sent to a 3rd party, board certified pathologist for review. He

characterizes each tissue type seen that qualifies as a “floater” per study criteria and counts the fragments identified.


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mitigating contamination in the h&e process

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