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Cytology Technical Help

Technical FAQ regarding issues such as processing, instrumentation, etc.

Questions

  1. If the fluid sample contains numerous erythrocytes of no diagnostic value, how to lyse them before processing?
  2. Is there a better way of dissolving mucus in bronchial specimens than using CytoLyt? Perhaps another product?
  3. I have enjoyed you website very much. I noticed recently that you have nothing on vaginal cytology for maturati o n index. Is there any info on this at cytopathnet?
  4. In processing serous effusions what is the most widely recommended or standard for actually how much specimen should be sampled to yield accurate diagnosis. We are having a hot debate with our chest docs- who think the entire spec needs to be looked at.
  5. Can anyone tell me what the acceptable or average ratio is for postive to negative HPV tests? Additionally, would HPV testing be recommended for a patient with LSIL or higher in the past 6 months who has been treated and now has an ASCUS pap? Thank you f
  6. "please refer me to the section in CLIA which governes the review of unsasisfactory GYN slides in cytology, and perhaps a brief synopsis. Daryn"
  7. What is a low-grade squamous intraepithelial lesion?
  8. Are there any adequacy criteria for non-genital specimens?
  9. I may not be searching corrrectly but I would like to get some information on HPV in hysterectomy patients.
  10. Cyto Shuttle?
  11. Does anyone frequently blend their ThinPrep specimens to break up chunks of inflammation, etc.? We usually use acetic acid technique for excess blood, which works well and seems to work for other things like that too, but one of our techs thinks we should blend them instead since it's faster.
  12. How would you explain unsatisfactory due to scant squamous cellularity?

Answers

Question: If the fluid sample contains numerous erythrocytes of no diagnostic value, how to lyse them before processing?
Answer 

You can find information regarding this question in our Forums section. Here is the direct link that you can paste in your browser if the link doens't work properly: http://cytopathnet.hostingmountain.com/modules.php?op=modload&name=XForum&file=viewthread&tid=14.

Question: Is there a better way of dissolving mucus in bronchial specimens than using CytoLyt? Perhaps another product?
Answer 

"We received several responses from the Cytopathnet-L listserer regarding your question, provided below:

Hello Jana:

  • FAQ Question: Is there a better way of dissolving mucus in

bronchial
specimens than using CytoLyt? Perhaps another product?

See: http://www.thinprep.com/85488/ms.htm(external link)

  • ""...how to make sure that the Harris Hematoxylin is working

properly
before the staining begin.""

Use buccal smears as QA probes. Stain 1 for the lab's usual time, rinse
in water, differentiate in HCl if done routinely, rinse in water, blue,
rinse in water, rinse in 95% ethanol, then absolute ethanol, clear in
xylene and coverslip.

Chromatin should be blue (i.e., qualitatively satisfactory formulation
is chemically competent
).

Slight cytoplasmic staining is expected and acceptable (i.e.,
quantitatively satisfactory). If excessive, too much is going in and
not enough is being removed. Put less stain in by staining for less
time or diluting the stain. Or remove more stain by differentiating in
HCL longer or use stronger HCL for same time. Simple.

Best regards.

Gary
Webmaster's note: reply from Gary Gill
--------------------------
At our hospital, we use the Saccomanno blending technique. Usually our bronchial specimens are received fresh, so we had saccomanno to the specimen and blend on high for about 5 seconds. Transfer to falcon tube and centrifuge. We then pour off the supernatant and resuspend in 5 - 10 ml of cytolyt and vortex to homogenize. Before I got here, they were doing nothing, and the bronchial specimens were thick and horrible.

WILL
Methodist Medical Center
Dallas, Texas
-------------------------

Partly for H&S reasons, as well as to mucolyse, we leave our bronchs & spits overnight in cytolyte. They seem to work OK for us.

Cheers

Mark
---------------------------
i haven't worked with bronchials or sputums for 20 years but i just wanted
to comment on something i noticed back then. saccomano blender broke up
most mucous.
But when i received bloody bronchial washings, after centrifuging, i noticed
that the blood went to the bottom of the tube, the mucus to the top and
there were chunks of tumor cells frequently, at the center between the blood
and the mucous. was this a common occurance and is it still used today?

Judy
-------------------------
Call Cytyc and ask about the product they use called DTT (DiThioThreithol
80mg). They gave me a sample to use in mucoid specimens and it worked very
well.

Julia
-------------------------
We collect our specimens fresh in Normosol and never had any problem with mucus

PG
--------------------------
We use a product called Sputolysin which was used by our microbiology department for years for TB specimens. It is made by a company called Calbiochem. Their website is www.calbiochem.com(external link) or phone 800-628-8470 (US & Canada).

Carol Green
New Zealand
---------------------------

"

Question: I have enjoyed you website very much. I noticed recently that you have nothing on vaginal cytology for maturati o n index. Is there any info on this at cytopathnet?
Answer 

"Good morning,

Thank you! We are delighted that you find Cytopathnet useful.

Cytopathnet does not have any information posted on vaginal cytology for maturation index. This is partly due to the fact that it is an infrequently used test which is quite unreliable when not used in the proper settings. I will, however, make sure that gets put on our suggestions list for information to add.

Thank you for your inquiry!!

Regards,

Jana Sullinger, MD
President/CEO
CYTOPATHNET
mailto:webmaster@cytopathnet.us
http://www.cytopathnet.us(external link)

"

Question: In processing serous effusions what is the most widely recommended or standard for actually how much specimen should be sampled to yield accurate diagnosis. We are having a hot debate with our chest docs- who think the entire spec needs to be looked at.
Answer 

"That is an excellent question. I think this question is perhaps best posted to our Online Forum area. I have posted the question there, and hopefully, will get some feedback. Thank you for your question.

Jana Sullinger, MD"

Question: Can anyone tell me what the acceptable or average ratio is for postive to negative HPV tests? Additionally, would HPV testing be recommended for a patient with LSIL or higher in the past 6 months who has been treated and now has an ASCUS pap? Thank you f
Answer 

"I am not sure that I understand fully the first part of your question. Are you actually asking about specificity/sensitivity of the test?

Regarding clinical management, the best resource for the latest information is the ASCCP. They have published several algorithms for how to handle a variety of scenarios. I would start there. If you particular scenario is not there, I would email them directly with your question. I may go ahead and contact them with your question as well, as I would like to be able to post back to this site.

Jana Sullinger, MD
Webmaster"

Question: "please refer me to the section in CLIA which governes the review of unsasisfactory GYN slides in cytology, and perhaps a brief synopsis. Daryn"
Answer 

"I have forwarded your question to Sandra Fite, who I am certain will be able to reply. I will update this when her response is received.

Jana Sullinger, MD"

Question: What is a low-grade squamous intraepithelial lesion?
Answer 

Low-grade squamous intraepithelial lesion or LSIL is a term used by the Bethesda System classification for cervicovaginal cytology used to describe cellular changes ranging from HPV (human papillomavirus) changes ("koilocytotic atypia) to mild dysplasia (CIN 1).

Question: Are there any adequacy criteria for non-genital specimens?
Answer 

The adequacy criteria for non-gyn specimens is refered in the Non-Gyn Guidelines. This excerpt from the Guidelines specifically refers to FNAB, ..."Although there are no universal criteria for adequacy, a multiparameter assessment is usually performed which includes cellularity, site-specific architectural features, and cellular elements." Another excert lists the following..."It is important to consider the adequacy of each sample and to note in the report if a specimen is suboptimal. “Insufficient for diagnosis”, “Non-diagnostic specimen”, or “Unsatisfactory”, are comments that can mean less than adequate samples. Commenting on the material that is submitted and its limitations is important to assist the clinician in patient follow-up. Factors
limiting the interpretation of the specimen, such as degeneration or obscuring elements should be noted.".

Although I don't have specific references, I was taught the following:

1) sputum: a minimum of 6 alveolar macrophages present to be considered adequate
2) body fluids, ie. peritoneal/pleural- presence of mesothelial cells for adequate sample
3) urine- don't recall any specific criteria, other than "no cells present" = unsatisfactory"

Hopefully, others reading this may have specific written criteria that they have found as "pearls" in various publication. I recall that there may be specific criteria provided in one of Dr. Tilda Kline's Non-Gyn? texts, but I can't recall the textbook.

Question: I may not be searching corrrectly but I would like to get some information on HPV in hysterectomy patients.
Answer 

PubMed? Query: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&term=HPV+in+hysterectomy+patients(external link)
1: J Am Board Fam Pract. 2000 Jul-Aug?;13(4):233-8. Related Articles, Links

Comment in:

* J Am Board Fam Pract. 2000 Nov-Dec?;13(6):470.

 

Routine vaginal cuff smear testing in post-hysterectomy patients with benign uterine conditions: when is it indicated?

Videlefsky A, Grossl N, Denniston M, Sehgal R, Lane JM, Goodenough G.

Department of Family and Preventive Medicine, Emory University School of Medicine, Grady Memorial Hospital, Atlanta, GA 30335, USA.

BACKGROUND: By the age of 60 years, an estimated 33% of women will have undergone a hysterectomy. Approximately 85% of these hysterectomies are performed for benign disease. The object of this study was to evaluate cytologic findings from vaginal cuff smears in patients who have undergone hysterectomy for benign uterine conditions. METHODS: We conducted a community-based retrospective study and follow-up of women with vaginal cuff cytologic smears who had had a hysterectomy for benign uterine conditions. A total of 220 women were randomly selected who had one or more vaginal cuff smears. The main outcomes measures were invasive carcinoma, dysplastic lesions, and infections detected by vaginal cuff smear testing. The setting was a large inner-city hospital. RESULTS: Ninety-seven percent of 220 women who underwent hysterectomy for benign uterine conditions and who were observed for an average of 89 months had no cytologic abnormalities on vaginal cuff smears. Cytologic evaluation found no invasive carcinomas. Dysplastic lesions were detected in 7 patients (3%). Seventy percent of patients (n = 154) had one or more infections; these infections included bacterial vaginosis (106), trichomoniasis (95), candidiasis (40), koilocytosis suggestive of human papilloma virus (HPV) infection (3), and cytopathic effect of herpes (4). The prevalence of koilocytosis was much higher in the patients with dysplasia (P = .0003). CONCLUSIONS: Most routine vaginal cuff cytology screening tests need not be performed in women who have had a hysterectomy for benign uterine conditions.

PMID: 10933286 PubMed - indexed for MEDLINE


Int J Cancer. 1997 Jul 29;72(3):412-5. Related Articles, Links

Click here to read

Distinct manifestations of human papillomaviruses in the vagina.

Sugase M, Matsukura T.

Department of Obstetrics and Gynecology, Nagano Red Cross Hospital, Japan.

To clarify the pathogenic relationships between human papillomavirus (HPV) and vaginal intraepithelial neoplasia (VAIN), we examined 71 vaginal biopsy specimens by histopathology and immunohistochemistry and analyzed the presence of HPV DNA by blot hybridization at Tm - 40 degrees C using an HPV 58 probe (PBM-58 method). We found 27 cases of VAIN in patients with previous hysterectomy or antecedent or concomitant cervical intraepithelial neoplasia (CIN) and 44 cases of VAIN in patients without any abnormal findings on the cervix and the vulva. Histopathologically, 53 of 71 cases were graded as VAIN I and 15 and 3 cases were VAIN II and III, respectively, while 59 cases showed positivity for HPV capsid antigen by immunohistochemistry. Using the PBM-58 method, all 71 VAIN cases harbored a single HPV type at more than 1,000 viral copies per cell. We identified 15 different types (HPV 16, 18, 30, 31, 35, 40, 42, 43, 51, 52, 53, 54, 56, 58 and 66). Furthermore, we molecularly cloned 7 novel prototypes (HPV 59, 61, 62, 64, 67, 69 and 71) from VAIN I. Our results are strongly indicative that HPVs are etiologic agents of VAIN, like in the case of CIN. The distinct manifestations of HPV infection in the vagina are discussed in comparison with those in the cervix.

PMID: 9247283 PubMed - indexed for MEDLINE


Question: Cyto Shuttle?
Answer 

Excerpt from CellPathNewsApril1995? http://www.cellpath.co.uk/pdfs/press_releases/CellPathNewsApril1995.pdf(external link)
The Cyto-shuttle, from Cancer Diagnostics Inc. of Virginia, USA, is a disposable cytology monolayer preparation filter comprising of a two part filter housing with a unique membrane assembly. Sample fluid is drawn through the Cyto-shuttle using an ordinary syringe and the cells in the fluid remain on the membrane until the surface is covered by a monolayer.
At this point the flow of liquid will automatically stop. The cell preparation can then be easily transferred to a slide and, after removing the membrane, cells can be fixed and stained by conventional procedures. As no additional equipment is required slides can be prepared almost anywhere.

Jana Sullinger, MD

Question: Does anyone frequently blend their ThinPrep specimens to break up chunks of inflammation, etc.? We usually use acetic acid technique for excess blood, which works well and seems to work for other things like that too, but one of our techs thinks we should blend them instead since it's faster.
Answer 

I know that we do not in our laboratory. We have had success with utilizing acetic acid for excess blood. Additionally, we have done a comparative study reprocessing the samples as SurePath?, with very good results.

I will add your question to our general forums, unless I missed it that you already posted there. Perhaps others may have some input.

Jana Sullinger, MD

Question: How would you explain unsatisfactory due to scant squamous cellularity?
Answer 

In our laboratory, we usually use the terminology "Unsatisfactory due to scant celluarity." We do not specify whether squamous or glandular component. Depending on the situation, for example, lets say there are primarily endocervical cells on the smear, but no squamous component, we would most likely use the same "Unsatisfactory due to scant cellularity", but clarify with a comment. We usually also call the doctors office to confirm site in those situations, as sometimes they only sampled the endocervix. In that situation, the sample would be satisfactory, although we would most likely make some comment.

Using a strict definition including Cytomorphologic Criteria:
Unsatisfactory due to scant squamous cellularity. Groups of endocervical cells are seen as well as debris. An adequate liquid based preparation should have an estimated minimum of 5,000 well-visualized/preserved squamous cells.

I hope that helps answer your question.

Jana Sullinger, MD


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