Chemistry: analytical and immunological testing – Cancer
Reexamination Certificate
2000-08-08
2001-09-04
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Cancer
C436S063000, C436S174000, C435S040500, C435S040510
Reexamination Certificate
active
06284543
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to cell staining. Specifically this invention relates to staining a Pap slide specimen, including conventional smears and ThinPrep Pap test slides.
2. Background and Discussion of the Prior Art
Almost 5,000 women die each year from cervical cancer in the United States. The cervico-vaginal Papanicolaou test or Pap test is a powerful tool for detecting cancerous and precancerous cervical lesions. The Pap test smear has been credited with reducing mortality from cervical cancer by as much as 70%.
The Pap test involves a staining method. This staining method includes a polychromatic reaction which seeks to display the many variations of cellular morphology to show degrees of cellular maturity and metabolic activity. The four main steps of the Pap test staining method are: (1) slide fixation, (2) nuclear staining with hematoxylin, (3) cytoplasmic staining, generally with counterstains orange G and EA, and (4) clearing and mounting. The term “Pap staining method” or “Pap test staining method” as used hereinbefore and hereinafter throughout the specification refers generally to the aforesaid method.
The Pap test staining method has been generally used on conventional cervico-vaginal Pap smear specimens. The conventional Pap test smear however had false negative rates ranging from 10-15%, and up to 90% of those false negative rates were due to limitations of staining and slide preparation of such specimens. More recently the Cytyc Corporation developed the ThinPrep Pap test. Instead of smearing the cells on a slide, the cells are collected in a transport medium, from which a slide with a filter preparation is obtained for the test.
The art provided several complex Pap test staining methods involving multiple steps of greatly varying times and clippings. Certain prior art short versions of Pap test staining methods took an inordinate amount of time of upwards to 20 minutes or more and were complex, which readily invited error by the laboratory technician. Many of these prior art Pap test staining short methods were generally directed to fine needle aspirated (FNA) specimens and to the conventional cervico-vaginal smears, and not to the ThinPrep Pap test specimen.
One such conventional prior art short method which sought to reduce the overall time, was the “Quick Papanicolaou Staining Procedure for Stat Specimens” developed by the Johns Hopkins Cytopathology Laboratory, Baltimore, Md., as follows:
1.
Tap water
5-10 dips (until surface is smooth)
2.
Gill's hematoxylin No. 2
1 min, including 10 initial dips
3.
Tap water
5 dips
4.
Scott's tap water substitute
15 sec
5.
Tap water
5 dips
6.
Stat OG/EA
1 min, including 10 initial dips
7.
95% ethanol
5 dips
8.
95% ethanol
5 dips
9.
Absolute ethanol
10 dips
10.
Absolute ethanol
10 dips
11.
Xylene
5 dips
12.
Xylene
5 dips
Coverslip
The Johns Hopkins' Method required 12 post-fixing, pre-coverslip steps, and took several minutes. The great disparity in procedure times for each step and sequence invited error by the laboratory technician and difficult the mechanization of the staining method.
Another attempt to reduce the Pap test staining method time was done specifically in connection with fine needle aspiration (FNA) specimens as disclosed in “Ultrafast Papanicolaou Stain—An Alternative Preparation for Fine Needle Aspiration Cytology” G. C. H. Yang and I. I. Alvarez, ACTA Cytol., vol. 39, no. 1, January-February 1995. The Yang-Alvarez FNA staining method was as follows:
A. Alcoholic Formalin Fixative:
Mixture of 65% ethanol and 4%
formaldehyde. It was convenient to
make 3L of fixative from 300 mL of
38-40% formaldehyde, 2,053 mL of
95% ethanol and 647 mL of
distilled water.
B. Staining Method:
1. Normal saline
30 seconds
2. 95% Ethanol (optional,
for storage/transport)
3. Alcoholic formalin
10 seconds
4. Water
6 slow dips
5. Richard-Allan Hematoxylin 2
2 slow dips
6. Water
6 slow dips
7. 95% Ethanol
6 slow dips
8. Richard-Allan Cytostain
4 slow dips
9. 95% Ethanol
6 slow dips
10. 100% Ethanol
6 slow dips
11. Xylene
10 slow dips
Mount and coverslip
The Yang-Alvarez FNA staining method required a relatively complicated fixative mixture solution, 8 post-fixing, pre-coverslip steps, and provided improvement in the resultant stained specimen, but again the laboratory technician was faced with greatly disparate dipping sequences and times, and as such invited error. That is, the laboratory technician had to change from a 10 second fixing to a 6 dip hydrating and then to a 2 dip hematoxylin staining. These disparate sequencing times invited error. Further the Yang-Alvarez staining method was FNA specimen specific and not for cervico vaginal specimens.
One attempt was made to apply the Yang-Alvarez FNA approach to cervicovaginal smears as disclosed in “Ultrafast Papanicolaou Protocol for Cervicovaginal Smears,” G. C. H. Yang et al., ASC Abstracts, Nov. 1, 1995. This modification of the Yang-Alvarez FNA staining method to cervicovaginal smears is as follows:
1.
Dist. water
3 min
2.
Dist. water
3 min
3.
Dist. water
3 min
4.
Hexatox II
20 sec
5.
Tap water
until clear
(discard dirty water
fill with clean water)
6.
95% alcohol
6 dips
7.
Cytostain
40 sec-5 min
8.
95% alcohol until clear
(discard dirty alcohol,
fill with clean alcohol)
9.
100% alcohol
6 dips
10.
100% alcohol
6 dips
11.
Xylene
6 dips
12.
Xylene
6 dips
The Yang et al adaption or modification of the Yang-Alvarez FNA staining method required 12 separate post-fixing, pre-coverslip steps, and also required more than 15 minutes, all in disparate sequence times ranging from a 6 dip step taking about 6 seconds to a cytostain staining step taking upwards of 5 minutes. Again this method was not laboratory technician friendly in that it invited errors because of the disparate dip sequencing as well as in the number of steps and in the disparate step times, and difficult the use of automatic stainer machines.
The art desired a simple, faster and laboratory technician friendly Pap test staining method. The art also desired a Pap test staining method used for conventional pap smears and which was particularly useful for ThinPrep Pap test specimens. The art further desired a Pap test staining method as aforesaid which consequently is adaptable for handling large numbers of Pap test slide specimens with minimal or no errors, and will facilitate the use of automatization for the staining method. The art still further required a method as aforesaid with an improved stained specimen, particularly an improvement in background and contrast stain quality. The present invention addresses and provides those art desired improvements.
SUMMARY OF THE INVENTION
A method for staining Pap test slide specimens for conventional pap test smears, and also particularly ThinPrep Pap test specimens, which method provides hydration, staining and dehydration steps of about the same number of dips and times. Each step is about 10 to 15 dips of about 1 second for each dip. The post staining dehydration requires at least about three separate alcohol steps with each step of the same 10 to 15 one second dips. The hematoxylin and cytostain staining steps are each also 10 to 15 one second dips. The present Pap test staining method requires only 10 post-fixation, precoverslip steps, with each step of minimal and yet equal times of about 10 to 15 seconds, with the overall time being from 100 to 150 seconds. The method is laboratory technician friendly in eliminating the step sequence time disparity and in reducing the sequence time and total time, and consequently the likelihood of error, which will facilitate the use or adaption of automatic stainer machines. The method also provides an improved stained specimen, particularly one of improved background and contrast stain quality.
The present invention is an improvement over the used Yang-Alvarez staining method for FNA which required a fixation step dependent on a mixture of pure formaldehyde (38-40%), water and alcohol; which could be a problem if the laboratory has only 10% buffered formalde
Siegel Lackenbach
Wallenhorst Maureen M.
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