Improved Pap Testing Methods

In 1928, Dr. George Papanicolaou reported that analysis of individual cervical cells, collected by a simple swab technique could be used to detect cervical cancer1. Specifically, he showed that cancer cells displayed changes in cell size and shape compared to healthy cells that were visible under a microscope. In his widely cited paper from 1941 he demonstrated that this test could be used to detect early-stage cervical cancer including in women that were not yet showing symptoms [1]. This test became known at the Pap test, or Pap smear, and has been highly effective in reducing the rates of cervical cancer and associated mortality in the United States [3, 4].

The Pap test helped initiate an entirely new branch of diagnostic medicine known as cytopathology, the diagnosis of disease by studying individual cells. The Pap test had a major impact on women’s health. Doctors could now identify cervical cancer by analyzing cells collected from the surface of the cervix bypassing more expensive and invasive tissue biopsy procedures. By the 1960s the Pap test was being implemented in private practice and public clinics across the US and in other countries2.

However, the expanded use of the Pap test presented many challenges. Cytopathology was still a new field, and its development was tied to the growth of Pap testing [3]. There was a shortage of physicians and technicians3 trained in analyzing cells, and experts in this area, such as Dr. Stern, were rare. Although Pap testing was easy to execute, diagnosing a sample as cancerous or non-cancerous was difficult for inexperienced analysts due to a lack of standardization in the medical literature and problems with sample preparation.

Dr. Stern recognized these issues and dedicated her research to improving and standardizing the detection of cervical cancer. Dr. Stern’s work made it easier for physicians and technicians to identify abnormal cells and to increase the accuracy of Pap testing for cervical cancer diagnosis [5]4.

Problems with the Conventional Pap Test

By the late 1970s the Pap test had been used for more than 20 years in the United States, but few changes had been made to the technique during this time. Yet there were clear problems that needed to be addressed.

Many physicians were concerned by false negatives in Pap testing as some patients with cervical cancer were misdiagnosed as healthy. These false-negative results were often due to improper sample collection, processing, or analysis, with rates as high as 33% in some laboratories [5, 6]. Of course, false positives from the Pap test, in which healthy patients were incorrectly diagnosed with cervical cancer, was also a concern, and had equally high rates. This led to unnecessary testing and anxiety on the part of patients as well as inefficient use of hospital resources [3].

To fully understand these concerns, it is important to know how the Pap test worked. This test is very straightforward, making it feasible to implement in labs worldwide.

Briefly, cells are collected from the cervix, lightly smeared onto a glass slide, and placed in a fixative solution that prevents cells from degrading and preserves cell size and shape. The slides are air dried and then stained using a series of dyes that help distinguish cell features. Finally, Pap test slides are analyzed by a technician or cytopathologist. The frequency of abnormal and normal cells is noted, and patients are followed up as needed.

This process seems simple, but each step is prone to multiple errors that can impact the results. These are the main problems physicians faced:

1. Cell Damage
If cells are pressed too firmly onto the glass slide, they may burst or become distorted, thereby changing test results. If too many cells are lost or damaged, it can complicate analysis of a sample due to a lack of material.

2. Immune Cells in the Sample
The cells for Pap tests are collected directly from the cervix, but the cervix doesn’t contain just one type of cell. Immune cells are located throughout every tissue, constantly interacting with the environment to ward off infections and disease. When physicians take a cervical scraping for a Pap test, there are a large number of immune cells in the sample that complicates the analysis of cervical cells.

3. Cell Clumping or Overlap
Even if the cells are transferred perfectly, there may be clumping with cells lying on top of one another, making it impossible to analyze the shape of individual cells.

4. Cell Counts
Early-stage cervical cancer patients have mostly healthy cells. Abnormal, cancerous cells are rare. Thus, it is crucial to examine as many cells as possible to accurately diagnose patients and avoid false negative results.

5. Long Processing Times
With the conventional Pap test, each sample had to be individually processed, stained and analyzed by trained cytopathology staff, requiring a considerable amount of hands-on time. The lack of knowledgeable staff3 along with the increasing number of samples4 contributed to long processing times and placed additional stress on overworked cytopathology staff.

6. Human Error
The above problems, combined with inexperienced cytopathology staff and poorly defined stages of cervical cancer, led to Pap testing results with high false negative and false positive rates [3, 5, 6]5.

Dr. Stern had been working in the fields of cytopathology and cervical cancer since the late 1940s and was well aware of these concerns from running Pap testing clinics in Los Angeles County.

In an effort to standardize testing results and provide guidance for physicians, she designed and validated a 100-point scale for scoring cervical cell abnormalities in Pap tests and biopsies [7]. This scale ranged from healthy to invasive cancer and included very early changes in cell size and shape that were difficult for non-cytopathologists to identify.

However, this scaling system did not solve all the problems associated with the conventional Pap test. Cell damage was still a concern, as was removing immune cells and other contaminating cell types. New methods were needed to make samples cleaner, accelerate Pap test analysis, and make the results more reliable.

Collaboration with JPL to Improve Pap Testing

In the mid 1970s, Dr. Stern and her colleagues at UCLA, Claire McLatchie and Dr. Dorothy Rosenthal6, connected with scientists at the Jet Propulsion Laboratory (JPL) to begin working on an automated Pap test.

Dr. Stern and colleagues were interested in providing clinicians with a digital analysis that would ease the burden of conventional hands-on analysis on cytopathology staff. Although the role of cytotechnicians and cytopathologists could not be completely eliminated, they reasoned it would be helpful to flag abnormal cell samples for additional hands-on analysis, while the remaining healthy samples, which comprised the majority, could be marked as normal7.

The scientists at JPL, Drs. Kenneth Castleman and Benjamin White, were experts in microscopy and digital imaging analysis. Their long-term objective was to develop a reliable, automated system using microscopic analysis of individual cells to detect disease. They based this digital processing system on Dr. Stern’s 100-point scale with the goal of developing a computer program that labeled cells in the same way as expert cytopathologists or cytotechnicians. The researchers devised a two-stage plan as follows:

1. Developing cell processing techniques to remove non-cervical cells and disperse cervical cells into a single layer on slides. This process had to be gentle enough to preserve cell shape and size but firm enough to break up cell clumps.

2. Working with JPL to develop an automated, digital measuring system. Cells that represented different categories along Dr. Stern’s cervical cancer scale would be measured by JPL’s system. These digital measurements would be compared with the conventional hands-on measurements to identify distinct stages of cancer.

Optimizing Cell Processing Methods to Improve Pap Testing

The first step in this project was to develop a technique that made individual cervical cells visible for automated measurements. Ideally, this method would also remove immune cells and mucus.

Dr. Stern reviewed contemporary work on cell preparation for cytopathology analysis [2, 8, 9] and adopted newer strategies. The main differences from the conventional Pap test described above are:

• Instead of smearing cells directly onto a glass slide, the swab or brush containing the cervical sample is placed in a vial containing a fixative solution.

• To enrich cervical cells, the sample is strained across a nylon mesh filter capturing large cervical cells, while smaller immune cells and mucus flow through the filter and are discarded.

• To break up cell clumps, captured cervical cells are gently mixed using a syringe.

• Finally, cells are applied to another filter and transferred to a glass slide for Pap staining and analysis.

The difference between the two methods can be visualized by looking at slides.
Figure 1A shows a Pap test prepared using the conventional method. The red box shows small immune cells and the purple box shows a clump of cervical cells.

Figure 1B shows a Pap test prepared using Dr. Stern’s new technique. There are very few infiltrating immune cells, and the cervical cells are separated and easily visualized. Dr. Stern’s group found that 92% of immune cells were removed from the sample using their procedure, and only 2% of cervical cells were lost over the filter [2].



Dr. Stern’s new technique was a major scientific advance taking methods that had been applied to other cell types and using them to develop a technique that allowed doctors to more easily analyze cervical cells.

Automated Digital Image Analysis of Pap Tests

With this method in hand, Dr. Stern and her colleagues went to JPL to develop a digital imaging system [10, 11]. Although automated Pap tests were not unheard of at the time, none had been successful.

To validate their system, Dr. Stern’s lab analyzed 7,000 individual cervical cells, both manually and using the computer system developed by JPL [11]. Cells were derived from 105 normal and 96 abnormal cervical scrapings with each sample prepared using Dr. Stern’s technique.

The cells were assigned to categories along Dr. Stern’s scale during the manual analysis. They were then compared to the digital measurements both within and across cell categories. The results were very promising. In fact, the computer system could detect more small changes and rank samples at a more precise level than the original hands-on cellular scale [11]. These data supported the additional development of automated and computerized Pap test systems.

Long-term Impact and Conclusions

The most important contribution of Dr. Stern’s research was the new cell processing technique, not Pap testing or digital analysis8. This technique helped establish an entirely new version of the Pap test at the cutting-edge of modern cytology, now known as the Liquid-Based Pap test9.

The major improvements in this technique are as follows:

• Cells did not have to be immediately smeared onto a glass slide. Instead, doctors could simply collect the cervical cells using a swab or brush and place them in a vial.

• The capped vials were more easily transferred from clinic to lab than glass slides and could be stored for longer periods of time prior to processing and analysis.

• By removing cell clumps, mucus and non-cervical cells, samples were much easier to analyze. This allowed physicians to more efficiently and accurately label samples as abnormal vs. normal and improved turnaround times.

Other researchers took notice. The coming decades would see advances making this process faster and more consistent across laboratories. The first Liquid-Based Pap test, ThinPrep, was FDA approved in 1996, and used a technique similar to Dr. Stern’s.

Liquid-Based Pap tests are now the gold standard of Pap testing in the United States and around the world. In addition to the advantages listed, these Liquid-Based Pap tests can be combined with testing for Human Papillomavirus (HPV), one of the leading causes of cervical cancer [12]. Dr. Stern’s work helped pave the way to these modern cytopathology technologies, transforming cancer diagnostics and saving countless lives.



Footnotes:

1Although the Pap test is named for Dr. Papanicolaou, it was first presented by Romanian physician Dr. Aurel A. Babes in 1927, a year before Dr. Papanicolaou presented his own work in 1928. Dr. Babes did not continue working on this test and only published a single paper. Thus, the test was ultimately named for Dr. Papanicolaou, who continued working on the test and promoted its use as a screening tool (see References: [3, 4]).

2The American Cancer Society played an important role by promoting promoted the use of the Pap Test in the early 1960s.

3Pap tests are analyzed by trained cytotechnicians and/or cytopathologists. A cytotechnician may also be referred to as a technician or a cytologist and has a bachelor’s or master’s degree. A cytopathologist is a physician who has performed their residency in pathology and focused on the diagnosis of disease by tissue or cell analysis. The terms physician, doctor, and cytopathologist are used throughout the text. From the 1940s through the 1960s cytotechnicians or cytopathologists may not have been accessible for Pap test analysis depending on location in the US. This resulted in medical personel without special training analyzing Pap test slides.

4The accuracy of conventional vs. liquid-based Pap tests is still debated by some experts [13]. Nevertheless, the liquid-based test has allowed for more standardized testing and currently accounts for 90% of Pap tests in the US [5].

5In the 1970s, to help overcome inconsistencies in Pap testing results, the American Cancer Society recommended women have a Pap test once a year. If a cancer diagnosis was missed by an inaccurate test, it would likely be detected the following year. Because cervical cancer often develops slowly over the course of at least ten years, detecting cancer a year late would usually not result in a serious problem. With the advent of improved Pap tests, the American Cancer Society now recommends women have a Pap test performed every three years.

6Dr. Dorothy Rosenthal was interviewed as part of Elliott’s Scientific American piece on Dr. Stern and provided valuable insight on the impact of Dr. Stern’s work on the Pap test with JPL as well as the challenges associated with working in a new field, cytopathology.

7Dr. Rosenthal was interviewed in 2017.

8Despite multiple efforts by researchers and companies, automated Pap test analysis was not commercially successful. This was due to complications in standardizing imaging analysis, as well as significant up-front costs. According to Dr. Rosenthal, if a system was both prone to error and expensive, hospitals were not interested.

9The term ‘liquid-based’ refers to the fact that cells are taken out of a solution and placed onto a slide rather than smeared directly onto the slide.


References:

1. Papanicolaou, G.N. and H.F. Traut, The diagnostic value of vaginal smears in carcinoma of the uterus. 1941. Arch Pathol Lab Med, 1997. 121(3): p. 211-24.
2. Rosenthal, D.L., et al., A simple method of producing a monolayer of cervical cells for digital image processing. Anal Quant Cytol, 1979. 1(2): p. 84-8.
3. Tambouret, R.H., The evolution of the Papanicolaou smear. Clin Obstet Gynecol, 2013. 56(1): p. 3-9.
4. Shaw, P.A., The History of Cervical Screening I: The Pap. Test. Journal SOGC, 2000. 22(2): p. 110-114.
5. Gibb, R.K. and M.G. Martens, The impact of liquid-based cytology in decreasing the incidence of cervical cancer. Rev Obstet Gynecol, 2011. 4(Suppl 1): p. S2-S11.
6. Nanda, K., et al., Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review. Ann Intern Med, 2000. 132(10): p. 810-9.
7. Stern, E., et al., A cytological scale for cervical carcinogenesis. Cancer Res, 1974. 34(9): p. 2358-61.
8. Barrett, D.L. and E.B. King, Comparison of cellular recovery rates and morphologic detail obtained using membrane filter and cytocentrifuge techniques. Acta Cytol, 1976. 20(2): p. 174-80.
9. Husain, O.A., B.A. Page-Roberts, and J.A. Millet, A sample preparation for automated cervical cancer screening. Acta Cytol, 1978. 22(1): p. 15-21.
10. Rosenthal, D.L., et al., Endocervical columnar cell atypia coincident with cervical neoplasia characterized by digital image analysis. Acta Cytol, 1982. 26(2): p. 115-20.
11. Stern, E., et al., An expanded cervical cell classification system validated by automated measurements. Anal Quant Cytol, 1982. 4(2): p. 110-4.
12. Sherman, M.E., et al., Cervical specimens collected in liquid buffer are suitable for both cytologic screening and ancillary human papillomavirus testing. Cancer, 1997. 81(2): p. 89-97.
13. Davey, E., et al., Effect of study design and quality on unsatisfactory rates, cytology classifications, and accuracy in liquid-based versus conventional cervical cytology: a systematic review. Lancet, 2006. 367(9505): p. 122-32.