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A Self-Sampling Method to Obtain Large Volumes of Undiluted Cervicovaginal Secretions

BOSKEY, ELIZABETH R. PhD, MPH,*; MOENCH, THOMAS R. MD; HEES, PAUL S. PhD; CONE, RICHARD A. PhD*†

Article

Background Studies of vaginal physiology and pathophysiology sometime require larger volumes of undiluted cervicovaginal secretions than can be obtained by current methods. A convenient method for self-sampling these secretions outside a clinical setting can facilitate such studies of reproductive health.

Goal The goal was to develop a vaginal self-sampling method for collecting large volumes of undiluted cervicovaginal secretions.

Study Design A menstrual collection device (the Instead cup) was inserted briefly into the vagina to collect secretions that were then retrieved from the cup by centrifugation in a 50-ml conical tube.

Results All 16 women asked to perform this procedure found it feasible and acceptable. Among 27 samples, an average of 0.5 g of secretions (range, 0.1–1.5 g) was collected.

Conclusions This is a rapid and convenient self-sampling method for obtaining relatively large volumes of undiluted cervicovaginal secretions. It should prove suitable for a wide range of assays, including those involving sexually transmitted diseases, microbicides, vaginal physiology, immunology, and pathophysiology.

This study describes a rapid and convenient method of self-sampling for relatively large volumes of undiluted cervicovaginal secretions.

From the *Thomas C. Jenkins Department of Biophysics, Johns Hopkins University; ReProtect, LLC; and Department of Medicine, Johns Hopkins University, Baltimore, Maryland

Received August 7, 2001,

revised March 18, 2002, and accepted July 8, 2002.

Supported by NIH grants T32-GM-0-7231 and P01-AI-45967.

Correspondence: Richard A. Cone, PhD, Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218. E-mail: cone@jhu.edu

CERVICOVAGINAL SECRETIONS are often used in reproductive health studies. In the past, women have expressed preferences for self-sampling procedures over samples obtained by clinicians. 1 Furthermore, self-sampling procedures have been shown to provide similar diagnostic accuracy to more traditional testing methods for the diagnosis of chlamydial infection, 2–4 group B streptococcal infection, and human papillomavirus infection. 5

Cervicovaginal secretions are usually collected in one of four ways: swab, tampon, filter paper, or lavage. 6 Each has benefits, but all four usually require that the sample be diluted at some point during the procedure. Dilution reduces the sensitivity of most assays, and the extent of dilution is often difficult to determine, making quantification of the initial in vivo concentrations difficult. The most frequently used methods often require the intervention of a clinician, reducing the convenience and acceptability to subjects, especially for volunteers being recruited for studies, which primarily take place outside a clinic setting.

Convenient self-sampling methods that can be done quickly and easily at the time of a study visit benefit both investigators and study participants. The self-sampling method described here uses a commercially available menstrual collection device to obtain a relatively large volume of cervicovaginal secretions, with no need for dilution. The procedure requires minimal instruction and can be done at home as well as in the clinic.

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Materials and Methods

Participants

Written informed consent was obtained from each participant, under protocols approved by the human subjects review boards of The Johns Hopkins University and The Johns Hopkins Bayview Medical Center. The 16 participants were between 19 and 41 years of age and were studied at random times between menses.

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Collection of Vaginal Secretions

Participants were provided with an individually wrapped commercial menstrual-collection device that is similar to a contraceptive diaphragm (Instead cup; Ultrafem, Missoula, MT), the manufacturer's packaged instructions, and an empty sterile preweighed 50-mL conical polypropylene centrifuge tube (Sarstedt no. 62547205 or equivalent). They were instructed to insert the cup into the vagina, wait about 5 seconds, remove it, place it into the centrifuge tube, cap the tube, and return the sealed tube to the investigator. When the collections were performed in a restroom on site, the capped sample tube was handed directly to the investigator. When the collection was performed off-site, the participants placed the sample tube containing the cup and secretions in a small plastic bag, along with the wrapper from the device, and stored the bag in a home freezer until returning it to the investigator. This storage method minimized drying of the sample and enabled an accurate determination of the sample weight. The tubes were centrifuged at 2000 rpm (600 g) for 15 minutes to retrieve and pool the vaginal fluid from the cup. The empty menstrual cup was then removed and discarded, and the mass of the retrieved sample was determined by reweighing the tube and sample.

The efficiency of fluid removal by centrifugation was determined, for a subset of participants, by weighing the cup before and after centrifugation and calculating the fraction of the total sample that was removed from the cup.

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MRI of the Vaginal Area Sampled by the Device

The volunteer inserted the device while seated in a bath (to prevent insertion of air along with the device). Magnetic resonance images were obtained on a Resonex RX5000 0.4-T clinical imaging system. After the optimal scanning plane was established with scout scans, a high-resolution T1-weighted spin-echo image (repetition time = 400 ms; echo time = 20 ms; number of averages = 2) was acquired through the plane of the collector device rim.

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Results

Collected Volume

On site, 22 samples were collected from 12 women. An average of 0.5 g of undiluted vaginal fluid (range, 0.1–1.5 g) was collected from each cup. This amount is comparable to the total amount of fluid (0.76 ± 0.04 g) a clinician can collect by swabbing the entire vaginal surface 7 and is about 10-fold larger than the volume typically obtained with a single vaginal swab, which averages about 0.03 to 0.05 g. 8,9 An additional set of five samples was collected to determine whether storing the sample frozen before centrifugation interfered with sample retrieval. Five women performed the sampling technique at home, froze the samples, and returned them to the study site at a later time (mean sample size, 0.3 g; range, 0.1–0.7 g). This was done to simulate the use of the technique in a study that required multiple or specially timed samples. Freezing did not appear to affect sample retrieval, and in a separate set of tests it reduced evaporative loss (drying) of the sample. Experiments with physiological saline documented that there was little evaporative loss from the capped tubes (<2 mg/d by evaporation) at room temperature and that storage in a freezer further reduced evaporative loss (<0.1 mg/d).

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Retrieval Efficiency

We used eight samples from four women to examine the fraction of secretions that failed to be removed from the cup by centrifugation. We found that on average, 10% of the secretions remained on the cup after centrifugation (range, 0–33%). Sample retrieval was not detectably altered when the sample was centrifuged for only 1 minute at 170 g, which shortens the procedure and may reduce stratification of the sample (i.e., pelleting of cells and bacteria).

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Discussion

This method was developed to assist in the collection of undiluted vaginal secretions for ongoing physiology studies in our laboratory. The large volume of these cup-collected samples enabled us to measure the D/L optical isomer ratio of vaginal lactate and thereby confirm that bacteria are the primary source of vaginal acidity. 10 Also, the sample volumes are sufficiently large to enable measurements of the viscoelasticity of undiluted cervicovaginal secretions (study in progress), a property that dilution markedly alters. Gram stains, vaginal pH determinations, and assays of any of the components of cervicovaginal secretions are readily done with these large-volume samples, and, of course, large undiluted samples facilitate the study of any component in these secretions that is present at low concentrations.

Only three participants were previously familiar with the Instead device; the other 13 participants had no prior experience with its use. None of the women reported difficulty following the package instructions for inserting and removing the device. The Instead cup is similar to a conventional contraceptive diaphragm, but it is uniquely suitable for this method since it is inexpensive, disposable, and nonabsorptive and has a very flexible dome with a large surface area to which vaginal secretions tend to adhere. The rim of the Instead cup is flat in cross-section, which enhances the efficacy with which the rim “squeegees” secretions as it is removed from the vagina. Finally, the cup is packaged in an individual wrapper, which may provide additional comfort and confidence for women. Simply inserting and removing the cup coats it with vaginal secretions, cells, and bacteria that are rapidly and easily collected by centrifugation. After removal, the cup is placed in a sterile, dry centrifuge tube that is sealed when capped, reducing the likelihood of evaporation and requiring no dilution for retrieval. This capped tube is easily transportable.

A primary advantage of this method is the relatively large volume of fluid collected—up to 1.5 g, a substantially greater volume than can be collected from a swab, which typically collects between 0.01 and 0.1 g. 8,9 The MRI image (Figure 1) illustrates how the cup completely opens within the vagina, to a diameter of 70 mm, and thereby contacts and retrieves vaginal fluid from a large area of the epithelium, an area significantly larger than is contacted by a tampon and much larger than is contacted by a swab or strip of filter paper. Thus, in terms of the total vaginal area sampled, the Instead cup method is most comparable to a cervicovaginal lavage but does not require dilution of the sample.

Fig. 1

Fig. 1

Since the cup method provides an averaged sample from a large vaginal area, it is not appropriate for studies that require pure cervical secretions or secretions from a specific location. For such studies, swabs and filters are more suitable. 6 The cup method should be especially useful for ex vivo studies that require undiluted secretions or for studies in which a rapid, convenient self-sampling procedure that can be done by study participants at home or in the clinic would be efficacious.

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References

1. Mercer BM, Taylor MC, Fricke JL, Baselski VS, Sibai BM. The accuracy and patient preference for self-collected group B Streptococcus cultures. Am J Obstet Gynecol 1995; 173: 1325–1328.
2. Wiesenfeld HC, Heine RP, Rideout A, Macio I, DiBiasi F, Sweet RL. The vaginal introitus: a novel site for Chlamydia trachomatis testing in women. Am J Obstet Gynecol 1996; 174: 1542–1546.
3. Domeika M, Drulyte O. Use of PCR for the detection of genital Chlamydia trachomatis infection on self-obtained mailed vaginal samples. Acta Obstet Gynecol Scand 2000; 79: 570–575.
4. Domeika M, Bassiri M, Butrimiene I, Venalis A, Ranceva J, Vasjanova V. Evaluation of vaginal introital sampling as an alternative approach for the detection of genital Chlamydia trachomatis infection in women. Acta Obstet Gynecol Scand 1999; 78: 131–136.
5. Coutlee F, Hankins C, Lapointe N. Comparison between vaginal tampon and cervicovaginal lavage specimen collection for detection of human papillomavirus DNA by the polymerase chain reaction. The Canadian Women's HIV Study Group. J Med Virol 1997; 51: 42–47.
6. Quesnel A, Cu-Uvin S, Murphy D, Ashley RL, Flanigan T, Neutra MR. Comparative analysis of methods for collection and measurement of immunoglobulins in cervical and vaginal secretions of women. J Immunol Methods 1997; 202: 153–161.
7. Stone A, Gamble CJ. The quantity of vaginal fluid. Am J Obstet Gynecol 1959; 78: 279–281.
8. Bartlett JG, Onderdonk AB, Drude E, et al. Quantitative bacteriology of the vaginal flora. J Infect Dis 1977; 136: 271–277.
9. Onderdonk AB, Polk BF, Moon NE, Goren B, Bartlett JG. Methods for quantitative vaginal flora studies. Am J Obstet Gynecol 1977; 238: 777–781.
10. Boskey ER, Cone RA, Whaley KJ, Moench TR. Origins of vaginal acidity: high D/L lactate ratio is consistent with bacteria being the primary source. Hum Reprod 2001; 16: 1809–1813.
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