Gyenocologic Pap Test Collection Procedure (2022)


University ofVirginia Health System

Department of MedicalLaboratories

Division ofCytopathology

RevisedOctober 2010

Revised September2012



A Pap test is a screening test thatcan also be a diagnostic test. Its primary purpose is to identifypatients who have cellular changes that place them at risk for thedevelopment of cervical cancer. The Pap test is a highly complexlaboratory test requiring careful patient preparation, skill inclinical collection, highly skilled and controlled laboratoryprocessing, and professional interpretation. Like all laboratorytests, the Pap test is not perfect. A Pap test is best viewed as amoderately sensitive, highly specific test with an established falsenegative rate of no less that 5%.

In cases of routine screening forcervical cancer and its precursors, cellular material can be obtainedfrom the uterine cervix using a variety of sampling devices. Mostclinics at the University of Virginia Health Sciences Center use thespatula and/or an endocervical brush. The cervix and the area adjacentto the cervix (fornices) must be fully visible. The ecto- andendocervix should be sampled separately. The material obtained can berinsed into liquid medium (ThinPrep Pap test), our preferred method,or, only exceptionally, spread on one or two glass slide slides(conventional Pap smear).

ThinPrep Pap test supplies areavailable from the Hospital Storeroom: Item ID 92189 tray of 25Thinprep Pap test vials and Item ID 92190 bag of 25 collector sets(spatulas and brushes). Cervex broom collectors and conventional Paptest supplies are available through Laboratory Customer Service at924-LABS. Carefully read the manufacturer's instructions for properapplications and all contraindications prior to using thecytobrush.


1. The patient should NOT make anappointment for her Pap test during her menstrual period. The preferredtime for the examination is two weeks after the first day of her lastmenstrual period.

2.The patient should beinstructed NOT to use vaginal medications, vaginal contraceptives,lubricants, or douches for 48 hours before her appointment. Intercourseis not recommended the night before the examination.


1.The physician should not useany lubricant during his examination prior to collecting thecellsample.

2.Every effort is made toidentify and sample the squamo-columnar junction and/or transformationzone since the majority of squamous lesions arise within this area.

3.Excess blood, mucus, orinflammatory exudate may be gently blotted away with a gauzepad. Do not scrape or wash this material away since such actions mayadversely affect the subsequent cellular sample.

4.The Pap test should always betaken prior to other testing (e.g. cultures, tissue sampling,application of acetic acid, etc.)

The ThinPrep Pap test requires uniquespecimen collectors and a special collection fluid. Some physiciantraining in proper collection techniques is also needed. To arrangetraining, contact Laboratory Customer Service at 924-LABS. In addition,an instructional video on ThinPrep Pap test collection is availablefrom the Cytology Laboratory (924-2770) and on-line from HologicCorporation (see #9 below).


1.Assemblecollection devices and ThinPrep Pap test vial.

2.Obtain anadequate sampling of the ectocervix using aplastic spatula. (A wooden spatula is notsuitable as the cellular material is more difficult to rinse into thecollection fluid.)

3.Rinse thespatula into the solution vial by swirling the spatula vigorously inthe vial 10 times. Discard the spatula.

4.Obtain anadequate sampling from the endocervix using an endocervical brushdevice. Insert the brush into the endocervical canal until only thebottom-most fibers are exposed. Slowly rotate ¼ or ½turn in one direction. DO NOT OVER ROTATE.

5.Rinse the brush in the solution vial by rotatingthe device in the solution 10 times whilepushing against the vial wall. Swirl the brushvigorously to further release material. Discard the brush.

6.Tighten the cap so that the torque line on the cappasses the torque line on the vial.

7.Record the patient’s name and history number on thevial or attach a printed patient identification label. Complete acytopathology test request form (in preprinted paper form or test orderprinted from Epic).

8.Sendlabeled specimen vial and completed request form to the laboratory forprocessing. No refrigeration of the specimen is necessary.

9.On-line video


NOTE: Currently 99.9% of Pap tests are ThinPrep, which is theexpected sample type.

1.Using a lead pencil, write thepatient's name and/or medical record number on the frosted end of eachglass slide to be used. Use a diamond point pencil to label plain glassslides. All slides submitted to the laboratory must be clearly labeledwith the patient identification information at the time of specimencollection. Paper labels affixed to the slides are NOT an acceptablemeans of slide identification. Federal regulations require thatunlabeled slides must be returned to the sender foridentification and correction. Laboratory personnel cannotcorrect this error.

2.Have spray fixative nearby toallow for immediate fixation of the prepared slides. Review and followthe manufacturer's instructions for correct application.

3.Under direct vision, sample theectocervix and endocervix separately using the appropriate samplingdevice. Prepare one combined slide or two separate slides.

a. To prepare a singlecombined smear, sample the endocervix first using the endocervicalbrush. Insert the brush into the external os and gently rotatethe device using ¼ or ½ turn to obtain the cell sample(over rotation may cause bleeding). Do not smear, but allow thematerial to remain on the brush. Scrape the ectocervix with thespatula and spread the material rapidly onto the upper end of theslide. Quickly roll the endocervical brush through theectocervical material to the end of the slide. Perform thistechnique as rapidly as possible to prevent drying artifacts.Immediately spray fix by thoroughly soaking the cellular sample whileholding the spray fixative container about 6-8 inchesfrom the slide. Allow spray fixative to evaporate.

b. To prepareseparate slides, rotate the spatula with pressure over the entireectocervix. Spread the cellular material evenly and rapidly across theglass slide being careful to avoid the labeled area. Immediately sprayfix by thoroughly soaking the cellular sample while holding the sprayfixative container about 6-8 inches from the slide.Allow spray fixative to evaporate.

For the second slide, insert theendocervical brush into the external os and obtain the cell sample aspreviously described. Remove the brush and roll it across theslide to remove the cellular material. Rubbing or scraping the brushacross the slide creates artifacts that may hamper evaluation.Immediately spray fix by thoroughly soaking the cellular sample whileholding the spray fixative container about 6-8 inches from the slide.Allow spray fixative to evaporate.

4.Place fixed slides in anappropriate container designed to prevent breakage for transport to thelaboratory. Complete a cytopathology test request form (in preprintedpaper form or test order printed from Epic).

5.Deliver specimen slidesand request form to the laboratory at the convenience of the submittingphysician or department.

IN PATIENTS AT RISK FOR ENDOMETRIALDISEASE, CERVICAL SAMPLES ARE USUALLY INADEQUATE TO SENSITIVELY DETECTENDOMETRIAL LESIONS. Aspiration of material from the vaginal poolmay provide some information; however, more direct sampling ispreferred. There are several procedures for obtaining endometrialmaterial, all of which require specialized skills and equipment.Consultation with clinicians in the Department of Obstetrics andGynecology who are familiar with such procedures is recommended priorto specimen collection.


A cytopathology test request form (in preprinted paper form or testorder printed from Epic) containing the following required informationmust accompany each specimen:

  1. Patient name, patient history number(medical record number), and date of birth (age is not sufficient as ameans of patient identification).
  2. Name of the ATTENDING physician orauthorized nurse practitioner requesting the test. A resident may belisted only if accompanied by the name of a staff physician.
  3. Clinic, floor, or service generatingthe specimen.
  4. Date of specimen collection.
  5. ICD-9 code indicating the medicalnecessity for the test. The code should be included in the space/fieldprovided. Medicare regulations for ICD-9 coding are written on the backof the multipart preprinted test request form.
  6. Type or source of specimen (check boxon form or use drop down box in Epic).
  7. Test order: check the appropriate boxon the paper form or use the on-line dropdown list in Epic to order Paptest screening alone or Pap test with HPV testing. Explanations for HPVtest ordering) are on the back of the top copy of the paper requestform.
  8. All pertinent clinicalhistory/findings or other data relating to the requestedevaluation.

For preprinted versions of theCytopathology Request form, computer generated registration labels arethe preferred method for providing patient identification information(i.e. name, history number, date of birth) for outpatients.

Clinical history or findings includethe information listed below. This information is important foraccurate interpretation of smears and should correlate with theassigned ICD-9 code provided on the request form.

1.All previous disease processes(e.g. dysplasia, cancer, etc.) and/or therapy (e.g. radiotherapy,cryo/laser therapy, estrogen replacement therapy, etc.) that couldinfluence the cytologic interpretation of the Pap test. Case numbersfrom previous cytology or surgical specimens are helpful, but shouldNOT replace written descriptions of previous findings.

2.Currentpatient symptoms or complaints.

3.Abnormalfindings discovered during the physical examination.

4.Menstrual history

  • a. Indicate menstrual status bychecking the appropriate box on the request form.
  • b. The most recent date fornormal menses or expected bleeding is written on the “LMP”dateline.
  • c.For post-menopausalpatients on estrogen replacement therapy, the date for normalwithdrawal bleeding is given on the LMP date line.
  • d.All ABNORMAL bleeding inpost-menopausal patients should be indicated in descriptive terms inthe clinical history/findings section of the request form or bychecking the appropriate box. Do not use the LMP date line for thisinformation.

All of theinformation discussed above is essential for the efficient processingof the specimen and the timely reporting of results. Missing vital datamay delay completion of the case.


The University of Virginia Cytology Laboratory uses the 2001Bethesda System for reporting the cytologic interpretations ofgynecologic Pap specimens. A description of this system is givenin the following pages.

Results are usually available within five to seven working daysafter receipt of the specimen in the laboratory. Inpatient andstat cases are read as quickly as possible, usually the next workingday after receipt in the laboratory.



In 1988 the Divisionof Cancer Prevention and Control at the National Cancer Instituteconvened a workshop of experts and consultants to review existingterminology for the reporting of cervical/vaginal diagnoses and torecommend effective methods for standardizing diagnosticreporting. As a result of this workshop, the Bethesda System(TBS) was published as a guideline for uniform terminology andreporting of cervical/vaginal diagnoses. Under this system thefinal report consists of three major components 1) a statement ofadequacy, 2) a general categorization/ descriptive diagnosis, and 3)recommendations for follow-up procedures, if necessary. A secondworkshop was held in 1991 at the National Cancer Institute for furtherdiscussion and review of TBS. Some minor changes andclarifications of TBS resulted from that meeting.

The 2001 Bethesda Conference provided areview of the application of TBS over the previous 10 years. After muchdiscussion, significant revision of reporting terminology was made. Themost significant change was the combination of the “within normallimits” category and the “benign cellular changes” category under theheading of “Negative for Intraepithelial Lesion or Malignancy.”Infectious organisms are reported directly with the “Negative”interpretation. Benign findings are considered optional and arereported as additional findings under the “Negative” heading. Therevised version was published as a reporting system for cervicalcytology.

The UVA Cytology Laboratory uses aslightly modified version of TBS for reporting cervicalinterpretations. The modifications are generally in specific wordingand in the inclusion of additional descriptive diagnosis in order tofacilitate patient management by clinicians at our institution.The intent, content, and scope of TBS remain intact.


Satisfactory forInterpretation:

Satisfactory gynecologic specimens aredefined as specimens having sufficient numbers of well-preserved,well-stained, unobscured epithelial cells such that a cytologicinterpretation can be rendered. No conditions exist (e.g.excessive inflammation or blood, poor preservation, etc.) that preventthe cytologic interpretation.

Relevant clinical information and/orhistory are considered necessary for accurate evaluation of Papsmears. These data may clarify otherwise uncertain cytologicfindings. Therefore, the provision of pertinent patientinformation that may influence the interpretation of the smears isnecessary for a "satisfactory" specimen.

Some specimens may have conditions thathamper but do NOT prevent a reasonable cytologic interpretation.The cellular material remains sufficient to render an interpretationalthough that interpretation may be somewhat limited in scope orspecificity. Reasons for the limitations are stated in the report underthe heading “Statement of Adequacy.”

Unsatisfactory for Interpretation:

Unsatisfactory specimens are defined asspecimens in which conditions exist (e.g. too few epithelial cells,excessive inflammation or blood, poor preservation, etc.) that preventa reasonable cytologic interpretation. Rendering a cytologicinterpretation on the specimens would not meet basic standards of care.In this category the statement of adequacy or lack thereof, becomes thedescriptive interpretation. All identifiable limiting factors forthe unsatisfactory results are stated in the report. No cytologicinterpretation is given.

If abnormal cells are detected,regardless of other factors, the specimen is NEVER categorized asunsatisfactory.

The laboratory has procedures in placeto avoid reporting unsatisfactory results whenever possible. Prior torecording a specimen as unsatisfactory, the residual material in thespecimen vial is evaluated for physical findings that may indicate thatadditional processing could potentially improve sample adequacy. Ifmerited, additional slides are made. In addition, all specimens inwhich basic criteria for adequacy are not met are sent for a secondcytotechnologist review and sign out. If the two cytotechnologistsagree, the unsatisfactory results are reported. If the two reviewers donot agree, the case is referred to the pathologist for finalinterpretation.


The interpretation element of thereport is largely self-explanatory. The descriptive terminologyused in our laboratory is listed on pages 8-10. TBS limits theterm "atypical cells" to those cases in which the cytologic findingsare of undetermined significance. Whenever possible, the use ofthe term should be further clarified when a high-grade lesion cannot beexcluded. The old numerical Papanicolaou classification system is notconsidered acceptable in the modern practice of diagnosticcytopathology.


Suggestions for follow-up areoptional. When included in the report, they should be concise andconsistent with clinical follow-up guidelines published by professionalorganizations (e.g., ASCCP). TBS does not contain guidelines forpatient management.


The results of any ancillary testingperformed on the specimen will be given either in the final report oras an addendum to that report. The results will provide a briefdescription of the test method and the test outcome so that clinicianscan easily understand them. Refer to the section on HPV testing pages10-11.


I.Statement of adequacy

Satisfactory for interpretation –limiting factors listed as needed

Unsatisfactory specimen - requiresexplanation, give all limiting factors

Limiting factors for satisfactoryspecimens:

Scant numbers of epithelial cells

Endocervical/transformation zonecomponent absent/scant

Poor fixation or preservation

Partially obscuring inflammatoryexudate

Partially obscuring blood

Partially obscuring bacterialovergrowth

Mechanical distortion

Cellular degeneration

Pertinent clinical information that mayaffect cytologic interpretation was not provided.

Explanations for unsatisfactoryspecimens:

Insufficient numbers of epithelialcells for adequate cytologic interpretation

Completely obscuring inflammatoryexudate

Completely obscuring blood

Completely obscuring bacterialovergrowth

Poor fixation or preservation of theepithelial cells prevents adequate cytologic evaluation

Mechanical distortion of the epithelialcells prevents adequate cytologicevaluation

Cellular degeneration prevents adequateevaluation of the epithelial cells

II.Descriptive interpretation/results categories

Negative for Intraepithelial Lesion orMalignancy

Infections (reported with theNegative interpretation):

Fungal organisms consistent withCandida species

Bacteria morphologically consistentwith Actinomyces species

Trichomonas vaginalis infection

Cellular changes indicative ofcytomegalovirus

Cellular changes indicative of Herpessimplex virus

Additional cytologic findings(reporting optional):

Benign cellular changes associated withan inflammatory process

Atrophic cervicitis/vaginitis

Benign cellular changes associated with ionizing radiation

Benign cellular changes associated witha reactive/reparative process

Benign cellular changes associated withatrophy

Benign cellular changes, associatedcause not identified

Benign cellular changes associated withpresence of intrauterine contraceptive device

Tubal metaplasia present

Effects of nonsteroidal estrogenexposure

Parakeratosis (reported only whenpresent in quantity)

NOTE: This entity may occur alonein response to various stimuli, or may beassociated with squamous neoplasia.

Anucleated squamous cells present(reported only when present in quantity)

NOTE: This entity may occur alonein response to various stimuli, or may beassociated with squamous neoplasia.

Endometrial material present inpostmenopausal woman

Endometrial material present, recommendclinical correlation

NOTE: This is reported in women40 years or older when not accounted for by patient history.

Pregnancy elements present

Endocervical-type cells present invaginal smear

Epithelial cellsabnormalities:

Atypical squamous cells present

Atypical squamous cells, cannot excludea high-grade intraepithelial lesion

Low-grade squamous intraepitheliallesion (mild dysplasia)

High-grade squamous intraepitheliallesion (moderate dysplasia)

High-grade squamous intraepitheliallesion (severe dysplasia)

Squamous cell carcinoma (statement ofgrade and type may be made)

Atypical endocervical cells present

Atypical endometrial cells present

Atypical glandular cells present

Atypical endocervical cells present,favor neoplastic

Atypical glandular cells present, favorneoplastic

Endocervical adenocarcinoma-in-situ

Suspicious for malignancy

Squamous cell carcinoma

Adenocarcinoma (if possible site oforigin suggested)

Adenosquamous carcinoma

Small cell undifferentiated orneuroendocrine carcinoma

Malignant mixed mulleriantumor


Metastatic carcinoma

Other malignancies (identify type ifpossible)

Other diagnoses (freetext)

Comments forinterpretations:

Paucity of atypical cells preventsdefinitive diagnosis

Predominance of cocci/bacilliconsistent with shift in vaginal flora

III.Suggestions for follow-up

Based on cytologicfindings, recommend repeat Paptest

Recommend clearing ofinflammation followed by repeat Pap test

Recommend treatmentfor the infection followed by repeat Pap test

Recommend repeat Paptest at mid-cycle

Recommend treatmentwith topical estrogen followed by repeat Pap test

Other recommendations(free text)

IV. HormonalEvaluation

(Performed only on lateral vaginal wall specimens, requiresspecific request from ordering physician/nurse practitioner)

MI/ / (interpretationrequired)

hormonal evaluationnot possible - cervical specimen or inflammation present orinsufficient patient history


1.Gilbert FE, Hicklin MD, Inhorn SL, et al. Standards of Adequacyof Cytologic Examination of the Female Genital Tract. Am JClin Path 1974;61:285-286.

2.Lundbery GD, ed. Quality Assurance in Cervical Cytology - ThePapanicolaou Smear. JAMA 1989;262:1672-1679.

3.Greening SE: The Adequate Papanicolaou Smear Revisited.Diagn Cytopathol 1985;1:55-56.

4. VooysPG, Elias A, van der Graaf Y, et al. Relationships Between theDiagnosis of Epithelial Abnormalities and the Composition of CervicalSmears. Acta Cytol 1985;29:323-328.

5.Lundberg GD, ed. The 1988 Bethesda System for ReportingCervical/Vaginal Cytologic Diagnoses. JAMA1989;262:931-934.

6. BroderS. The Bethesda System for Reporting Cervical/Vaginal CytologicDiagnoses - Report of the 1991 Bethesda Workshop. JAMA1992;267:1982.

7. The Bethesda System forReporting Cervical/Vaginal Cytologic Diagnoses. ActaCytol1993;37:115-124.

8. Solomon D, Davey D,Kurman R, et al. The 2001 Bethesda System – terminology forreporting results of cervical cytology. JAMA2002;287:2114-2119.

9. Solomon D, Nayar R(editors). The Bethesda System for Reporting Cervical Cytology(second edition). Springer, New York, 2004.


The Molecular Diagnostics Laboratory,in conjunction with the Cytopathology Laboratory, offers HPV High-RiskDNA with genotyping on the ThinPrep Pap Test. This test isdesigned to identify women infected with any of 14 high-risk HPV types(16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) that maybe associated with cervical cancer or its precursors, includinginformation on high risk HPV genotypes. TheRoche Cobas4800provides clinicians with a result that differentiatesHPV 16 and HPV 18 from the 12 other HPV high-risk types. HPVtesting via theRoche Cobas 4800has been found tobe more sensitiveand more specific than cytology alone indetecting persistent HGSIL or cancer in women with 1 of the 14high-risk genotypes. Physicians ordering the ThinPrep Pap Testhave the option of selecting ancillary HPV testing to be performed onthe original Pap test specimen submitted for a patient.HPV testing on ThinPrep samples should be ordered withinthree weeks from the date of specimen collection. Because of storagelimitations within the laboratory, specimen vials are not kept beyondthe three week time period.

The testing is performed in the Molecular Diagnostics division ofthe Medical Laboratories on material from the ThinPrep Pap testsubmitted to the Medical Laboratories for cervical cancerscreening.


The most recent version of thepreprinted cytopathology test request form and Epic on-line test orderprovide specific items for ordering HPV testing. In addition,information on test ordering is provided on the back of the pre-printedform.

Epic HPV TestOrders

At the time the ThinPrep sample issubmitted for cytologic evaluation, HPV testing can be ordered from thedropdown list for Epic on-line ordering.

1. HPV PrimaryScreening (means the Primary Screening FDA algorithm)

o If HPVPRIMARY SCREENING TEST is selected, the followingalgorithm will be used:

· If POSITIVEfor HPV 16 or HPV 18: No further testing will beperformed. Per the ASCCP guidelines, patients testing positivefor HPV 16 and/or HPV 18 should be considered for immediate colposcopy,regardless of the concurrent cytology result.

· If NEGATIVEfor all HR HPV types: No further testing will beperformed. Patients testing negative for HR HPV types have a<1% chance of HSIL lesion.

· If HR HPVPOS, NOT HPV 16 or HPV 18: Samples will beautomatically reflexed for PAP smear. Clinicians will not need toorder reflex testing or request a separate PAP smear. Per ASCCPguidelines, patients positive for HPV types other than 16 and 18 shouldbe considered for immediate colposcopy if concurrent cytology isabnormal at a threshold of ASCUS or above

2. HPV-CoTest with Pap:Refer immediately for HPV testing once the Pap test has been run.

3. HPV-reflex for ASCUS:Perform HPV testing only if the cytologic interpretation is ASCUS orAGUS.

4.Pap Test only, NO HPV performed.

5.HPV-Non-cervical/vaginal specimens.

Pre-printed CytopathologyRequest forms HPV Test Orders

At the time the ThinPrep sample issubmitted for cytologic evaluation, HPV testing can be ordered HPVtesting can be ordered by checking the appropriate box.

oThinPrep Pap with Reflex HPV Test: Perform HPV testingonly if the cytologic interpretation is ASCUS or AGUS.

oThinPrep Pap with HPV Test Regardless ofInterpretation: Perform HPV testing even if the interpretationis “negative” (usually in patients with a prior history of abnormalfindings on previous Pap tests or as part of a follow-up for previouslytreated intraepithelial lesions).

oThinPrep Pap with HPV Screening for Women 30 andolder: Refer immediately for HPV testing once the Pap test hasbeen run. This order is totally independent of the cytology results ofthe Pap test.

Pap tests with unsatisfactoryinterpretations are not appropriate for HPV testing.

The cytopathology report will indicatein the “notes” section whether or not HPV has been ordered and ifsufficient and/or acceptable specimen exists to perform HPV testing.Note: If you have ordered HPV testing and acomment regarding that order is NOT on the final report, please contactthe Cytology Laboratory immediately to verify that the test wasordered. If the order was missed by the laboratory staff, it canbe ordered immediately.

If HPV testing has not been ordered onthe original specimen request or the clinician wants to change the typeof HPV test order, an add-on request can be made.A written order signed by the requesting physician/nurse for therequest can be faxed to the Cytology Laboratory(434-924-0217) or an Epic on-line order can be faxedso that the HPV request can be initiated. Such a request shouldbe made within 5 days of the sign-out date of thecytopathology final report. In the event that sufficient and/orappropriate sample is not available, the laboratory will notify therequestor. The person taking the request will not be able to giveyou this information at the time the request is made.


Final results of HPV testing,regardless of algorithm requested, will be available in “Cytology, GYN”under the “Pathology” tab in EPIC. Clients without access to EPICwill receive HPV results via method used to receive all otherlaboratory test results.

Please contact the Clinical Microbiology Director, Dr.Melinda Poulter, at PIC 3677 with questions. Dr. Mark Stoler isavailable at PIC 4316 to discuss algorithmic or medical implementationquestions.


Wright, T., Stoler, M., Sharma, A.,Zhang, G., Behrens, C., Wright, T.L., Evaluation of HPV-16 andHPV-18 Genotyping for the Triage of Women with High-Risk HPV+ Cytology,Negative Results. AM J Clin Patholo2011;136:578-596.


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