Vol. 14 •Issue 11 • Page 46
Analysis of Body Fluids
Literature findings of new applications of body fluids are reviewed.
Recent literature has revealed several new and intriguing applications of body fluids. In particular, scientists are expanding on diagnostic and forensic applications of semen, urine, cerebral spinal fluid (CSF) and vaginal fluid.
Semen
The analysis of semen plays an important role in the clinical laboratory, particularly in assessing fertility and certain forensic cases. Some especially interesting articles are reviewed here. More than 20 years ago, Anne Jecquier was a prime mover in suggesting the need for quality assurance (QA) in andrology laboratories. She proposed this year that the quality assurance schemes for semen analysis may no longer be needed. She goes further to suggest that sperm function tests have limited predictive value in terms of fertility assessment.1
In an accompanying article,2 Holt agreed that since the QA schemes have brought about higher technical standards in laboratories, Jecquier’s assertion is possibly true. However, he feels that “vigilance is still needed in discriminating between unproductive investment of time and energy in the refinement of tests that may offer little information about fertility, and maintaining technical standards.”
Thus, although it may not matter in practice whether a sperm concentration is estimated as 100 or 200 million/ml, distinguishing between 25 million and 100 million /ml would probably influence a clinician’s treatment decisions.
The use of prostate specific antigen (PSA) as evidence of the presence of semen in forensic cases has been well established.3 In the Levine study, the detection of seminal PSA was compared to the identification of spermatozoa in swabs obtained from the vagina of deceased females. Agreement occurred between the two methods in 67 of the 80 cases (84 percent), including 18 cases where both the PSA was positive and sperm were observed microscopically. Enough discrepancies existed between the two methods (16 percent) to justify the use of both methodologies, if possible, to make the determination of sexual activity prior to death.
Also to determine the significance of semen analysis, 227 anonymous samples divided into four groups (normospermia, oligospermia, azoospermia and controls) after a semen analysis were subjected to three fast detection semen tests: Diff-Quick fast coloration, Phosphatesmo Km Paper for acid phosphatases (AP) detection and PSA-Check 1 for prostate specific antigen (PSA) detection.4 Unlike cytological studies, the results obtained with AP and PSA were not influenced by spermatozoa concentration. PSA detection results remained constant up to 72 hours and were more reliable after 48 hours than those obtained by AP detection.
Saliva, Urine and Blood
Other body fluids useful in diagnostic and forensic cases include saliva, urine and, obviously, blood. The reliability of drug analytes in saliva was studied using the Cedia and Emit tests for cannabinoids. Saliva (n = 97) and 103 urine samples (n = 103) from 31 opioid-dependent patients were tested for methadone.5 The results in saliva were comparable to those in urine. For most other drugs, including opioids and barbiturates, results were less favorable. For cannabinoids and benzodiazepines, results were unsatisfactory. Urine testing using immunoassays or gas chromatography (GC) is still the most reliable non-invasive test method. Drug testing in saliva may be of relevance as an additional method in substitution treatment and as an experimental tool in epidemiological road surveys.
An analysis for cannabinoids, amphetamine and its derivatives (opiates and cocaine) also was performed in urine and serum (IA/GC/MS) and saliva by GC-MS. Police and medical officer observations of symptoms of impairment were rated and evaluated using a threshold value for the classification of driving inability. Accuracy in correlating drug detection in saliva and serum was better than 90 percent for all substances and also better than 90 percent in urine and serum except for THC (71 percent). Of the cases with saliva positive for any drug, 97 percent of the corresponding serum samples were also positive for at least one drug; of drug-positive urine samples this was only 82 percent. In 119 of 146 cases, impairment symptoms above threshold were observed in 82 percent. Saliva appears superior to urine in correlating with serum analytical data and impairment symptoms of drivers under the influence of drugs of abuse.
A third study looked at on-site vs. laboratory evaluation using GC/MS on both and saliva.6 Samples were tested on amphetamines (AMP), cannabinoids (THC), opiates (OPI) and cocaine metabolites (COC). Both positive and negative samples were tested. A total of 800 persons and eight on-site devices for urine and two for saliva testing were included in this study. Good results were obtained for the urine on-site devices, with accuracy of 93-99 percent for amphetamines, 97-99 percent for cannabinoids, 94-98 percent for opiates and 90-98 percent for benzodiazepines. However, differences in the ease of performance and interpretation of test result were observed. It was possible to detect amphetamines and opiates in saliva by the on-site devices, but the benzodiazepines and cannabinoids did not fulfill the needs of sensitivity.
Since 1998, driving under the influence of drugs such as amphetamine, cannabis, cocaine, heroine and morphine is sanctioned due to Section 24a of the Road Traffic Regulations of Germany. From December 2000 to June 2002, 751 roadside tests with the immunochemical test device Toxiquick were conducted on 302 drivers (273 male and 29 female) on oral fluid samples obtained.7 The results of the tests are compared to the results obtained through measurement of corresponding blood samples by GC/MS. In 75 percent, the roadside test produced correct results and, therefore, provided helpful assistance to the police officers. Except for cannabinoids, the number of false negative results was relatively small, whereas false positive results ranged between 32 percent for opiates and 11 percent for benzoylecgonine.8
The Oratect® (Branan) and Uplink® (OraSure) were evaluated for their ability to detect amphetamines, cocaine, opiates and cannabinoids.9 An additional device, Drugwipe (Securtec), was evaluated for the detection of cocaine and cannabinoids.9 In general, the Branan and OraSure devices detected amphetamine, methamphetamine, opiates and cannabinoid metabolite (THC-COOH) well, but all three devices performed poorly in detecting Delta9-tetrahydrocannabinol (THC). The ability to accurately and reliably detect cocaine was dependent on the individual device.
In still another study, blood alcohol concentration (BAC) was measured by using both AST (QED A350; STC Technologies, Bethlehem, PA) and blood serum levels in 100 trauma patients admitted to the emergency department of an urban hospital.10 All 41 patients who tested positive for BAC on AST also tested positive on serum. Of the remaining 61 patients, 59 tested negative on both tests, while two patients with BACs of <30 mg/dL tested negative on the AST. Blood in the oral cavity did not appear to affect the accuracy of the test.
The detection times of drugs of abuse depend mainly on the dose and sensitivity of the method used and on the preparation and route of administration, the duration of use (acute or chronic), the matrix analyzed, the molecule or metabolite looked for, the pH and concentration of the matrix (urine, saliva), and the variation in metabolic and renal clearance. In general, the detection time is longest in hair, followed by urine, sweat, saliva and blood.
In blood or plasma, most drugs of abuse can be detected at the low nanogram per milliliter level for one or two days. In urine, the detection time of a single dose is 1.5 to four days. Drugs of abuse can be detected in urine of chronic users for approximately one week after last use, and in extreme cases even longer in cocaine and cannabis users. Drugs of abuse can be detected in saliva for five to 48 hours at a low nanogram per milliliter level. The duration of detection of GHB is much shorter. After a single dose of 1 or 2 ng of flunitrazepam, the most sensitive methods can detect 7-aminoflunitrazepam for up to four weeks in urine.11
Drug testing and information presented on eight company Internet sites that sold home drug-testing products that also contained a “parent’s section” were identified.12 Descriptions of products and their prices, as well as recommended indications for testing, consent, collection procedures and follow-up of positive and negative test results were researched. A variety of drug-testing products is available, including breath and saliva tests for alcohol, a multidrug panel hair test, and a host of laboratory and instant urine tests. Prices range from $2.75 for a single alcohol test to $89 for a multidrug combination urine/hair package. A total of 14 indications for home drug-testing were cited; all sites claimed that drug testing was a way to know with certainty whether a child has used drugs. Only one Web site made a clear statement against testing an adolescent against his or her will. Little information was presented on valid specimen collection procedures and the risks of false positive and false negative tests. Only half of the sites recommended that parents consult a professional if a test is positive.
Vaginal Fluid
Analysis of vaginal fluids is also critical in some forensic cases. Its validity for diagnostic purposes is well understood. In one study, a fluid-based, direct-to-vial method of thin-layer gynecologic cytology (ThinPrep Pap Test) was reported to be more effective than the conventional Pap test for the detection of squamous intraepithelial lesions.13 This retrospective analysis evaluated the validity of the findings on the thin-layer method using case material at a large independent laboratory and represented a comparison of performance of both methods over an identical period. Use of ThinPrep showed a greater than 100 percent increase in the detection rate of squamous intraepithelial lesions. A significant decrease was observed in the false negative proportion. A marked improvement (233 percent) occurred in the detection of high-grade squamous intraepithelial lesions in high-risk cases and a decrease in the atypical squamous cells of undetermined significance to squamous intraepithelial lesion ratio from 3.1 to 1.5 in period 2. The article drew the conclusion that ThinPrep is better than the conventional Pap test in detecting squamous intraepithelial lesions and is a superior screening test in detection of precancerous changes of the cervix.13
Annual screening for Chlamydia trachomatis infection is recommended for sexually active women 15 to 25 years old and for women older than 25 if they have a new or multiple sex partners and have not used condoms during the previous three months. Annual screening for cervical abnormalities using the Pap smear has achieved a substantial reduction in morbidity and mortality from cervical cancer. Screening for Neisseria gonorrhoeae infection has likely contributed significantly to the reduction in the rates of gonococcal infection.
One study14 evaluated the preservation of C. trachomatis and N. gonorrhoeae DNAs from ThinPrep liquid media and tested the feasibility of using a clinical specimen of this medium for the detection of cytologic abnormalities, C. trachomatis and N. gonorrhoeae. The study also evaluated ligase chain reaction (LCR) performed on PreservCyt and LCR performed on a cervical specimen. It also compared the LCR performances on culture, PCR on a cervical specimen, urine and a vaginal specimen (a multiple-site infection status standard), and transcription-mediated amplification (for C. trachomatis only) from 255 sexually active adolescent women. The agreement between LCR performed on PreservCyt and LCR from a cervical swab in LCx transport medium was high.14
A fluid-based Pap test has been established to improve sample collection and preparation.15 Its study was the first large-scale investigation in Japan to examine the feasibility of using fluid-based Pap specimens to detect human papillomavirus (HPV) using Hybrid Capture II and polymerase chain reaction (PCR). Three thousand patients were enrolled in the study; results corresponded well with those of conventional Pap smears (97 percent concordance). The sensitivities of cervical neoplasia detection using the fluid-based Pap test (74 percent) and Hybrid Capture II (76) were not significantly different. Among the cervical intraepithelial neoplasia three and squamous cell carcinoma specimens, HPV 16 and HPV 52 were predominantly detected using the PCR method. Although some DNA samples extracted from the fluid-based specimens were degraded, PCR and direct sequencing could be performed without difficulty even after one year of specimen storage.
A rapid PCR-based assay was designed to quantify and type HSV in cervicovaginal lavage (CVL) fluid of subjects attending a Genito-Urinary Medicine (GUM) clinic.16 Vaginal swabs, CVL fluid and venous blood were collected. Quantitative detection of HSV was conducted using real-time PCR with HSV specific primers and SYBR Green I. Fluorogenic TaqMan Minor Groove Binder (MGB) probes designed around a single base mismatch in the HSV DNA polymerase I gene were used to type HSV in a separate reaction. The Kalon test was used to detect anti-HSV-2 IgG antibodies in serum. Seventy consecutive GUM clinic attendees were studied. Twenty-seven subjects (39 percent) had detectable HSV DNA in CVL fluid; HSV-2 alone was detected in 19 (70 percent) subjects, HSV-1 alone was detected in 4 (15 percent) subjects and both HSV types were detected in 4 (15 percent) subjects. Eleven out of 27 subjects (41 percent) with anti-HSV-2 IgG had detectable HSV-2 DNA in CVL fluid. Seven subjects (10 percent) were HIV-positive. Three of seven (42 percent) HIV-infected subjects and two of five subjects with GUD (40 percent) were secreting HSV-2. None of the subjects in whom HSV-1 was detected had GUD. Quantitative real-time PCR and Taqman MGB probes specific for HSV-1 or -2 were used to develop an assay for quantification and typing of HSV. The majority of subjects in which HSV was detected had low levels of CVL fluid HSV, with no detectable HSV-2 antibodies and were asymptomatic.
Using vaginal fluid Gram stain, 129 pregnant women were screened for bacterial vaginosis at 24 to 29 weeks’ gestation.17,18 Two smears were collected from each woman during the same prenatal visit: the first was prepared from a self-obtained vaginal swab and the second from a physician-obtained speculum examination. Vaginal pH was recorded for each swab. When compared with the physician-obtained smear, the ability of the self-obtained Gram stain to diagnose bacterial vaginosis had a sensitivity of 77 percent, specificity of 97 percent, positive predictive value of 71 percent and negative predictive value of 97 percent.
Several ovulation indicators were studied in a university-based natural family planning center.19 Fifteen parous women during 29 ovulatory cycles detected cervicovaginal fluid at the vulva. They self-aspirated their upper vaginal fluid, described it, and kept it for later checking. They also took basal body temperature, collected timed first morning urine samples for estrone and pregnanediol glucuronide enzyme immunoassays, and submitted to serial ovarian transvaginal ultrasound scans. The women perceived ovulation from cervicovaginal fluid at the vulva in 76 percent or 97 percent of cycles on the basis of their visual description of vaginally extracted fluid in 76 percent or 90 percent, which rose to 90 percent or 97 percent for the instructor’s description, and in 76 percent or 86 percent with a rapid drop in glucuronide ratio. Basal body temperature was less precise (71 percent or 79 percent). From the data, the authors concluded that evaluation of cervicovaginal fluid changes is an accurate ovulation indicator. These data should be of help to those couples attempting conception as well as to physicians working with them.
David Plaut is a chemist and statistician in Plano, TX.
References
1. Jecquier A. Is quality assurance in semen analysis still really necessary? A clinician’s viewpoint. Hum Reprod 2005; 20(8):2039-42.
2. Holt W. Debate continued: Is quality assurance in semen analysis still really necessary? A spermatologist’s viewpoint. Hum Reprod 2005;Jul 8.
3. Levine B, Titus JM, Moore K, et al. Use of prostate specific antigen in the identification of semen in postmortem cases. Am J Forensic Med Pathol 2004;25(4):288-90.
4. Khaldi N, Miras A, Botti K, et al. Evaluation of three rapid detection methods for the forensic identification of seminal fluid in rape cases. J Forensic Sci 2004;49(4):749-53.
5. Toennes SW, Kauert GF, Steinmeyer S, et al. Driving under the influence of drugs: Evaluation of analytical data of drugs in oral fluid, serum and urine, and correlation with impairment symptoms. Forensic Sci Int 2005;10:152.
6. Gronholm M, Lillsunde P. A comparison between on-site immunoassay drug-testing devices and laboratory results. Forensic Sci Int 2001; Sept. 15;121(1-2):37-46.
7. Biermann T, Schwarze B, Zedler B, et al. On-site testing of illicit drugs: The use of the drug-testing device “Toxiquick” J Anal Toxicol 2004;Mar;28(2):132-4.
8. Gunnar T, Ariniemi K, Lillsunde P. An analytical procedure for the simultaneous sensitive identification, screening and quantitation. J Mass Spectrom 2005; 40(6):739-53.
9. Crouch DJ, Walsh JM, Flegel R, et al. An evaluation of selected saliva point-of-collection drug-testing devices. J Anal Toxicol 2005; 29(4):244-8.
10. Degutis LC, Rabinovici R, Sabbaj A, et al. The saliva strip test is an accurate method to determine blood alcohol concentration in trauma patients. Acad Emerg Med 2004; 11(8):885-7.
11. Verstraete AG. Detection times of drugs of abuse in blood, urine, and saliva. Ther Drug Monit 2004; 26(2):200-5.
12. Levy S, Van Hook S, Knight J. A review of Internet-based home drug-testing products for parents. Pediatrics 2004;113(4):720-6.
13. Limaye A, Connor AJ, Huang X, et al. Comparative analysis of conventional Papanicolaou tests and a fluid-based thin-layer method. Arch Pathol Lab Med 2003;127(2):200-4.
14. Koumans EH, Black CM, Markowitz LE, et al. Comparison of methods for detection of Chlamydia trachomatis and Neisseria gonorrhoeae using commercially available nucleic acid amplification tests and a liquid pap smear medium. Clin Microbiol 2003;41(4):1507-11.
15. Masumoto N, Fujii T, Ishikawa M, et al. Papanicolaou tests and molecular analyses using new fluid-based specimen collection technology in 3000 Japanese women. Br J Cancer 2003;88(12):1883-8.
16. Aryee EA, Bailey RL, Natividad-Sancho A, et al. Detection, quantification and genotyping of Herpes Simplex Virus in cervicovaginal secretions by real-time PCR: a cross sectional survey. Virol J 2005;2(1):61.
17. Strauss RA, Eucker B, Savitz DA, et al. Diagnosis of bacterial vaginosis from self-obtained vaginal swabs. Infect Dis Obstet Gynecol 2005;13(1):31-5.
18. Aryee EA, Bailey RL, Natividad-Sancho A, et al. Detection, quantification and genotyping of Herpes Simplex Virus in cervicovaginal secretions by real-time PCR: A cross sectional survey. Virol J 2005;2(1):61.
19. Alliende ME, Cabezon C, Figueroa H, et al. Cervicovaginal fluid changes to detect ovulation accurately.
