- Email: [email protected]
Age-related reference intervals for free and total Prostate-specific antigen in a Singaporean population
Pathology (2000 ) 32, pp. 245– 249
AGE-RELATED REFERENCE INTERVALS FOR FREE AND TOTAL PROSTATE-SPECIFIC ANTIGEN IN A SINGAPOREAN POPULATION SHARON SAW
AND
TAR -CHOON AW
Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074
Summary Cancer of the prostate is the sixth most frequently found cancer in Singapore. Prostate-specific antigen (PSA) is the most clinically useful tumour marker available today for the diagnosis and management of prostate cancer. To enhance the value of PSA as a screening test we developed agespecific intervals for our ethnic population. The measurement of free PSA was included in the study to calculate the free:total ratio which enhances the differential diagnosis of prostate cancer from benign prostatic hyperplasia or prostatitis. The total PSA upper limits of 10-year intervals, beginning at 30-years-old, were 1.4, 1.7, 2.3, 4.0, 6.3 and 6.6 mg/l. Free PSA cut-off limits were 0.4, 0.5, 0.5, 1.0, 1.5 and 1.6 mg/l. The free:total ratio of PSA was not age dependent. Abbott AxSym standardised their calibration material for both free and total PSA assays with the Stanford 90:10 reference material. This laboratory has implemented these age-specific reference intervals and are currently following up their pick-up rate in the detection of prostate cancer. Key words: Prostate-specific antigen, reference interval, age, AxSym. Abbreviations: PSA, prostate specific antigen; TPSA, total prostate specific antigen; FPSA, free prostate specific antigen; FTR, free:total ratio; BPH, benign prostatic hyperplasia. Accepted 12 May 2000
INTRODUCTION The rising incidence of prostate cancer is a cause for concern. In Singapore it is now the sixth most frequently found cancer. Prostate-specific antigen ( PSA) is the most clinically useful tumour marker in the diagnosis of prostate cancer. Although PSA is prostate-tissue specific, elevations in PSA levels are not prostate cancer specific, but organ specific, therefore also found in benign prostatic hyperplasia ( BPH) and prostatitis.1,2 It is now well established that the utilisation of total prostate-specific antigen ( TPSA) can be enhanced with the use of age-related reference intervals.3 These intervals will increase the sensitivity of screening younger men and increase the specificity of screening in older men. Of late it has also become more apparent that the reference intervals are also race dependent.3– 11 Diagnostic ability can be further enhanced in distinguishing prostate cancer from BPH by using a reflex testing function for free PSA ( FPSA) and calculating the free:total PSA ratio ( FTR). Patients with BPH have a higher proportion of the free form, while patients with prostate
cancer have more of the complexed form. However, if FPSA is used in patients with TPSA within the normal interval ( below 4.0 mg/l) then the use of FTR makes PSA a more sensitive test. If it is used in the more traditional “grey zone” of 4.0–10.0 mg/l then its use makes PSA more specific.12 This function of reflex testing with FPSA to calculate the FTR could now incorporate the age-specific reference intervals and adjust the lower interval accordingly. This data would require validation to determine if there was an improved pick up rate for prostate cancer. TPSA assays were calibrated against the Hybritech Tandem-R assay, which was taken by default as the “gold standard”. Now many total assays are using the Stanford 90:10 reference material to calibrate their assays, bringing the standardisation process nearer its target.13,14 However, differences in antibodies used ( monoclonal or polyclonal), different antibody avidities, and total assays with or without an equimolar response to the free and a1 -antichymotrypsin bound PSA still leave the process of complete standardisation of the assay a long way off. Free PSA assays are even further from their target with no agreement between manufacturers as to the calibrator to be used. Hence, laboratories involved in PSA testing must be knowlegeable about the assays they perform and that age- and racedependent intervals are also analyser dependent. Thus great care must be taken in adopting suitable reference intervals for specific assays and analysers. Today many manufacturers of immunoassay analysers offer PSA on their menu. We chose the AxSym to establish suitable reference intervals for our population and to implement these intervals into the working laboratory.
METHODS Study population Five hundred and thirteen consecutive healthy adult males were assessed for free and total PSA levels. The sample group consisted of subjects of Chinese, Malay or Indian origin (approximate percent distribution, 78:14:8) which comprise the ethnic Singaporean population. The subjects presented with no evidence of prostate history or current abnormalities that may interfere with normal function. All procedures conformed to the Helsinki Declaration of 1975 and the 1996 revision. Imprecision The total imprecision of free and total PSA assays was measured using pooled serum samples of five or six different concentrations covering the dynamic range. The imprecision analysis was performed on nine occasions over five consecutive days.
ISSN 0031–3025 printed/ISSN 1465– 3931 online/00/040245 – 05 © 2000 Royal College of Pathologists of Australasia
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Assays Free and total PSA AxSym (Abbott Diagnostics, Abbott Park, IL) total PSA and free PSA assays were utilised in this study. Principle Both assays used a murine monoclonal capture and murine monoclonal signal antibody. Anti-PSA-coated microparticles, assay diluent and the patient sample are incubated together in a reaction vessel allowing the formation of an antigen– antibody complex. An aliquot of this mixture is transferred to a matrix cell where the microparticles bind irreversibly to the matrix. Unbound material is removed by washing. Anti-free or anti-total PSA: alkaline phosphatase conjugate is applied to the matrix cell forming an antibody – antigen– antibody complex. Unbound material is removed by washing. The substrate 4-methylumbelliferyl phosphate is added to the complex resulting in the production of a fluorescence which is quantitated by the optical assembly. Calibration The total PSA is referenced against the Stanford 90:10 PSA reference material. It is an equimolar assay meaning that the assay responds equally to both free PSA and PSA-ACT regardless of the altered ratios found in prostate cancer and therefore suitable for use in free/total measurements. PSA complexed with a2 -macroglobulin is not detectable by this system. The free PSA assay is also referenced to the Stanford 90:10 PSA reference material.
Statistical analysis The non-parametric rank and percentile analysis of untransformed data is used to calculate the reference intervals. One-tailed 95th percentile is used for free and total PSA, and a two-tailed 95th percentile is used for the FTR. Linear regression is used for relating PSA result with age. All analysis is performed on a Microsoft Excel spreadsheet .
RESULTS As expected, both total and free PSA increased with age ( Fig. 1). PSA concentration increases with age and obtains a linear correlation coefficient of 0.44 and 0.42 for TPSA and FPSA respectively. The FTR has no relationship with age ( r = 0.03). Table 1 shows the 95th percentile intervals for the subjects in intervals of 10 years. The median value for each age group shows TPSA and FPSA increasing with age. There was no change in the median values for the FTR for the different age groups as well as the total study cohort. The one-tail 95% percentile range for the whole group was 0.0– 3.60 mg/l for TPSA and 0.0–0.80 mg/l for FPSA. The mean age ± SD for the 513 subjects in this study was 54 ± 14 years. Classified by age, the subjects between 30 and 39 years had no cases of a total or free result higher than 3.60 or 0.80 mg/l, respectively; however, using the age-specific intervals outlined in Table 1 three subjects had an elevated
TABLE 1
Age-specific reference intervals for total and free PSA and the free:total ratio in the Singaporean population
Age (years) 30– 39 40– 49 50– 59 60– 69 70–79 80– 89 All
Fig. 1 (A) Serum total prostate-specific antigen concentration as a function of patient age. A scattergram of all 513 subjects including the 95th percentile nomogram. (B) Free prostate-specific antigen concentration as a function of patient age. A scattergram of all 513 subjects including the 95th percentile nomogram. (C) Free:total prostate-specific antigen ratio as a function of patient age. The scattergram includes the 2.5th and 97.5th nomograms to represent the mid-95th percentile range.
n
Total PSA (mg/l)
Median
Free PSA (mg/l)
Median
F:T Ratio
Median
79 135 107 91 73 19 513
0–1.36 0–1.73 0–2.25 0–4.05 0–6.30 0–6.60 0–3.60
0.61 0.74 0.86 1.36 1.68 1.76 0.89
0–0.39 0–0.49 0–0.53 0–1.02 0–1.47 0–1.60 0–0.80
0.14 0.22 0.23 0.33 0.37 0.47 0.25
0.09– 0.48 0.08– 0.56 0.11– 0.60 0.12– 0.45 0.10– 0.47 0.18– 0.45 0.10– 0.55
0.27 0.28 0.29 0.26 0.29 0.27 0.28
247
AGE-RELATED REFERENCE INTERVALS FOR PSA
TABLE 2 The comparison of age-specific reference intervals for TPSA assayed on Abbott IMx in a number of different racial groups conducted by a number of different authors. Total PSA concentration (mg/l) Age range (years)
40– 49 50– 59 60– 69 70–79
Singaporean 9
– 3.5 3.8 6.0
American 7
Japanese 4
American, 5 White
White
Black
2.5 3.5 3.5 3.5
2.0 4.0 4.5 5.5
2.0 3.0 4.0 5.0
total and one subject an elevated FPSA. In the 40–49-years group, four subjects had an elevated total ( seven by agespecific intervals) and one subject had an elevated FPSA ( seven by age-specific intervals). In the 50–59-years group, one subject had an elevated total ( six by age-specific intervals) and no subject had an elevated FPSA ( six by agespecific intervals). In the 60–69-years group, six subjects had an elevated total (five by age-specific intervals) and seven subjects had an elevated FPSA ( five by age-specific intervals). In the 70–79-years group, 12 subjects had an elevated total ( four by age-specific intervals) and 14 subjects had an elevated FPSA ( four by age-specific intervals). In the 80– 89-years group, three subjects had an elevated total ( two by age-specific intervals) and four subjects had an elevated FPSA (one by age-specific intervals). Imprecision studies on AxSym using TPSA concentrations ranging from 0.100 to 30.032 mg/l, obtained a total CV £ 7.97%. FPSA concentrations ranging from 0.049 to 5.075 mg/l, obtained a total CV £ 6.88%. Table 2 shows data from five studies which examined different population cohorts to establish age-specific reference intervals. All these studies used the Abbott IMx to measure PSA. Table 3 includes our own study on AxSym as well as four other studies. All these PSA analyses were conducted on different analysers other than Abbott IMx. The intervals obtained from this study demonstrated that the subjects below the age of 60 years generally had a lower total PSA cut-off than all the other published studies. The 60–69-years group was equivalent to the Japanese4 and European 11 population studies, but lower than that of all other studies except that of Tay9 and Morgan’s7 American white. However, the 70–79-years group cut-off was found
1.5 2.5 4.5 7.5
American 8 White
Black
Latin
Asian
2.3 3.8 5.6 6.9
2.7 4.4 6.7 7.7
2.1 4.3 6.0 6.6
2.0 4.5 5.5 6.8
to be higher than all the studied populations except the American white studies of Oesterling,3 Anderson 5 and all the populations studied by DeAntoni.8
DISCUSSION The choice of PSA assay is very important in the diagnosis and management of prostate cancer. As this comes under the jurisdiction of laboratory medicine and not the ordering clinician it is up to the laboratory director to implement the most accurate and reproducible test available. To date the process of standardising the PSA assay is still actively in progress. Hence when choosing an assay, the method of calibration must be available for reference. In this study both assays are calibrated against the Stamford 90:10,13,15 which is currently the accepted standard for total PSA assays. Free PSA has no single reference standard at the moment. The AxSym total PSA assay uses a monoclonal format and is an equimolar response assay. This should eliminate the problem of falsely elevated results, found in cases with an elevated free fraction, as a result of increased access of the polyclonal antibody in the previous monoclonal– polyclonal format. The IMx, is a frequently published analyser to study PSA uses a monoclonal–polyclonal format. However, details of calibration are not given. The relationship between the new monoclonal TPSA on AxSym and the IMx is unpublished. This study on AxSym brings the number of different analysers having been used to measure PSA to at least six.3– 11 Age-specific reference intervals are now well established in improving the sensitivity and specificity of prostate
TABLE 3 The comparison of age-specific reference intervals in different racial groups, including this study. All analyses were conducted on different analysers other than Abbott IMx. Total PSA concentration (mg/l)
Age range (years) 40– 49 50– 59 60– 69 70–79
Singaporean AxSym
Taiwanese 1 0 Chinese RIACT*
European 11 Enzymunn
American, 3 white Tandem-R
American 6 white Immunofluoro
1.7 2.3 4.0 6.3
1.9 2.4 4.8 5.9
2.0 3.0 4.0 4.5
2.5 3.5 4.5 6.5
2.0 3.0 4.0 5.5
*RIACT, solid-phase two-site immunoradiometric assay (CIS bio international, France).
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SAW AND AW
cancer diagnosis in men.16 The use of the age-specific intervals shown in Table 1 should improve the sensitivity of the assay in the younger subjects, seen as an increase in the number of subjects which should be followed up. The TPSA results of these subjects were outside the ninety-fifth percentile for their age group. A subject in the 40–49-years group with a PSA of 3.0 mg/l would be followed up with a rectal examination and an ultrasound. This follow-up may result in an early diagnosis of prostate cancer, which can be treated and result in a good long-term outcome for the subject. Without these intervals the cancer would have gone undiagnosed. 3 In the older subjects the age-specific reference intervals lessen the number of subjects requiring follow-up. Age increases the prostate volume and secretions,17 thus intervals adjusted for these increases are necessary. This should minimise unnecessary diagnostic procedures in subjects unlikely to benefit in terms of improved time outcome due to their already advanced age, and that, if a cancer is detected it is unlikely to be of little significance within the subjects lifetime.3,17,18 Table 2 is a summary of data from the literature which give age-specific reference intervals in different populations using the Abbott IMx. The three studies of American white populations have a cut-off ranging from 3.5 to 6.9 and 7.5 mg/l.5,7,8 Which intervals are correct and suitable for adoption by a laboratory using the IMx? Differences found between the populations in Table 3 are not unexpected. The analysers are different, therefore the antibodies are varied with calibration information lacking. These studies indicate that differences in analysers will result in differences in results as well as the differences observed in various populations. Therefore, in principle no direct comparison can be made between the populations without taking the analyser and the assay specifications into consideration. Differences are also found in the methods used by the authors of these studies in calculating their reference intervals; either rank and 95th percentile of untransformed or log-transformed data, or 95% confidence interval. In 1994 a similar study was conducted using the ethnic Singaporean population as subjects. This study obtained the reference intervals of: 50–59 years, 3.51 mg/l; 60–69 years, 3.78 mg/l; 70–79 years, 6.02 mg/l.9 The 50s group range is high compared to the current study, whereas the 60s group is lower than any of the published intervals, while the 70s group is slightly lower than the present study. The important point to note is that the study was conducted on IMx, which makes the ranges suitable for other analyses on the IMx, but not other analysers. The study by Morgan et al. 7 included black and white subjects with the white subjects with the age range of 50–79 having a PSA cut-off of 3.5 mg/l. This is almost half the result obtained by Oesterling3 in his 70–79-years study group. Morgan’s study used the IMx for PSA analysis whereas the Oesterling study was conducted using Tandem-R. The age-specific reference intervals should be incorporated into the reflex testing of free PSA to obtain the free:total PSA ratio. However, the lack of consensus on standardisation for the free PSA assay makes the establishment of FTR cut-off levels for distinguishing cancer from normal or BPH difficult. The levels of free PSA varies between manufacturers due to different standardisation protocols. This inhibits the transfer of the cut-off levels of the FTR between analysers.
At the moment we are very much at the mercy of the manufacturers when standardisation of the PSA assay is still underway. Currently, laboratories should adopt analyserspecific and age- specific reference intervals to suit their own population until such a time when the standardisation process is complete and all manufacturers have complied with the requirements. Then population studies should be comparable between analysers. Ultimately, it is at the discretion of the attending clinician whether to act according to the age-specific intervals or to base the patient follow-up on the “universal” cut-off of 4.0 mg/l for TPSA.19 It is unclear how knowlegeable clinicians are regarding differences in the PSA assays or the issue of standardisation, in particular the non-specialists. Hence, the responsibility of the laboratory in establishing appropriate intervals for the analyser in use and the ethnic population and to be actively involved in their implementation is critical. The study of El-Galley et al. 20 confirmed that age-specific PSA intervals should be used in the early detection of cancer, with their goal being to increase the sensitivity in younger men who benefit more from treatment and decrease the biopsy rate in older subjects who would not benefit significantly from aggressive treatment. In conclusion, this laboratory has implemented the reference intervals for total PSA obtained in this preliminary study into our daily work. The laboratory will continue to monitor the use of these intervals and their outcome in our screening programme for the detection of prostate cancer. Address for Correspondence: S.S., Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074. Email [email protected] g
References 1. Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine E. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. New Engl J Med 1987; 317: 909–16. 2. Oesterling JE. Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. J Urol 1991; 145: 907– 23. 3. Oesterling JE, Jacobsen SJ, Chute CG, Guess HA, Girman CJ, Panser LA, Lieber MM. Serum prostate-specific antigen in a communitybased population of healthy men: Establishment of age-specific reference ranges. J Am Med Assoc 1993; 270: 860–4. 4. Oesterling JE, Kumamoto Y, Tsukamoto T, Girman CJ, Guess HA, Masumori, et al. Serum prostate-specific antigen in a communitybased population of healthy Japanese men: lower values than for similarly aged white men. Br J Urol 1995; 75: 347– 53. 5. Anderson JR, Strickland D, Corbin D, Byrnes JA, Zweiback E. Agespecific reference ranges for serum prostate-specific antigen. Urology 1995; 46: 54–7. 6. Oesterling JE, Jacobsen SJ, Klee GG, Pettersson K, Piironen T, Abrahamsson P-A, et al. Free, complexed and total serum prostate specific antigen: the establishment of appropriate reference ranges for their concentrations and ratios. J Urol 1995; 154: 1090– 5. 7. Morgan TO, Jacobsen SJ, McCarthy WF, Jacobson DJ, McLeod DG, Moul JW. Age-specific reference ranges for serum prostate-specific antigen in black men. New Eng J Med 1996; 335: 304–10. 8. DeAntoni EP, Crawford ED, Oesterling JE, Ross CA, Berger ER, McLeod DG, et al. Age- and race-specific reference ranges for prostate-specific antigen from a large community-based study. Urology 1996; 48: 234–9. 9. Tay KP, Chin CM, Lim PH, Chng HC. Prostate screening –the Singapore experience. Int J Urol 1996; 3: 102–7. 10. Chia HK. Age-related free PSA, total PSA and free PSA/total PSA ratios: establishment of reference ranges in Chinese males. Anticancer Res 1997; 17: 1361– 6. 11. Boeringer Mannheim. Interpretation of total PSA. Distribution of PSA in a reference group. Elecsys PSA handout.
AGE-RELATED REFERENCE INTERVALS FOR PSA
12. Vashi AR, Oesterling JE. Percent free prostate-specific antigen: Entering a new era in the detection of prostate cancer. Mayo Clin Proc 1997; 72: 337–44. 13. Stamey TA. Second Stanford conference on international standardization of prostate-specific antigen immunoassays: September 1 and 2, 1994. Urology 1995; 45: 173–84. 14. Stamey TA. Progress in standardization of immunoassays for prostatespecific antigen. Urol Clin North Am 1997; 24: 269–73. 15. National Committee for Clinical Laboratory Standards. Primary reference preparations for standardization and calibration of immunochemical assays for serum prostate specific antigen (PSA): Approved guidelines. NCCLS Document I/LA 19-A. Wayne, PA:NCCLS, 1997. 16. Henderson RJ, Eastham JA, Culkin DJ, Kattan MW, Whatley T, Mata J, et al. Prostate-specific antigen (PSA) and PSA density: racial differences in men without prostate cancer. J Natl Cancer Inst 1997; 89: 134– 8.
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17. Babaian RJ, Miyashita H, Evans RB, Ramirez EI. The distribution of prostate specific antigen in mwn without clinical or pathological evidence of prostate cancer: relationship to gland volume and age. J Urol 1992; 147: 837–40. 18. Catalona WJ, Smith DS, Wolfert RL, Wang TJ, Rittenhouse HG, Ratliff TL, Nadler RB. Evaluation of percentage of free serum prostate-specific antigen to improve specificity of prostate cancer screening. J Am Med Assoc 1995; 274: 1214–20. 19. Catalona WJ, Richie JP, Ahmann FR, Hudson MA, Scardino PT, Flanigan RC, et al. Effect of patient age on early detection of prostate cancer with serum prostate-specific antigen in the early detection of prostate cancer: Results of a multicenter clinical trial of 6,630 men. J Urol 1994; 151: 1283– 90. 20. El-Galley RES, Petros JA, Sanders WH, Keane TE, Galloway NTM, Cooner WH, Graham SD Jr. Normal range prostate-specific antigen versus age-specific prostate-specific antigen in screening prostate adenocarcinoma. Urology 1995; 46: 200–4.
AGE-RELATED REFERENCE INTERVALS FOR FREE AND TOTAL PROSTATE-SPECIFIC ANTIGEN IN A SINGAPOREAN POPULATION SHARON SAW
AND
TAR -CHOON AW
Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074
Summary Cancer of the prostate is the sixth most frequently found cancer in Singapore. Prostate-specific antigen (PSA) is the most clinically useful tumour marker available today for the diagnosis and management of prostate cancer. To enhance the value of PSA as a screening test we developed agespecific intervals for our ethnic population. The measurement of free PSA was included in the study to calculate the free:total ratio which enhances the differential diagnosis of prostate cancer from benign prostatic hyperplasia or prostatitis. The total PSA upper limits of 10-year intervals, beginning at 30-years-old, were 1.4, 1.7, 2.3, 4.0, 6.3 and 6.6 mg/l. Free PSA cut-off limits were 0.4, 0.5, 0.5, 1.0, 1.5 and 1.6 mg/l. The free:total ratio of PSA was not age dependent. Abbott AxSym standardised their calibration material for both free and total PSA assays with the Stanford 90:10 reference material. This laboratory has implemented these age-specific reference intervals and are currently following up their pick-up rate in the detection of prostate cancer. Key words: Prostate-specific antigen, reference interval, age, AxSym. Abbreviations: PSA, prostate specific antigen; TPSA, total prostate specific antigen; FPSA, free prostate specific antigen; FTR, free:total ratio; BPH, benign prostatic hyperplasia. Accepted 12 May 2000
INTRODUCTION The rising incidence of prostate cancer is a cause for concern. In Singapore it is now the sixth most frequently found cancer. Prostate-specific antigen ( PSA) is the most clinically useful tumour marker in the diagnosis of prostate cancer. Although PSA is prostate-tissue specific, elevations in PSA levels are not prostate cancer specific, but organ specific, therefore also found in benign prostatic hyperplasia ( BPH) and prostatitis.1,2 It is now well established that the utilisation of total prostate-specific antigen ( TPSA) can be enhanced with the use of age-related reference intervals.3 These intervals will increase the sensitivity of screening younger men and increase the specificity of screening in older men. Of late it has also become more apparent that the reference intervals are also race dependent.3– 11 Diagnostic ability can be further enhanced in distinguishing prostate cancer from BPH by using a reflex testing function for free PSA ( FPSA) and calculating the free:total PSA ratio ( FTR). Patients with BPH have a higher proportion of the free form, while patients with prostate
cancer have more of the complexed form. However, if FPSA is used in patients with TPSA within the normal interval ( below 4.0 mg/l) then the use of FTR makes PSA a more sensitive test. If it is used in the more traditional “grey zone” of 4.0–10.0 mg/l then its use makes PSA more specific.12 This function of reflex testing with FPSA to calculate the FTR could now incorporate the age-specific reference intervals and adjust the lower interval accordingly. This data would require validation to determine if there was an improved pick up rate for prostate cancer. TPSA assays were calibrated against the Hybritech Tandem-R assay, which was taken by default as the “gold standard”. Now many total assays are using the Stanford 90:10 reference material to calibrate their assays, bringing the standardisation process nearer its target.13,14 However, differences in antibodies used ( monoclonal or polyclonal), different antibody avidities, and total assays with or without an equimolar response to the free and a1 -antichymotrypsin bound PSA still leave the process of complete standardisation of the assay a long way off. Free PSA assays are even further from their target with no agreement between manufacturers as to the calibrator to be used. Hence, laboratories involved in PSA testing must be knowlegeable about the assays they perform and that age- and racedependent intervals are also analyser dependent. Thus great care must be taken in adopting suitable reference intervals for specific assays and analysers. Today many manufacturers of immunoassay analysers offer PSA on their menu. We chose the AxSym to establish suitable reference intervals for our population and to implement these intervals into the working laboratory.
METHODS Study population Five hundred and thirteen consecutive healthy adult males were assessed for free and total PSA levels. The sample group consisted of subjects of Chinese, Malay or Indian origin (approximate percent distribution, 78:14:8) which comprise the ethnic Singaporean population. The subjects presented with no evidence of prostate history or current abnormalities that may interfere with normal function. All procedures conformed to the Helsinki Declaration of 1975 and the 1996 revision. Imprecision The total imprecision of free and total PSA assays was measured using pooled serum samples of five or six different concentrations covering the dynamic range. The imprecision analysis was performed on nine occasions over five consecutive days.
ISSN 0031–3025 printed/ISSN 1465– 3931 online/00/040245 – 05 © 2000 Royal College of Pathologists of Australasia
246
Pathology ( 2000 ), 32, November
SAW AND AW
Assays Free and total PSA AxSym (Abbott Diagnostics, Abbott Park, IL) total PSA and free PSA assays were utilised in this study. Principle Both assays used a murine monoclonal capture and murine monoclonal signal antibody. Anti-PSA-coated microparticles, assay diluent and the patient sample are incubated together in a reaction vessel allowing the formation of an antigen– antibody complex. An aliquot of this mixture is transferred to a matrix cell where the microparticles bind irreversibly to the matrix. Unbound material is removed by washing. Anti-free or anti-total PSA: alkaline phosphatase conjugate is applied to the matrix cell forming an antibody – antigen– antibody complex. Unbound material is removed by washing. The substrate 4-methylumbelliferyl phosphate is added to the complex resulting in the production of a fluorescence which is quantitated by the optical assembly. Calibration The total PSA is referenced against the Stanford 90:10 PSA reference material. It is an equimolar assay meaning that the assay responds equally to both free PSA and PSA-ACT regardless of the altered ratios found in prostate cancer and therefore suitable for use in free/total measurements. PSA complexed with a2 -macroglobulin is not detectable by this system. The free PSA assay is also referenced to the Stanford 90:10 PSA reference material.
Statistical analysis The non-parametric rank and percentile analysis of untransformed data is used to calculate the reference intervals. One-tailed 95th percentile is used for free and total PSA, and a two-tailed 95th percentile is used for the FTR. Linear regression is used for relating PSA result with age. All analysis is performed on a Microsoft Excel spreadsheet .
RESULTS As expected, both total and free PSA increased with age ( Fig. 1). PSA concentration increases with age and obtains a linear correlation coefficient of 0.44 and 0.42 for TPSA and FPSA respectively. The FTR has no relationship with age ( r = 0.03). Table 1 shows the 95th percentile intervals for the subjects in intervals of 10 years. The median value for each age group shows TPSA and FPSA increasing with age. There was no change in the median values for the FTR for the different age groups as well as the total study cohort. The one-tail 95% percentile range for the whole group was 0.0– 3.60 mg/l for TPSA and 0.0–0.80 mg/l for FPSA. The mean age ± SD for the 513 subjects in this study was 54 ± 14 years. Classified by age, the subjects between 30 and 39 years had no cases of a total or free result higher than 3.60 or 0.80 mg/l, respectively; however, using the age-specific intervals outlined in Table 1 three subjects had an elevated
TABLE 1
Age-specific reference intervals for total and free PSA and the free:total ratio in the Singaporean population
Age (years) 30– 39 40– 49 50– 59 60– 69 70–79 80– 89 All
Fig. 1 (A) Serum total prostate-specific antigen concentration as a function of patient age. A scattergram of all 513 subjects including the 95th percentile nomogram. (B) Free prostate-specific antigen concentration as a function of patient age. A scattergram of all 513 subjects including the 95th percentile nomogram. (C) Free:total prostate-specific antigen ratio as a function of patient age. The scattergram includes the 2.5th and 97.5th nomograms to represent the mid-95th percentile range.
n
Total PSA (mg/l)
Median
Free PSA (mg/l)
Median
F:T Ratio
Median
79 135 107 91 73 19 513
0–1.36 0–1.73 0–2.25 0–4.05 0–6.30 0–6.60 0–3.60
0.61 0.74 0.86 1.36 1.68 1.76 0.89
0–0.39 0–0.49 0–0.53 0–1.02 0–1.47 0–1.60 0–0.80
0.14 0.22 0.23 0.33 0.37 0.47 0.25
0.09– 0.48 0.08– 0.56 0.11– 0.60 0.12– 0.45 0.10– 0.47 0.18– 0.45 0.10– 0.55
0.27 0.28 0.29 0.26 0.29 0.27 0.28
247
AGE-RELATED REFERENCE INTERVALS FOR PSA
TABLE 2 The comparison of age-specific reference intervals for TPSA assayed on Abbott IMx in a number of different racial groups conducted by a number of different authors. Total PSA concentration (mg/l) Age range (years)
40– 49 50– 59 60– 69 70–79
Singaporean 9
– 3.5 3.8 6.0
American 7
Japanese 4
American, 5 White
White
Black
2.5 3.5 3.5 3.5
2.0 4.0 4.5 5.5
2.0 3.0 4.0 5.0
total and one subject an elevated FPSA. In the 40–49-years group, four subjects had an elevated total ( seven by agespecific intervals) and one subject had an elevated FPSA ( seven by age-specific intervals). In the 50–59-years group, one subject had an elevated total ( six by age-specific intervals) and no subject had an elevated FPSA ( six by agespecific intervals). In the 60–69-years group, six subjects had an elevated total (five by age-specific intervals) and seven subjects had an elevated FPSA ( five by age-specific intervals). In the 70–79-years group, 12 subjects had an elevated total ( four by age-specific intervals) and 14 subjects had an elevated FPSA ( four by age-specific intervals). In the 80– 89-years group, three subjects had an elevated total ( two by age-specific intervals) and four subjects had an elevated FPSA (one by age-specific intervals). Imprecision studies on AxSym using TPSA concentrations ranging from 0.100 to 30.032 mg/l, obtained a total CV £ 7.97%. FPSA concentrations ranging from 0.049 to 5.075 mg/l, obtained a total CV £ 6.88%. Table 2 shows data from five studies which examined different population cohorts to establish age-specific reference intervals. All these studies used the Abbott IMx to measure PSA. Table 3 includes our own study on AxSym as well as four other studies. All these PSA analyses were conducted on different analysers other than Abbott IMx. The intervals obtained from this study demonstrated that the subjects below the age of 60 years generally had a lower total PSA cut-off than all the other published studies. The 60–69-years group was equivalent to the Japanese4 and European 11 population studies, but lower than that of all other studies except that of Tay9 and Morgan’s7 American white. However, the 70–79-years group cut-off was found
1.5 2.5 4.5 7.5
American 8 White
Black
Latin
Asian
2.3 3.8 5.6 6.9
2.7 4.4 6.7 7.7
2.1 4.3 6.0 6.6
2.0 4.5 5.5 6.8
to be higher than all the studied populations except the American white studies of Oesterling,3 Anderson 5 and all the populations studied by DeAntoni.8
DISCUSSION The choice of PSA assay is very important in the diagnosis and management of prostate cancer. As this comes under the jurisdiction of laboratory medicine and not the ordering clinician it is up to the laboratory director to implement the most accurate and reproducible test available. To date the process of standardising the PSA assay is still actively in progress. Hence when choosing an assay, the method of calibration must be available for reference. In this study both assays are calibrated against the Stamford 90:10,13,15 which is currently the accepted standard for total PSA assays. Free PSA has no single reference standard at the moment. The AxSym total PSA assay uses a monoclonal format and is an equimolar response assay. This should eliminate the problem of falsely elevated results, found in cases with an elevated free fraction, as a result of increased access of the polyclonal antibody in the previous monoclonal– polyclonal format. The IMx, is a frequently published analyser to study PSA uses a monoclonal–polyclonal format. However, details of calibration are not given. The relationship between the new monoclonal TPSA on AxSym and the IMx is unpublished. This study on AxSym brings the number of different analysers having been used to measure PSA to at least six.3– 11 Age-specific reference intervals are now well established in improving the sensitivity and specificity of prostate
TABLE 3 The comparison of age-specific reference intervals in different racial groups, including this study. All analyses were conducted on different analysers other than Abbott IMx. Total PSA concentration (mg/l)
Age range (years) 40– 49 50– 59 60– 69 70–79
Singaporean AxSym
Taiwanese 1 0 Chinese RIACT*
European 11 Enzymunn
American, 3 white Tandem-R
American 6 white Immunofluoro
1.7 2.3 4.0 6.3
1.9 2.4 4.8 5.9
2.0 3.0 4.0 4.5
2.5 3.5 4.5 6.5
2.0 3.0 4.0 5.5
*RIACT, solid-phase two-site immunoradiometric assay (CIS bio international, France).
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Pathology ( 2000 ), 32, November
SAW AND AW
cancer diagnosis in men.16 The use of the age-specific intervals shown in Table 1 should improve the sensitivity of the assay in the younger subjects, seen as an increase in the number of subjects which should be followed up. The TPSA results of these subjects were outside the ninety-fifth percentile for their age group. A subject in the 40–49-years group with a PSA of 3.0 mg/l would be followed up with a rectal examination and an ultrasound. This follow-up may result in an early diagnosis of prostate cancer, which can be treated and result in a good long-term outcome for the subject. Without these intervals the cancer would have gone undiagnosed. 3 In the older subjects the age-specific reference intervals lessen the number of subjects requiring follow-up. Age increases the prostate volume and secretions,17 thus intervals adjusted for these increases are necessary. This should minimise unnecessary diagnostic procedures in subjects unlikely to benefit in terms of improved time outcome due to their already advanced age, and that, if a cancer is detected it is unlikely to be of little significance within the subjects lifetime.3,17,18 Table 2 is a summary of data from the literature which give age-specific reference intervals in different populations using the Abbott IMx. The three studies of American white populations have a cut-off ranging from 3.5 to 6.9 and 7.5 mg/l.5,7,8 Which intervals are correct and suitable for adoption by a laboratory using the IMx? Differences found between the populations in Table 3 are not unexpected. The analysers are different, therefore the antibodies are varied with calibration information lacking. These studies indicate that differences in analysers will result in differences in results as well as the differences observed in various populations. Therefore, in principle no direct comparison can be made between the populations without taking the analyser and the assay specifications into consideration. Differences are also found in the methods used by the authors of these studies in calculating their reference intervals; either rank and 95th percentile of untransformed or log-transformed data, or 95% confidence interval. In 1994 a similar study was conducted using the ethnic Singaporean population as subjects. This study obtained the reference intervals of: 50–59 years, 3.51 mg/l; 60–69 years, 3.78 mg/l; 70–79 years, 6.02 mg/l.9 The 50s group range is high compared to the current study, whereas the 60s group is lower than any of the published intervals, while the 70s group is slightly lower than the present study. The important point to note is that the study was conducted on IMx, which makes the ranges suitable for other analyses on the IMx, but not other analysers. The study by Morgan et al. 7 included black and white subjects with the white subjects with the age range of 50–79 having a PSA cut-off of 3.5 mg/l. This is almost half the result obtained by Oesterling3 in his 70–79-years study group. Morgan’s study used the IMx for PSA analysis whereas the Oesterling study was conducted using Tandem-R. The age-specific reference intervals should be incorporated into the reflex testing of free PSA to obtain the free:total PSA ratio. However, the lack of consensus on standardisation for the free PSA assay makes the establishment of FTR cut-off levels for distinguishing cancer from normal or BPH difficult. The levels of free PSA varies between manufacturers due to different standardisation protocols. This inhibits the transfer of the cut-off levels of the FTR between analysers.
At the moment we are very much at the mercy of the manufacturers when standardisation of the PSA assay is still underway. Currently, laboratories should adopt analyserspecific and age- specific reference intervals to suit their own population until such a time when the standardisation process is complete and all manufacturers have complied with the requirements. Then population studies should be comparable between analysers. Ultimately, it is at the discretion of the attending clinician whether to act according to the age-specific intervals or to base the patient follow-up on the “universal” cut-off of 4.0 mg/l for TPSA.19 It is unclear how knowlegeable clinicians are regarding differences in the PSA assays or the issue of standardisation, in particular the non-specialists. Hence, the responsibility of the laboratory in establishing appropriate intervals for the analyser in use and the ethnic population and to be actively involved in their implementation is critical. The study of El-Galley et al. 20 confirmed that age-specific PSA intervals should be used in the early detection of cancer, with their goal being to increase the sensitivity in younger men who benefit more from treatment and decrease the biopsy rate in older subjects who would not benefit significantly from aggressive treatment. In conclusion, this laboratory has implemented the reference intervals for total PSA obtained in this preliminary study into our daily work. The laboratory will continue to monitor the use of these intervals and their outcome in our screening programme for the detection of prostate cancer. Address for Correspondence: S.S., Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074. Email [email protected] g
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