Measurement of Prostate-specific Antigen by Use of a ... - CiteSeerX

Clinical Chemistry 48:8 1272–1278 (2002)

Cancer Diagnostics: Discovery and Clinical Applications

Measurement of Prostate-specific Antigen by Use of a Novel Blood Collection and Analytical System Barbara R. Grzeda,* Tuan Le Bui, Cheryl N. Warner, Tracy L. Pirucki, Lisa M. Dewey, Milan Babich, and Jack A. Maggiore Background: Prostate-specific antigen (PSA) is widely used in the detection and monitoring of prostate cancer. We developed a system for the self-collection and transport of capillary whole blood for PSA analysis, with the goal of reducing phlebotomy visits and, thus, increasing the access and utilization of PSA in prostate cancer screening and monitoring. Methods: The blood collection device [BIOSAFE Blood Transport System (BTSTM)] collects 70 ␮L of blood through a heparin-coated material into 200 ␮L of stabilizing solution. The diluted whole blood is used for measurement of PSA by a modified version of the Hybritech® Tandem-MP PSA Assay. Results were compared for matched samples of professionally and selfcollected BTS blood and for matched BTS samples sera from blood collected by venipuncture. Imprecision for the whole-blood PSA measurement was estimated from analysis of whole-blood controls in duplicate, twice per day, over 20 days. Results: BTS samples (n ⴝ 140) collected by a qualified healthcare professional compared with serum samples yielded the regression equation: y ⴝ1.02x ⴙ 0.04 (Sy円x ⴝ 0.35; r ⴝ 0.99). Comparison of the results for samples (n ⴝ 128) collected by the patient without professional assistance with serum samples yielded: y ⴝ 1.08x ⴙ 0.02 (Sy円x ⴝ 0.31; r ⴝ 0.99). The between-run CVs at 0.069, 0.53, 2.9, and 10.7 ␮g/L were 21%, 6.0%, 3.5%, and 3.8%, respectively. PSA was stable in BTS samples stored for 21 days at 18 –24 °C and for 7 days at 37 °C. Conclusion: The BIOSAFE BTS system allows accurate and convenient measurement of circulating PSA by a precise method for diluted whole blood. © 2002 American Association for Clinical Chemistry

BIOSAFE Laboratories, Inc., 8600 W. Catalpa Ave., Chicago, IL 60656. *Author for correspondence. Fax 773-693-0410; e-mail bgrzeda@ ebiosafe.com. Received January 28, 2002; accepted May 21, 2002.

The prostate is the leading site for cancer incidence, accounting for 31.0% of new cancer cases in men (1 ). The current detection system for prostate cancer involves both a digital rectal exam and prostate-specific antigen (PSA) screening. Although the prevalence of prostate cancer has increased over the past two decades, the survival rates have dramatically improved; the increase in PSA screening has been offered as an explanation for both these occurrences. Prostate cancers are detected more frequently when men are screened for PSA, but generally cases are detected earlier when curative treatment is available (1– 6 ). Radical prostatectomy is performed for clinically localized tumors, and the success of the treatment is typically monitored by continual PSA measurement checked quarterly the first year and at intervals of 4 – 6 months in succeeding years (7–10 ). PSA monitoring after radical prostatectomy requires an assay that can at least reliably detect PSA concentrations ⬍0.4 ␮g/L (11 ). However, the use of ultrasensitive PSA assays in prostate monitoring has been advocated as a way to improve detection of prostate cancer relapse (12–14 ). The monitoring of PSA is one such setting in which ongoing testing is required, and frequent phlebotomy visits, up to four times a year, can be cumbersome. Currently, prostate cancer screening is available via large-scale mail-in programs using capillary whole blood spotted onto filter-paper cards (15 ). This method of collection enables patients to collect their own capillary blood samples in their homes and receive laboratorydetermined results. However, capillary whole blood collected onto filter-paper cards has not been shown to reliably detect PSA concentrations ⬍0.35 ␮g/L. The monitoring of patients after radical prostatectomy requires a more sensitive assay (12–14 ). To accomplish the goal of developing an at-home self-collection monitoring system for PSA, certain conditions are necessary: ease of use, high assay sensitivity, and accurate results. The BIOSAFE Blood Transport System (BTSTM) was specifically designed with the intention of simplifying blood collection for analytical purposes without compromising the sensi-

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tivity necessary for clinical monitoring. To ensure that a layperson can collect an adequate sample, the BTS contains a built-in control indicator to ensure that a precise amount of blood is collected and that a quality sample is delivered to the laboratory. The BTS provides the means for determining a low PSA concentration by maintaining the sample in a stabilizing solution. Maintaining the whole-blood sample in a liquid state rather than dried on a filter-paper card allows a more precise and larger sample volume to be analyzed. The whole-blood sample is essentially preeluted, and no further dilution of the sample is necessary. The BTS collects a precisely metered amount of capillary whole blood; a handle is then turned by the collector, introducing the metered blood into the stabilizing solution (see Fig. 1). The turning of the handle also seals the sample, now maintained in a stabilizing solution, in the BTS. The BTS can then be shipped to a central laboratory via regular mail for PSA analysis.

Materials and Methods materials The Hybritech Tandem-MP PSA assay was purchased from Beckman Coulter. Sodium azide and polyoxyethylene-(20)-sorbitan monooleate were purchased from EM Science Industries. Hydrochloric acid, bilirubin cali-

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brator (in a bovine albumin base), Lipid Lin-Trol Set®, sodium chloride (ACS grade), flutamide {2-methyl-N-[4nitro-3-(trifluoromethyl)-phenyl]propanamide}, sulfamethoxazole (4-amino-N-[5-methyl-3-isoxazolyl]benzonesulfonamide), doxorubicin hydrochloride, leuprolide, cyclophosphamide (monohydrate), doxycycline, diethylstilbestrol, megestrol acetate (17␣-acetoxy-6-methyl4,6-pregnadiene-3,20-dione), trimethoprim (2,4-diamino5-[2,4,5-trimethoxybenzyl]-pyrimidine), terazosin hydrochloride, methotrexate, paclitaxel (semisynthetic), albumin, water-soluble ␤-estradiol (cyclodextrin-encapsulated), estrone (1,3,5,10-estratrien-3-ol-17-one), luteinizing hormone-releasing hormone (acetate salt), and heparin (sodium salt from porcine intestinal mucosa) were purchased from Sigma Diagnostics. Ciprofloxacin hydrochloride was purchased from Serologicals Proteins, Inc. Tris was purchased from J.T. Baker Chemical Company.

preparation of reagents After collection, capillary whole blood was diluted in the PSA stabilizing solution (patent pending), which consists of deionized water, 50 mmol Tris, and 0.5 g/L sodium azide brought to a pH of 7.2 with hydrochloric acid. The BTS devices are precoated with a solution of polyoxyethylene-(20)-sorbitan monooleate and heparin solution, which is allowed to dry to produce a film that prevents coagulation and promotes sample flow into the metering chamber.

preparation of calibrators and controls

Fig. 1. BIOSAFE BTS. Actual dimensions, 55 ⫻ 26 ⫻ 32 mm. (A), blood is dropped into fill cup from finger nick. (B), view indicator turns red, signaling that sufficient blood has been collected. The BTS handle is rotated counterclockwise, which meters the blood to the dilution reservoir. (C), the handle is locked into the closed position, which seals the diluted blood in the reservoir for transportation to the laboratory. The device is depicted semitransparent to facilitate viewing of the path of blood.

A five-point calibration curve was constructed for the modified PSA assay with calibrators containing 0.0, 0.5, 2.0, 10.0, and 25.0 ␮g/L PSA. Tandem-MP PSA serum assay calibrators, with the exception of the 50 ␮g/L calibrator, were diluted 1:7 (50 ␮L of calibrator plus 300 ␮L of PSA stabilizing solution) with the PSA stabilizing solution to produce calibrators at the same dilution as the whole-blood BTS samples. Liquid controls were prepared by diluting the Hybritech Tandem-MP PSA serum assay controls 1:7 with the PSA stabilizing solution. Modified PSA calibrators and liquid controls were prepared fresh daily. Whole-blood controls were prepared by collecting venous blood samples from a male volunteer in 10-mL Vacutainer® Tubes (Becton Dickinson), three containing SST® gel and clot activator and four containing 143 units of sodium heparin. The SST tubes were allowed to clot, and all tubes were centrifuged at 1300g. The separated serum was decanted for later use. The plasma and buffy coats from the four heparinized tubes were removed and discarded. The remaining red blood cells were manually washed three times with isotonic saline to remove any lingering plasma. The red blood cells were then transferred to plastic freezer vials and placed in a ⫺20 °C freezer for 4 h to permit lysis. After this period, the tubes containing the washed and

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lysed red blood cells were removed from the ⫺20 °C freezer and allowed to thaw at room temperature. The serum that been decanted from the SST tubes was combined with the liquid controls packaged within the Hybritech Tandem-MP PSA assay at differing ratios to produce various clinically relevant PSA concentrations. These serum/liquid control samples were thoroughly mixed, aliquoted, and retained for later testing with the Hybritech Tandem-MP PSA serum assay. The remainder of the sample was combined with the washed and lysed red blood cells to simulate a prepared whole-blood sample with a hematocrit of 45%. This prepared whole-blood sample was combined with the PSA stabilizing solution to produce a 1:7 plasma dilution, assuming a 45% hematocrit, to produce a whole-blood control. These whole-blood controls were aliquoted into freezer vials for storage at ⫺20 °C for up to 1 year.

blood (70 ␮L) into the stabilizing solution (200 ␮L) when the handle is turned, yielding a 1:7 plasma dilution, assuming a 45% hematocrit. According to the study protocol, the patients were asked to follow the written directions for self-collection of capillary whole blood into the BTS without assistance, except for a provided telephone help line to BIOSAFE Customer Support. After each patient completed self-collection, a healthcare professional also collected a sample from the patient volunteer into a BTS. All BTS devices were shipped to BIOSAFE Laboratories, Inc. in specialized leak-proof bags (lowdensity polyethylene; Vonco, Inc.) via first class mail (US Postal Service) and analyzed for PSA in capillary whole blood, using the analytical procedure described below. This study was designed to ensure that no teaching or training occurred before collection by a lay user to better assess the ease of use of the collection device and understanding of labeling and instructions.

populations A three-site clinical trial was performed in hospital-based ambulatory care facilities to assess the clinical feasibility and utility of the BTS Device for PSA analysis. Male volunteers were asked to participate in the study, which included collection of three samples in this order: venous blood, a self-collected capillary blood sample into a BTS, and a professionally collected capillary blood sample into a BTS. The study was approved by the Institutional Review Boards at all three sites in compliance with the Declaration of Helsinki. The exclusion criteria for the study included: females, men ⬍30 or ⬎85 years of age, hemophiliacs, individuals taking any anticoagulant medication, and individuals with medical conditions that at the discretion of the Study Director may limit their ability to participate in the study. Samples of prostate cancer patients (11.5%), patients who had benign prostatic disease (9.5%), and healthy controls (78.4%) were sought and consented before participation. There were a total of 148 participants, ranging in age from 30 to 83 years, with a median age of 56. Volunteers were also asked to complete a short survey comparing the methods of collection. All samples were analyzed at BIOSAFE Laboratories, Inc., using the Hybritech Tandem-MP PSA assay in either its serum format or in a format modified for use with whole blood. The survey results were tabulated to evaluate patient performance and preference.

samples Venous whole-blood samples were collected while patients were seated. Venous blood samples were collected in 10-mL SST Vacutainer Tubes. The serum was separated rapidly by centrifugation for 10 min at 1300g and analyzed for PSA with the Hybritech Tandem-MP PSA serum assay within 24 h of collection. A SurgiLanceTM Lancet (SurgiLance Pte Ltd) was used to puncture the finger to facilitate capillary blood flow. Capillary whole blood was collected into the BIOSAFE BTS. The BTS delivers a metered amount of capillary

analytical procedure The sample reservoir, which stores the blood and stabilizing solution in transit, was punched with a handheld stylus. A funnel molded to the dimensions of the sample reservoir was attached to the sample reservoir after it had been punctured, and the end of the funnel was placed in a test tube where the sample was collected. Capillary whole-blood samples were removed from the BIOSAFE BTS Device by centrifugation for 5 min at 1300g into a 12 ⫻ 75 mm test tube. Cellular debris and unlysed cells do not interfere with the whole-blood PSA protocol. Therefore, to ensure that the proper plasma dilution was met, the samples were vortex-mixed for at least 5 s after centrifugation. A modified version of the Hybritech Tandem-MP PSA serum assay was used to analyze these capillary whole-blood specimens as described below.

whole-blood psa protocol

Test strips and holders were labeled, and 75 ␮L of PSA calibrators, controls, and patient samples were pipetted into corresponding microplate wells, in duplicate. To each well was added 75 ␮L of assay conjugate containing two PSA-specific monoclonal antibodies, one enzyme-labeled and the other biotin-labeled. The microplate was shaken at room temperature for 1 h at 700 rpm and then was washed six times with prepared Hybritech Tandem-MP PSA wash concentrate in a microplate autowasher (BIOTEK Instruments) to remove any unbound antibody. All excess wash solution was removed by blotting. One hundred microliters of the Hybritech Tandem-MP PSA serum assay substrate, p-nitrophenyl phosphate in a stabilizing buffer containing a preservative, prepared according to the manufacturer’s protocol, was added to each well. The microplate was incubated at room temperature with rotation at 700 rpm for 1 h while the substrate reacted with the bound biotin; the substrate/antibody reaction was then quenched with 100 ␮L of EDTA in a buffer containing a preservative. The microplate was read

Clinical Chemistry 48, No. 8, 2002

in a spectrophotometer (MRX Revelation; Dynex Technologies) at 405– 650 nm. The amount of substrate turnover was directly proportional to the amount of total PSA in the capillary whole-blood sample. All serum specimens were analyzed using the standard Hybritech Tandem-MP PSA serum assay with a doubleblind approach to ensure the validity of the study.

statistical analysis A total of 140 professionally collected BTS samples and 128 self-collected BTS samples were analyzed by the modified version of the Hybritech Tandem-MP PSA assay, and the corresponding serum samples were analyzed using the comparable serum methodology. All methods were compared by linear regression analysis using Analyze-It® Statistical Software, Ver. 1.44.

method validation Precision. Within-run and between-day variance was determined by analyzing four different whole-blood control samples (0.069, 0.53, 2.9, and 10.7 ␮g/L) in duplicate over 20 days, two analytical runs per day, with a minimum of 2 h between runs. BIOSAFE BTS precision. Device precision was determined by filling 20 BIOSAFE PSA BTS devices with venous whole blood collected from the same male patient and analyzed for PSA within the same analytical run. Linearity. The linearity of the capillary whole-blood assay was determined by assaying prepared whole-blood samples, as prepared below. A serum sample (0.7 ␮g/L) was combined with the Hybritech Tandem-MP PSA assay high control (37.0 ␮g/L) in a 1:15 ratio (200 ␮L of the low-concentration specimen plus 2800 ␮L of the high-concentration specimen). This enriched sample was serially diluted with the original serum. Portions of the prepared enriched serum samples were retained and analyzed using the Hybritech Tandem-MP PSA serum assay for purposes of comparison. The remainder of the enriched samples were combined with washed and lysed heparinized red blood cells (prepared as described above) to simulate a hematocrit of 45%. These whole-blood samples were then diluted with the PSA stabilizing solution in an equivalent ratio to samples collected in the BTS device. Solutions with expected values of 0.14, 0.18, 0.26, 0.46, 1.1, 2.3, 4.7, 8.6, and 16.7 ␮g/L were analyzed using the whole-blood PSA protocol, and linearity results (observed vs expected) were graphed (not shown). Recovery. The low-concentration heparinized plasma sample (⬍1.0 ␮g/L) was prepared undiluted or combined with a heparinized plasma sample with a high PSA concentration (45.0 ␮g/L) in serial dilutions (1:2, 1:4, 1:8, 1:16, and 1:32). An aliquot of each dilution was analyzed using the Hybritech Tandem-MP PSA serum assay. The

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remainder of each dilution was combined with washed, lysed heparinized red blood cells (prepared as described above) to simulate a hematocrit of 45%. These prepared samples were then diluted with PSA stabilizing solution to replicate the dilution made in the BTS and analyzed using the whole-blood PSA protocol. The percentage of recovery was calculated by dividing the observed PSA concentrations by the expected concentrations and multiplying by 100. Interference. The influence of the interfering substances hemoglobin, bilirubin, triglycerides, and albumin in the matrix on the PSA whole-blood assay was assessed. Interference by chemotherapeutic agent was also evaluated, using cyclophosphamide, diethylstilbestrol, watersoluble ␤-estradiol, estrone, doxorubicin hydrochloride, methotrexate, megestrol acetate, and paclitaxel. The effects of the following antibiotics were evaluated: ciprofloxacin hydrochloride, sulfamethoxazole, trimethoprim, and doxycycline, as were luteinizing hormone-releasing hormone and its agonists leuprolide and flutamide, and the alpha-blocker terazosin hydrochloride. PSA quantification in whole blood was evaluated by analyzing samples to which various concentrations of these interferents had been added. The interference study samples for all but hemoglobin were prepared by collecting venous blood samples, obtaining the serum, and combining it with washed and lysed heparinized red blood cells (prepared as described above) to simulate a prepared whole-blood sample with a hematocrit of 45%. The prepared whole-blood samples were diluted with PSA stabilizing solution to replicate the dilution made in the BTS and analyzed using the wholeblood PSA protocol. For hemoglobin, washed and lysed heparinized red blood cells were added to the serum sample in simulated hematocrits of 0 – 80%. Detection limit. The detection limit of the PSA whole-blood assay was determined by assaying 20 replicates of the 0.0 ␮g/L calibrator from the Hybritech Tandem-MP PSA assay to which washed, lysed heparinized red blood cells (prepared as described above) were added to simulate a 45% hematocrit. The prepared sample was pipetted into 20 BTS devices and analyzed within the same analytical run, using the whole-blood PSA protocol. The minimum detectable concentration was determined based on the mean absorbance of the 20 replicates, plus 2 SD, and read from the calibration curve for that given run. Stability. PSA stability in the BTS was assessed using venous whole-blood samples collected from a male patient into a 10-mL evacuated tube and immediately pipetted into the BTS, before clotting occurred. Three patient samples, containing 0.65, 4.5, and 10.4 ␮g/L PSA, were used. The devices were closed and stored at room temperature (18 –24 °C) and 37 °C. Three devices for each PSA

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concentration were placed in a ⫺20 °C freezer immediately after collection; these samples provided baseline values. Each BTS device, one device per time limit and temperature, was placed in the ⫺20 °C freezer after storage at its respective temperature for the allotted time period (0 –21 days ⫾ 6 h). At completion of the stability study, all BTS stability devices were opened and assayed in duplicate on the same day, using the modified wholeblood PSA assay. For samples to be considered stable, the PSA values from each storage temperature were compared with their respective baseline values. These differences were used to calculate the slope of the regression line (change in PSA value divided by change in time in days) and compared with a zero slope by t-test to identify significant deviations from a zero change.

Results comparison of methods Linear regression analysis was performed on all paired serum and BTS samples. Twelve samples were unsuccessfully self-collected by patients, producing a slight disparity between the number of professionally collected and self-collected specimens. Comparison of serum to professionally collected BTS samples (n ⫽ 140) produced a linear regression equation of y ⫽ 1.02x ⫹ 0.04 (Sy兩x ⫽ 0.35), with a correlation coefficient (r) value of 0.99 (see Fig. 2A). The mean difference between the two methods was ⫺0.10 ␮g/L with a SD of 0.37 ␮g/L. When linear regression data for values ⬍1.0 ␮g/L were examined, which is the primary range of interest for post-radical prostatectomy monitoring, the Sy兩x was 0.14 (n ⫽ 81). Comparison of serum to self-collected BTS samples (n ⫽ 128) produced a linear regression equation of y ⫽ 1.08x ⫹ 0.02 (Sy兩x ⫽ 0.31; r ⫽ 0.99; see Fig. 2B). The mean difference between the two methods was ⫺0.14 ␮g/L, with a SD of 0.35 ␮g/L. Again, PSA values ⬍1.0 ␮g/L were analyzed by linear regression and gave a Sy兩x of 0.15 (n ⫽ 77).

precision The within-run SDs for concentration of 0.069, 0.53, 2.9, and 10.7 ␮g/L were 0.01, 0.02, 0.5, and 0.21 ␮g/L, respectively, giving CVs of 14%, 3.4%, 1.7%, and 2.0%. The between-run SDs for the same concentrations were 0.01, 0.03, 0.1, and 0.4 ␮g/L, yielding CVs of 21%, 6.0%, 3.5%, and 3.8%, respectively.

Fig. 2. Serum PSA concentrations, measured in the Tandem-MP serum PSA assay, vs professionally collected BTS whole-blood PSA (A) and self-collected BTS whole-blood PSA (B) concentrations, measured by the whole-blood PSA protocol. All concentrations are in ␮g/L. PC, professionally collected; SC, self-collected. The equations for the lines are: (A), y ⫽ 1.02x ⫹ 0.04 (Sy兩x ⫽ 0.35; n ⫽ 140); (B), y ⫽ 1.08x ⫹ 0.02 (Sy兩x ⫽ 0.31; n ⫽ 128).

recovery Recovery of PSA in whole-blood samples was 97.8 – 100.8% for PSA values between 2.2 and 12.4 ␮g/L.

biosafe bts precision

The imprecision (CV) shown by the BTS device (n ⫽ 20) was ⬍5.5% at a mean PSA concentration of 1.8 ␮g/L.

linearity Whole-blood PSA values were compared with serum values and analyzed by linear regression to give the equation: y ⫽ 1.02x ⫺ 0.15 (r ⫽ 0.99; Sy兩x ⫽ 0.37). The whole-blood PSA assay was linear between 0.14 and 16.7 ␮g/L

interference The hemoglobin interference study showed that wholeblood PSA concentrations varied by ⬍5.0% in a physiologic hematocrit range (25–75%). The mean hematocrit of patients tested in the clinical trial was 45.4%. Whole-blood PSA concentrations varied by no more than 5.5% at triglyceride concentrations of 0.217–9.87 mmol/L (19 – 866 mg/dL). The bilirubin and albumin interference studies

Clinical Chemistry 48, No. 8, 2002

both showed that whole-blood PSA concentrations varied by no more than 5.0% at bilirubin concentrations of 1.71–749.0 ␮mol/L (0.01– 4.38 mg/L) and albumin concentrations of 45–180 g/L (4.5–18.0 g/dL), respectively. Interference data from chemotherapeutic agents, antibiotics, hormonal therapies, and alpha-blockers showed that none of the agents tested yielded a detectable difference in PSA concentration compared with a baseline value.

detection limit The minimum detectable concentration of PSA in capillary whole blood was 0.063 ␮g/L as analyzed using the whole-blood PSA protocol.

stability For storage at the ambient temperatures tested, PSA stability was 21 days, whereas at 37 °C, the PSA concentrations was stable for 7 days. The stabilities of the low-, mid-, and high-concentration samples were evaluated over the course of three analytical runs on the same day. After 21 days of storage at ambient temperature, the mean differences in the slopes for the low-, mid-, and highconcentration samples were ⫺0.002, 0.144, and 0.066, respectively. After 7 days of storage at 37 °C, the mean differences in the slopes for the low-, mid-, and highconcentration samples were 0.003, 0.107 and ⫺0.033, respectively. t-Test analysis showed nonsignificant differences (P ⬎0.05) from a zero slope for each value. After 14 days of storage at 37 °C, the samples were considered unstable: the measured values were 0.51 ␮g/L for the low-PSA sample (0.65 ␮g/L), 2.64 ␮g/L for the mid-PSA sample (4.5 ␮g/L), and 5.0 ␮g/L for the high-PSA sample (10.4 ␮g/L).

Discussion The paramount position of PSA as a urologic tumor marker is well accepted and indisputable; it remains the best and most widely used tumor marker in urology (16, 17 ). However, PSA has the most clinically significant value when used as a means to monitor disease recurrence after treatment of prostate cancer, specifically radical prostatectomy (17–19 ). According to numerous studies, the utility of PSA as a tool for postsurgical monitoring is considerably enhanced when ultrasensitive immunoassays (detection limit ⬍0.1 ␮g/L) are used because conventional systems are not able to quantify the low concentrations of PSA in the sera of prostatectomized males (9, 20, 21 ). This use of PSA testing for continual monitoring and screening purposes implies a need for a convenient and reliable system to ensure compliance, without sacrificing the assay sensitivity and precision required to accurately assess postsurgical patients. Professionally or self-collected capillary whole blood collected into the BIOSAFE BTS yielded PSA results that strongly correlated with results obtained by the standard Hybritech Tandem-MP PSA serum assay. Whole-blood assay precision was comparable to that of the serum assay

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(22 ), with within-run CVs ⬍4.0% and between-run CVs ⬍7.3% for serum compared with ⱕ3.5% and ⱕ6.0% for whole blood for all PSA concentrations above the functional sensitivity (defined as the concentration at which the between-run is 20%). The stability of the BTS samples in simulated shipping conditions provides adequate assurance that delivery by mail, even up to 21 days for temperatures ⬍23 °C, will ensure accurate PSA determination. Some of the concerns sited in previous reports on the use of liquid whole blood involve safety concerns, transport delays, and instability at high temperatures (15 ). All of these concerns were addressed in our test method validation. All safety regulations involved in shipping a liquid sample have been met by the BTS shipping system. Transit time was evaluated during the clinical validation; the median number of days between sample collection and delivery was 4 days (range, 1–14 days), within the stability of the PSA samples as determined by our stability testing. It was also stated by Hoffman et al. (15 ) that a filter-paper protocol as currently constituted is not suitable for the serial monitoring of patients after radical prostatectomy for the recrudescence of prostate cancer, where small changes in PSA concentrations in the range of 0.4 ␮g/L, 0.1 ␮g/L, and even lower have clinical significance and must be reliably detected. It was also concluded that home monitoring in this clinical setting would have been ideal. A separate customer satisfaction survey (unpublished study), performed by BIOSAFE Laboratories, Inc., done on PSA at-home collections in a dried-blood sample format, found that 92% of patients preferred at-home collection. Of the patients surveyed, which included a sampling of patients with normal (⬍4.0 ␮g/L; n ⫽ 550) and increased (ⱖ4.0 ␮g/L; n ⫽ 520) PSA, 63% of the patients stated that their PSA results prompted them to see their physician. More than 93.0% of the patients surveyed would recommend the test to others, and ⬎28% responded that they were not likely to be tested for PSA without the convenience of a home-collection method. Of the 148 participants of this PSA clinical trial, 145 completed and returned the questionnaire (98% return rate). Of these, 97% found the instructions easy to read and understand, and 78% claimed that it was easy to collect a sufficient sample. The majority (⬎94%) stated that if available, they would use the BIOSAFE PSA BTS in their homes. When asked to compare the two methodologies of sample collection (venipuncture vs finger nick), 66% preferred the finger nick. These findings suggest that the BTS design is accepted by patients for at-home selfcollection when under the care of a physician. The patient population would benefit from a collection system that provides their physicians with valuable information while allowing ease of use and collection to ensure compliance with both screening and monitoring. Of the 117 apparently healthy male volunteers (i.e., with no history of prostate cancer, no clinical symptoms

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of prostate disease or previously increased PSA results) who participated in the clinical trials, 11 (9%) had increased PSA by BTS and confirmed with serum (range, 4.1–22.8 ␮g/L). Subsequent examination and consultation with the trial site investigators identified that four of these men (3% of all apparently healthy participants) had documented prostate cancer. In conclusion, the clinical utility of PSA testing and monitoring may be enhanced through the use of the BIOSAFE BTS. This novel collection system addresses the needs of patients who require lifelong monitoring of prostate cancer for the detection of metastases or cancer recurrence after surgical or therapeutic treatment.

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