Comparison of Fresh Frozen Serum to Proficiency Testing Material in College of American Pathologists Surveys: [alpha]-Fetoprotein, Carcinoembryonic Antigen, Human Chorionic Gonadotropin, and Prostate-Specific Antigen

Archives of Pathology & Laboratory Medicine,  Mar 2005  by Schreiber, William E,  Endres, David B,  McDowell, Geraldine A,  Palomaki, Glenn E,  Et al

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Context.-Most proficiency testing materials (PTM) contain an artificial matrix that may cause immunoassays to perform differently with this material than with clinical samples. We hypothesized that matrix effects would be reduced by using fresh frozen serum (FFS).

Objective.-To compare the performance of an FFS pool to standard PTM for measurement of α-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin (hCG), and prostate-specific antigen (PSA).

Design.-One FFS specimen and 4 different admixtures of PTM were distributed in the 2003 College of American Pathologists K/KN-A (for α-fetoprotein, carcinoembryonic antigen, hCG, and total and free PSA) and C-C (hCG only) Surveys.

Participants.-The number of laboratories that participated in the surveys varied from a low of 288 (free PSA, K/KN-A Survey) to a high of 2659 (hCG, C-C Survey).

Main Outcome Measures.-Method imprecision and method bias were compared between the FFS specimen and the standard PTM specimen with the closest value. Method imprecision was determined by calculating the coefficients of variation for each method and for all methods combined. Bias was defined as the proportional difference between peer-group mean and the median of all method means.

Results.-The FFS specimen gave significantly higher imprecision than PTM for the analytes α-fetoprotein, carcinoembryonic antigen, total PSA, and free PSA. For hCG, no substantial imprecision differences were observed in both surveys. Bias was significantly greater for the a-fetoprotein, carcinoembryonic antigen, and total PSA assays and significantly lower for the hCG and free PSA assays when comparing the FFS with the PTM.

Conclusions.-Fresh frozen serum did not provide consistently lower imprecision or bias than standard PTM in a survey of commonly ordered tumor markers.

(Arch Pathol Lab Med. 2005;129:331-337)

A key component of quality assurance in the clinical laboratory is participation in external proficiency testing programs. These programs allow individual laboratories to assess the relative accuracy of their methods in comparison with a peer group of other laboratories that subscribe to the survey. Such programs can also provide data on the comparability of different methods for the same analyte. This information is useful to laboratory and medical personnel, who must interpret test results obtained by different methods, as well as to the manufacturers who produce these assays.

The materials that are used by proficiency testing programs should, as much as possible, behave like genuine clinical specimens. Most proficiency testing materials (PTM) consist of treated human serum that is spiked with one or more analytes of interest to produce the desired concentrations. The artificial matrix may cause assays to perform differently with this material than with clinical samples. This difference is referred to as "matrix bias," the presence and magnitude of which is typically unknown for any given analyte/method combination. Consequently, the performance of a given method in an external survey may not reflect the results obtained when measuring patient specimens.

Immunoassays of serum proteins, such as hormones and tumor markers, introduce another form of bias. Many proteins that are secreted or shed into the circulation exist in more than one molecular form. Because of the nature of these analytes, there is no reference method that gives a universally accepted correct value for each analyte's concentration in serum. The amount measured by a given immunoassay depends on how the assay is standardized and the antigenic specificity of the antisera used. Performance of immunoassays in external proficiency surveys is thus subject to bias from the molecular nature of the spiked analyte (spike bias) as well as matrix bias.

One potential approach to reducing matrix and spike biases is to use pooled human serum as a PTM. To test this hypothesis, the College of American Pathologists (CAP) included a fresh frozen serum (FFS) sample in the first mailing of the 2003 K and C-C surveys. The questions to be answered were as follows:

* Is there a methodology bias between FFS and standard PTM?

* Is there a difference in imprecision between FFS and PTM?

* Do PTM specimens act like FFS specimens?

In this manuscript, we analyze the results of these surveys for 5 tumor markers: ct-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (hCG), and total and free prostate-specific antigen (PSA).

MATERIALS AND METHODS

Samples

The 2003 CAP K/KN-A and C-C surveys included a commutable FFS specimen and 4 different admixtures of PTM. Fresh frozen serum was prepared by Aalto Scientific (Carlsbad, Calif) using a modification of the NCCLS C37-A Guideline 13. Briefly, donor blood was collected into plastic bags that were immersed in ice water, then centrifuged at 1500g for 8 minutes at 4°C to obtain platelet-rich plasma. The plasma was transferred to sterile plastic centrifuge bottles and allowed to clot for 4 hours at room temperature. Following centrifugation for 18 minutes at 2100g at room temperature, the resulting serum was removed and flash frozen. Serum units were shipped frozen from the donor center to the processing center and stored at -70°C for up to 2 months prior to pooling. A more detailed description of the collection and preparation of FFS appears in an accompanying manuscript by Miller et al.1