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Technical Briefs |
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The group of tumor-associated antigens with superficial carbohydrate structure influencing cell–cell interaction processes and cell growth regulation is commonly named "mucins," characterized by large and heterogeneic molecular mass and the presence of O-linked carbohydrates. Mucins are released in extracellular space and can be found and measured in serum and other biological fluids. Among mucins, CA 19.9 is widely used for diagnosing gastrointestinal cancers (1), CA 125 for gynecological cancers (2), and CA 15.3 for breast cancer (3). Despite the wide use of these tumor markers in clinical laboratories, few data about their preanalytical phase exist.
We studied the stability of CA19.9, CA 125, and CA 15.3 for 72 h and the effect of serum collection differences between plain tubes and tubes with thixotropic gel. We used our findings to define practical and empirical guidelines for blood drawing, storage, transportation, and delay from venipuncture to analysis.
We collected blood by routine sampling, namely, in plain tubes (pink cap, 7-mL Vacutainer Tube; Becton Dickinson) and in gel-containing tubes (gold cap, 5-mL Vacutainer Tube), completely filling the tubes and centrifuging them at 4 °C for 10 min at 1560g. The samples were stored at room temperature until analyses (within 3 h from blood drawing) and afterwards at 4 °C.
We collected 20 pairs of samples in plain and gel-containing tubes for each of the three markers from outpatients who had already had one or more measurements in our laboratory for the marker in the previous 2 months. This way, we obtained values opportunely and regularly distributed in the linearity range of the method: 0–240 IU/mL for CA 19.9, 0–200 IU/mL for CA 15.3, and 0–500 IU/mL for CA 125.
For ethical reasons, only patients with a physician's request for a tumor marker (routinely assayed in plain tubes) and general clinical chemistry (routinely assayed in gel-containing tubes) tests were recruited.
CA 19.9, CA 125, and CA 15.3 were measured by the same operator with the automated system Immuno1 (Bayer) by immunoenzymometric methods. The tumor marker assays were also performed on the contents of 5 separator-gel tubes filled with doubly distilled water and 5 tubes filled with serum containing no immunoassayable mucins; these measurements were performed after 72 h of contact between liquids and gels. The results were evaluated statistically by using Student's paired t-test, with P <0.05 considered as significant.
We noted a conspicuous increase of the measured markers in the
gel-containing tubes after 24 h (Table 1
).
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The stability is an important finding for mucins because some laboratories do not perform these analyses but send the sample in tubes to other centers, and some laboratories that do perform the measurements do not perform them daily.
The gel-containing tubes are widely used for endocrinological tests (4). These tubes probably are frequently used also for mucins and, in general, for tumor markers, these assays ordinarily being performed in the same section and with the same methods as the endocrinological assays.
The relationship between thixotropic gel and analytes, especially after a long contact period, has not been completely described. What has been reported includes: a decrease of progesterone after storage for 1 to 2 h (5) or after 6 days (6), and no differences between plain and gel-containing tubes until 72 h after blood collection for thyrotropin, free and total thyroxine, and triiodothyronine (7).
We noted no differences in the mucin results between serum from plain tubes and that from gel-containing tubes immediately after blood drawing; in general, both tubes could be used for measurement of mucinous tumor markers. Also, storage of serum at 4 °C in centrifuged plain tubes did not significantly modify the mucins' values. Conversely, gel-containing tubes cannot be used for storing sera for these analyses because of spurious, apparent increases in concentration of these tumor markers after 24 h from blood drawing. The increase is time-dependent because the values are constantly rising until 72 h from basal measurement. A release of some "mucin-like" antigens from inert gel can be excluded because keeping the tubes in contact with the doubly distilled water and with sera without immunoassayable tumor markers for 72 h did not produce spurious results.
An interference with avidin–biotin binding in an IRMA by using serum separator collection tubes has been described, but that inhibition was induced by water-soluble silicone polymer used for coating the interior of the tubes (8). In our study, however, we had an increase and not a decrease of concentration; moreover, the gel-separator tubes did not contain a silicone-coated interior.
In general, we can expect an interference from barrier gel by an absorption of small molecules, as described for therapeutic drugs (9). Tumor markers, however, are big molecules and we can exclude any interaction with gel similar to that described for drugs. A release of cell particles from cell membrane should be also excluded because we observed an increase in concentration in gel-containing tubes where the separator is a mechanical barrier between the corpuscular parts of blood and the serum, whereas in plain tubes the two parts of blood are in contact continuously.
In conclusion, plain and gel-containing tubes can be used for measuring tumor markers CA 19.9, CA 125, and CA 15.3 in sera stored at 4 °C for no more than 24 h from blood collection. Use of sera from gel-containing tubes after this period is not recommended.
Footnotes
Serv. Integrato di Med. di Lab., H San Raffaele, Via Olgettina, 60–20132 Milano, Italy
References
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