The CLSI H21 standard lists causes for coagulation specimen rejection, including hemolysis, icterus, lipemia, clotted specimens, underfilled tubes, and overfilled tubes. In most applications, underfilling is defined as a specimen comprising less than 90% of the stated volume. Most blue-closure tubes contain 0.3 mL of 3.2% sodium citrate and are calibrated to collect 2.7 mL of whole blood to yield a precise ratio of 1 part anticoagulant to 9 parts blood. An underfilled tube raises the ratio of anticoagulant to blood, leading to falsely prolonged clot-based assay results, such as the PT and PTT. But what about overfilling? We received this question on 4-14-25: “If the tube stops filling when the vacuum is depleted, how does a blue-top get overfilled? Further, is there a limit to overfilling?”
Overfilling is uncommon, but the phlebotomist may collect a specimen by syringe and apply excessive force when transferring to the coagulation tube through a needle (unsafe) or a safe transfer device. Alternatively, the collector may remove the closure and the needle and express too much specimen through the syringe hub–a nonstandard method we used before the advent of transfer devices. I (Geo) have also learned of inadequately trained collectors who combine the contents of two underfilled tubes, thus doubling the amount of anticoagulant. I’ve seen no data that suggest an overfilling limit. In the comments section below, I invite participants to share their experiences with underfilled and overfilled tubes.
4-18-25: Dave McGlasson referenced his 2005 study, McGlasson DL, Kaczor DA, Krasuski RA, et al. Effects of pre-analytical variables on the anti-activated factor X chromogenic assay when monitoring unfractionated heparin and low molecular weight heparin anticoagulation. Blood Coagul Fibrinolysis. 2005;16:173-6. doi: 10.1097/01.mbc.0000164424.90545.6e. PMID: 15795534. The article is available from your medical library. Here is a summary: We determined whether the anti-activated factor X (anti-FXa) heparin assay is less affected by pre-analytical variables in monitoring patients on UFH and LMWH than the PTT. Forty-six subjects receiving either LMWH or UFH were randomly selected. Each study subject had two tubes drawn by atraumatic venipuncture. One tube had a blood to anticoagulant ratio of 9:1, the other had a short-draw of 6:1. All specimens had a PTT and anti-FXa assay performed on each specimen. The intentional short-draw tube did not affect the PTT or anti-FXa assay. Given Dr. Ali’s comment below and McGlasson’s findings, the 90% rule may be subject to variation related to specimen, reagent, and instrument type.
Hi George,
My comment is not directly related to experience with under- or overfilled tubes, but it is relevant to the impact of citrate on coagulation assays. The general assumption that relatively increased citrate concentration always leads to prolonged PT/APTT results does not hold true across all reagents or assay systems. Our R&D team recently investigated this through a systematic ex vivo study, and the findings will be presented as a poster at ISLH 2025 (Halifax, May 7–9).
Our results indicate that the effect of citrate variation is reagent-specific. Therefore, discussions about the impact of elevated citrate—whether due to underfilled tubes, polycythemia, or hematocrit greater than 55% should consider the specific assay system in use.
Factors such as clot detection method, sample-to-reagent volume ratio, the presence of negatively charged particles, and the accessible calcium or magnesium ions in the assay can also influence clotting time results. For sure, rejecting abnormalities like under- or overfilled tubes without interpretation bias can ensure consistent and reproducible outcomes if there is a good lot-to-lot blue-top tube performance.
Regards, -Ali
Agree with George. I think as a general guide, ≥10% above the marked collection line would suggest overfilling. If overfilled due to combination of 2 underfilled tubes, then the sample is unsuitable because of excess citrate to blood ratio, leading to prolonged clotting times and diluted (false low) factor levels. If overfilled because of syringe collection, then risk is inability to mix the sample correctly, which may lead to clotting in the sample. Depending on the level of in situ clotting, may lead to prolonged clotting times and reduced factors, or in some cases of ‘primed activation’ may lead to short APTTs.