Charles Sebastien, Guyotat Denis, Fontana Pierre, Tardy Bernard, Lecompte Thomas, Chalayer Emilie
INSERM UMR 1059, Equipe SAINBIOSE, Université Jean Monnet, Saint-Étienne, France.
Centre d'Investigation Clinique CIC 1408, CHU Saint Etienne, Saint-Étienne, France.
Front Cardiovasc Med. 2022 Oct 21;9:998687. doi: 10.3389/fcvm.2022.998687. eCollection 2022.
To perform Calibrated Automated Thrombography (CAT), the use of reduced plasma volumes (referred to as "MidiCAT") makes it possible to more efficiently use limited volumes of valuable biobanked plasma samples and decreases expenses for reagents. It is, however, unclear whether the MidiCAT procedure is suitable when thrombin generation (TG) is studied in the presence of added thrombomodulin (TG-TM). Moreover, a simplified centrifugation scheme would facilitate biobanking, if appropriate, for more sensitive coagulation studies. We aimed to compare the results of "MidiCAT" (halved plasma and reagent volumes) with those from regular CAT, in the absence or presence of TM, as well as to study the impact of a single-centrifugation scheme for plasma preparation before freezing.
Plasma samples were prepared from the citrated blood from 20 Geneva hospital diverse patients without gross coagulation abnormalities with a single- or double-centrifugation scheme. Samples were kept frozen at -80°C and thawed just before the TG assay in duplicate under two conditions: 1 pM tissue factor (TF) or 5 pM TF + TM.
(1) We externally validated "MidiCAT" and also extended the validation to TG-TM. Whatever the method (CAT or MidiCAT), intra-assay (assessed with duplicates) CV was below 6% (1 pM TF) or below 10% (5 pM TF + TM) for ETP. Agreement between the MidiCAT and CAT results was satisfactory; the p coefficients were above 0.95 for ETP and above 0.90 for most other parameters; biases for ETP were +10.0% (1 pM FT) and +13.5% (5 pM + TM). (2) The centrifugation scheme markedly affected the results obtained in the presence of TM, whereas the bias and limit of agreement (difference plots) were low for the no TM condition. The bias in the presence of TM was obvious, more marked with plasma samples sensitive to TM when double centrifuged: the lower the ETP-TM, the greater the relative difference between the ETP-TM of plasma samples prepared with just single centrifugation and the reference plasma samples. Thus, a single-centrifugation procedure, as is often used for plasma biobanking, is suitable for TG study only if it is not performed in the presence of TM.
为了进行校准自动血栓形成试验（CAT），使用减少的血浆体积（称为“微型CAT”）能够更有效地利用有限体积的珍贵生物样本库中的血浆样本，并降低试剂成本。然而，在添加血栓调节蛋白（TG-TM）的情况下研究凝血酶生成（TG）时，微型CAT程序是否适用尚不清楚。此外，如果合适的话，简化的离心方案将有助于生物样本库的建立，以进行更敏感的凝血研究。我们旨在比较“微型CAT”（血浆和试剂体积减半）与常规CAT在有无血栓调节蛋白（TM）情况下的结果，并研究冷冻前血浆制备采用单步离心方案的影响。
从20名日内瓦医院不同患者的枸橼酸盐血中制备血浆样本，这些患者无明显凝血异常，采用单步或两步离心方案。样本保存在-80°C冷冻，在TG检测前解冻，一式两份，在两种条件下进行检测：1 pM组织因子（TF）或5 pM TF + TM。
（1）我们对“微型CAT”进行了外部验证，并将验证扩展到TG-TM。无论采用何种方法（CAT或微型CAT），对于内源性凝血酶原酶（ETP），批内变异系数（通过重复检测评估）在1 pM TF时低于6%，在5 pM TF + TM时低于10%。微型CAT和CAT结果之间的一致性令人满意；ETP的p系数高于0.95，大多数其他参数的p系数高于0.90；ETP的偏差在1 pM TF时为+10.0%，在5 pM + TM时为+13.5%。（2）离心方案对存在TM时获得的结果有显著影响，而在无TM条件下偏差和一致性界限（差异图）较低。存在TM时的偏差很明显，当对TM敏感的血浆样本进行两步离心时更为显著：ETP-TM越低，单步离心制备的血浆样本与参考血浆样本的ETP-TM之间的相对差异就越大。因此，单步离心程序，如常用于血浆生物样本库的程序，仅在不存在TM的情况下适用于TG研究。
