Interpretive limitations associated with plasma volume shifts in the clinical assessment of hemostasis.

We read with interest the recent publication by Austin et al. (1), where the authors analyzed blood from healthy males before and after the Tier Social Stress Test for markers of coagulation activation. They then reconstituted the samples for changes in plasma volume (PV) using participants’ prestress plasma and saline and mathematically using the methods of Dill and Costill (2). They concluded that observed changes in coagulation were, in part, a consequence of stress and hemoconcentration and that the method of correction by Dill and Costill is not optimal for PV correcting coagulation times. They concluded by suggesting that saline reconstitution was a more ‘‘biologically relevant’’ method of correction. Our laboratory has performed an experiment examining the same phenomenon (3), using significantly more potent PV stressors to ‘‘prove the same concept. ’’ We also used the correction technique of Dill and Costill used by the present authors, although it yielded very different results. To that end, we have identified several discrepancies that warrant consideration for interpretation of these findings. We noted an intriguing finding where mathematical correction resulted in further shortening of coagulation times after the experimental stress-induced hemoconcentration. This is, as already stated, contrary to our findings and is also contrary to research they cited in their study (4). Darlington and colleagues (4) found that progressive dilutions from 0 to 90% of citrated plasma elongated coagulation times, which is in agreement with the results of our experiment and makes intuitive sense. We feel that the present authors’ divergent findings could be explained by mathematical error. The equation used by the authors, as stated on page 290 of the article, is correct and indeed proper for correcting the concentrations of plasma coagulation proteases and molecules such as fibrinogen and D-dimer; however, it is not the correct equation to use for the correction of coagulation times. The problem lies in data interpretation; an ‘‘increase’’ in coagulation results in a decrease in coagulation time across the contact factor pathway or tissue factor pathway and common pathway. Therefore, time needs to be ‘‘given back’’ to correct for loss of PV. The correct equation should incorporate a plus sign (+) and not, as is the case with the current publication, a minus (j) sign. This would then correctly account for the shift in PVand remove the artifactual decrease in activated partial thromboplastin time (aPTT) and percentage of prothrombin time (PT%) noted by the authors in the present publication. Indeed, if the reported $PV from baseline to poststress is used (3.5% T ,2.3%) and apply the correction mentioned earlier, the aPTT only reduces by 0.1 second (34.3 T 0.8 second from 34.4 T 0.8 second), thereby abrogating the reduction. Furthermore, ‘‘significant’’ shortening from baseline to uncorrected values was reported as 33.1 T 0.8 second, which is comfortably within the boundaries of a normal aPTT (5). Even with the incorrect mathematical correction artifactually shortening aPTT further still to 31.1 T 0.8 second, it remains clinically insignificant because it has long been established that normal values are 27 to 37 second (5) on nonheparinized participants. We feel that the authors fail to express this important fact adequately in their study, which is especially relevant given that the clinical focus underlying their publication is a possible link between acute stress and cardiovascular disease. The PT data were expressed as PT%, which is confusing given that the standard clinical expression for PT in the patient population is the international normalized ratio (INR). The study would be more accessible if the authors performed the INR calculation, or even expressed their PT data as raw times, given that their participants were free from coagulopathy. This would have made their data more comparable for the clinical reader, given that most point of care devices express PT in INR. However, their PT% data unfortunately suffer from the same artifactual error as aPTT and, again, did not fall outside the boundaries of normality (6). Beyond themathematical oversight, we have some additional, mostly biochemical, concerns that may further influence the interpretation of the findings. The authors used the participants’ own un-stressed plasma as a method of reconstitution. It has been suggested that acute psychological stress increases inflammation (7), and it is known that there is a bidirectional relationship between coagulation and inflammation (8). Furthermore, acute psychological stress has been shown to increase oxidative stress (9), and a relationship between oxidative stress and coagulation activation has long been suggested (10). Thus, reconstituting with the participants’ plasma-harvested prestress is actually inappropriate. Furthermore, the authors state that this study forms part of a larger flavanol intervention trial and take great care to point out that the intervention would not influence the present data. We are not sure that this will be the case, for the reasons outlined in the previous paragraph. Flavanols are potent phenolic antioxidants and, as such, have an outstanding capacity to scavenge free radicals, alter nitric oxide bioavailability (11), and thus affect shear stress and intravascular pressures. Because the brain noadrenergic system is activated by acute stress (12), an additional aspect to consider for reconstitution may have been to measure norepinephrine levels and to treat the prestress plasma with a norepinephrine ‘‘spike’’ to try and achieve a reconstitution fluid more similar in composition to the poststress plasma. Also, given the potentially exciting mechanistic value of these data, it is possible that the patient population may benefit from drugs that have the potential LETTERS TO THE EDITOR