**5. Issues on blood sample**

to the latest consensus diagnostic criteria [55]. Statistical analysis was done using JMP version 9.0.0 (SAS Institute Inc., Cary, NC, USA). The mean response of each experimental group was compared with its simultaneous control by unpaired Student's *t*-test, setting a significant difference at *P* < 0.05. To examine the plasma annexin A5 levels in diagnoses of AD, DLB, and MCI, logistic regression modeling was employed to construct receiver operator (ROC) curves.

The plasma level of annexin A5 was significantly increased in AD patients compared to that of a control group (*P*-value < 0.0001 in the logistic regression analysis), suggesting that annexin

**Figure 3.** Comparison of plasma levels of annexin A5 in AD, DLB, MCI, depression, and age-matched healthy control.

For quantification of plasma annexin A5, we used a previously established chemiluminescent enzyme immunoassay system with monoclonal antibodies against human annexin A5 [52] (see Section 4.1). Individual plasma annexin A5 concentration is plotted in (A). The probability of either AD, DLB, or MCI can be predicted by a logistic regression model with the plasma level of annexin A5. Receiver operating characteristic (ROC) curves are shown in (B)–(D). The areas under the curve are 86.3%, 83.8%, and 91.6% for AD (B), DLB (C), and MCI (D), respectively. AD, Alzheimer's disease; DLB, dementia with Lewy bodies; MCI, mild cognitive impairment.

As annexin A5 binds not only phospholipids but also Ca2+, it might have a role in protecting against Ca2+-induced damage by chelating elevated intracellular Ca2+. A defensive role against

A5 is a potentially positive biomarker for AD (**Figure 3**) [52].

74 Update on Dementia

Since annexin A5 is also expressed in peripheral blood lymphocytes [58, 59], the effect of physical stress (such as osmotic pressure and temperature changes) upon blood cells may induce leakage of annexin A5. In fact, if a prolonged period of time passes (such as 12 h) after collecting blood, prior to centrifugation, the amount of plasma annexin A5 increases compared with a shorter period (such as within 6 h) (data not shown). Therefore, blood samples should be centrifuged within a specified period of time after collection. In our study, we did this within 6 h after blood collection. However, the lack of consistent technical standard for blood sampling in plasma biomarker studies may induce complicated and inconsistent observations depending on the study groups [60]. With respect to some conditions, such as anticoagulant reagent (EDTA or others), needle gauge, and 6-h fasting, standards should be proposed. For plasma preparation, a time limit until plasma separation after blood sampling may be critical to avoid induction of unwanted component leakage. Centrifugation speed (gravity force), duration, temperature, and number of spins, sample storage conditions may also require specification, though most common plasma samples are stored immediately at a temperature of –80°C for long-term storage. There will also be a number of factors that apply to subjects (patients and other participants involved): such as demographics (age, sex, and race/ethnicity), life style, overall health conditions (chronic drug administration, dietary supplements), smoking, and alcohol consumption.
