Effect of storage on levels of nitric oxide metabolites in platelet preparations

Abstract

Background: Nitric oxide (NO), a potent signaling molecule, is known to inhibit platelet (PLT) function in vivo. We investigated how the levels of NO and its metabolites change during routine PLT storage. We also tested whether the material of PLT storage containers affects nitrite content since many plastic materials are known to contain and release nitrite.

Study design and methods: For nitrite and nitrate measurement, leukoreduced apheresis PLTs and concurrent plasma (CP) were collected from healthy donors using a cell separator. Sixty-milliliter aliquots of PLT or CP were stored in CLX or PL120 Teflon containers at 20 to 24°C with agitation and daily samples were processed to yield PLT pellet and supernatant. In a separate experiment, PLTs were stored in PL120 Teflon to measure NO generation using electron paramagnetic resonance (EPR).

Results: Nitrite level increased markedly in both PLT supernatant and CP stored in CLX containers at a rate of 58 and 31 nmol/L/day, respectively. However, there was a decrease in nitrite level in PLTs stored in PL120 Teflon containers. Nitrite was found to leach from CLX containers and this appears to compensate for nitrite consumption in these preparations. Nitrate level did not significantly change during storage.

Conclusion: PLTs stored at 20 to 24°C maintain measurable levels of nitrite and nitrate. The nitrite decline in nonleachable Teflon containers in contrast to increases in CLX containers that leach nitrite suggests that it is consumed by PLTs, residual white blood cells, or red blood cells. These results suggest NO-related metabolic changes occur in PLT units during storage.

Figure 1. Changes in nitrite content during storage

Leukoreduced (< 2×10⁴ leukocytes/60 mL) apheresis platelets were collected from healthy donors using the Trima Accel. Sixty mL of PLT or CP was stored in CLX (A and B, n=5) or PL120 Teflon (C and D, n=4) containers at 20–24°C with agitation. Nitrite (A and C) and nitrate (B and D) were measured in the PLT supernatant, pellets and CP using a chemiluminescence method. The percentage of CD62 positive cell was measured by flow cytometry (E, n=3). Data are means ± SEM.

Figure 2. Nitrite leaching out of CLX container

Sixty ml of plasma was stored in CLX containers (n=4) or in PL120 Teflon containers (n=4) at 20–24°C and aliquots from each container were taken every day to measure nitrite content with triiodide (I₃⁻) ozone-based chemiluminescence. Data are means ± SEM.

Figure 3. NO measurement during storage

PLT or CP aliquots were stored in PL120 Teflon containers at 20–24°C with agitation. Samples were taken every day and mixed with cPTIO (200 µM) to analyze NO level with EPR. Inverted central peak heights were shown, so the values are directly proportional to the amount of NO present (A). Aggregation (B) was measured by PLT response to two agonists (ADP and collagen, 10 µM and 10 µg/ml, respectively) via changes in light transmittance and the percentage of CD62 positive PLT (C) was measured by flow cytometry. Data are means ± SEM.