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Raman peeks into blood bags Spectroscopic techniques could provide clues to health of stored red blood cells CELIA HENRY ARNAUD, C&EN WASHINGTON
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very year in the US, about 6.8 million people donate blood. After a person donates blood, the clock starts ticking for its ingredients—the plasma, platelets, and red blood cells— that might be transfused into a patient. Just like any biological matter, they have a shelf life. They degrade. In the case of red blood cells, for instance, hospitals and blood banks have up to 42 days to use them as long as they’ve been refrigerated properly. That shelf life, though, is set by the blood bag manufacturers, not by measuring the quality of blood over time, but by measuring the time that transfused blood cells last inside a healthy person’s body. No one really knows the quality of blood stored for the full shelf life. Biochemically speaking, researchers don’t have a solid idea of what the indicators are of “good” blood versus “bad” blood. What blood experts do know is that, as blood ages, it acquires what is known as “the storage lesion,” the accumulation of changes to the red blood cells during storage, many of which are caused by oxidative
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damage. Currently there’s no way to test for this damage in blood that’s been stored inside a bag without opening the bag, compromising its sterility, and rendering the blood unusable. Even if there were testing methods, people still don’t know whether such changes in the blood correlate with poor patient outcomes. But an option could be available soon. Two independent teams—one led by Robin F. B. Turner and Michael W. Blades of the University of British Columbia (UBC) and the other led by Richard A. Dluhy of the University of Alabama at Birmingham (UAB)—are working to develop Raman spectroscopy as a method that can monitor blood inside storage bags without ever opening them. Turner and Blades became interested in analyzing stored blood after they met Dana V. Devine, a pathology professor at UBC and the chief scientist at Canadian Blood Services, Canada’s blood banking agency. About a decade ago, Devine says, transfusion medicine specialists started worrying about the quality of blood products in the late stages of their shelf lives. They thought blood recipients might have worse outcomes when they were given blood products that had been stored for 5 or 6 weeks instead of a couple of weeks. Prospective trials, such as the Age of Blood Evaluation trial, have allayed some of those concerns, showing that the age of transfused blood has little or no effect on patient outcomes (N. Engl. J. Med. 2015, DOI: 10.1056/nejmoa1500704). “That’s good news for organizations like mine,” Devine says. “But it also got us focused on what’s actually in that bag of red blood cells.” Sure, on average, fresh blood and weeks-old blood are both OK for patients under most circumstances, but the blood in some bags could still go bad earlier than the blood in others. Devine likens it to the expiration date on a carton of yogurt. Some yogurt remains good past the printed expiration date, whereas other
yogurt goes bad before that date. A color change or smell can indicate to consumers if they’ve gotten a bad yogurt, but for donated blood, it’s not easy to tell, especially because the bag needs to stay closed. Through their connection at UBC’s Centre for Blood Research, Devine teamed up with Turner and Blades to see whether the pair’s Raman methods could be useful. Their method of choice is called spatially offset Raman spectroscopy (SORS), which can collect Raman spectra from beneath translucent surfaces, such as the polymer that makes up blood storage bags. In SORS, an excitation beam is directed into a sample, and Raman scattered photons are collected from two locations—one at the same spot as the excitation beam and another at a location offset by a few millimeters. This strategy allows the collection of Raman spectra from molecules below the surface. Before the UBC team could analyze blood through a bag, it first needed to directly analyze blood removed from bags. “It was the best way for us to begin our learning curve about how the blood changes,” Turner says. “Our red cells have a lot of hemoglobin,” Turner says. Spectral bands from hemoglobin can mask other spectral markers the researchers would like to identify that correlate with the chemistry and physiology of blood cells. “We’re trying to figure out ways to see between the hemoglobin bands more clearly and get additional information from the suspended cells.” One promising marker of blood cells’ health is lactate. Blood cells are stored in a solution of glucose and other additives. When cells metabolize glucose under low-oxygen conditions, like those in a storage bag, they produce lactate as a waste product. In blood storage bags, that lactate collects in the supernatant liquid above the cells that have precipitated to the bottom. Preliminary work suggests that lactate levels change over the course of storage and that some stored blood units accumulate lactate more readily than others. Thus, lac-
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tate might be a biomarker for blood quality they can detect the rupture, or hemolysis, of red blood cells through a storage bag. In (Analyst 2016, DOI: 10.1039/c6an00373g). methemoglobin, the iron in the heme group Despite the challenges of hemoglobin, is in the ferric (+3) state rather than the Turner and Blades are also looking at it ferrous (+2) state of normal, oxygenated as a possible marker of storage-induced hemoglobin. Methemoglobin is attractive changes in blood. They’re primarily foas a potential marker because it’s usually a cusing on the ratio of deoxygenated to minor form of hemoglobin, so changes in oxygenated hemoglobin in samples. Raman spectra reveal changes in hemoglobin its abundance could make it a selective indicator of storage-related changes. oxygenation that appear to correlate with In a preliminary study, the team remorphological changes in the blood cells peatedly sampled blood from the same six (Analyst 2017, DOI: 10.1039/c7an00349h). donors at 7, 21, and 35 days of storage and As they’ve continued studying stored analyzed it with conventional Raman specblood, Turner and Blades have demontroscopy (Lab Med. 2018, DOI: 10.1093/ strated that they can indeed analyze blood labmed/lmy018). The researchers found directly in the storage bags by monitoring that levels of both oxyhemoglobin and the ratio of deoxyhemoglobin to oxyhemethemoglobin increased during the stormoglobin (Analyst 2018, DOI: 10.1039/ age period. c8an01509k). O “We’ve been able to But they’re not entirely satisfied with SORS OH show that the predominance of methemoglobin yet. One problem that N- N Fe2+ is an indicator of hemoTurner and Blades have N Nlysis and toxicity in red found is that commercial OH blood cells,” Dluhy says. SORS instruments are “We have to do a lot more optimized to analyze O studies correlating the more highly concentrated spectroscopy with the samples than the comHeme, a major component of ponents in blood storage red blood cells, is responsible biochemistry of what’s going on in the bag. bags. They would also for transporting oxygen. Hopefully, we’ll be able to like to be able to adjust The difference between do something like that in the offset. “We would like deoxyhemoglobin (shown) the near future.” to be able to turn some and oxyhemoglobin is that In another study, the knobs and optimize the oxyhemoglobin has oxygen UAB researchers used instrument. Because it’s a bound to the iron atom. diffuse resonance Raman commercial instrument, to analyze blood not in the storage bags it’s not easy to do that,” Turner says. themselves but in segments of polymer “We’re doing the best we can with what tubing connected to and made of the same we’ve got. We’ve learned a few tricks to material as the blood bags. They were able improve things, but it’s still not optimal.” to spectrally distinguish blood that had Dluhy and his team at UAB, including been stored 6–8 days from blood that had Rakesh P. Patel in the School of Medicine, been stored 35–40 days (Analyst 2018, DOI: have also tried SORS for analyzing stored 10.1039/c8an01135d). blood but found that it wasn’t yielding After the technology that both the enough signal for the molecular compoUBC and UAB teams are working on gets nents they wanted to study. The group to a level where it can be used routinely, has instead turned to a different Raman Devine imagines blood banks will regularly method called resonance Raman spectrosmonitor the status of their supplies. Blood copy, which uses a red excitation beam cells that look like they’re metabolizing at 632 nm excitation. This wavelength quickly could be moved to the front of the coincides with an electronic transition of inventory to be used before they go bad. hemoglobin and thus provides a resonant “There’s a clear need for label-free, enhancement in the signal that comes noninvasive, and sensitive techniques that from blood samples. To adapt the method can assess the quality of red blood cells for storage bags, Dluhy and his team defoduring storage,” Dluhy says. He thinks the cus the excitation laser beam and change early results indicate that Raman could detector settings to collect Raman scatmeet that need. But those early results tered photons from deeper in the sample. need to be reproduced, he says. “There They call their adapted technique diffuse needs to be a lot more correlative work resonance Raman spectroscopy. to convince not just spectroscopists but The UAB researchers are using diffuse biochemists, pathologists, and the medical resonance Raman to measure oxygenated community that this is something they can hemoglobin and another form of hemogloreliably use and trust.” ◾ bin called methemoglobin to see whether APRIL 15, 2019 | CEN.ACS.ORG | C&EN
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