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How the making of it matters
We started with the idea that red cells are resilient.
They live for 120 days, traversing capillaries and splenic meshes narrower than their unsprung diameters, while withstanding the rusty chemistry of their gas exchange mission.
Then we turned to an abundance of literature that has been focused — with a bias of dread — on the impact that ex-vivo storage might have on clinical outcomes.
What could be worse than being separated from all the cells served in the circulation, and instead being statically refrigerated in a sugary, additive-laden, plastic-leaching bag for 28 to 49 days?
Blood storage duration
We have an arms race of storage studies.
On the bench is a litany of findings predicting retired red cell function (at best) and the accumulation of things than can hurt people (at worst). Meanwhile, and despite alarms from some papers, the gold standard of randomized controlled trials speaks consistently of fresh blood being indistinguishable from standard storage.
What is fresh — the first day, week or fortnight? What is “not fresh,” “stale” or “old” — the final week or fortnight? How narrow and separated should those vintages be to see an in-the-bag — and then in-the-body — difference? How much do you give to see a result?
When comparing two storage profiles, how can we know if giving nothing is better or worse than the group with a bad outcome? How do we classify and think about the blood that is “standard issue,” when it is arguably a giant mound of dispensations that is neither the first nor the last week, but encompassing weeks 2 to 3, ± 4 ± 5? If “standard” is indistinguishable from “fresh,” is everyone convinced that the same holds in the less studied last 1 to 2 weeks?
The research investment to some is a settled deal. To others, it is a confusing or unconvincing tailspin.
But what happens when you consider the ways in which the whole-blood donation has been processed into the red blood cell unit (RBC)? Should this be a factor in how we view studies from a particular “production-style region?”
There is an American–Canadian divide on how red cells are made, and within Canada, a further two-way split. Additive/preservative solutions, both reaching for the 42-day mark, are either a base of CPD–SAGM (citrate, phosphate and dextrose; saline, adenine, glucose and mannitol) in Canada, or “AS” (–1, –3, –5) in the United States (CP2D with minor concentration variations in SAGM).
Separation begins with centrifugation, and this can be a sequence of “starting soft” or “starting hard.”
In the United States, a soft-to-hard spin sequence separates a platelet-rich plasma from red cells and is increasingly preceded by prestorage leukoreduction. In Canada, and in other “buffy coat production” nations, the spin starts hard.
Making of RBC
This is where Nancy Heddle, FCSMLS, MSc, and collaborators within the McMaster Transfusion Research Program and Canadian Blood Services took a look at production-related outcomes.
The question was not only about how in-hospital mortality varied with three phases of storage (first week/fresh [F], mid-range/8-35 days [M], and old/36-42 days [O]), but how it varied by processing.
Roughly half of Canada’s blood gets made one way or another, with the first called “red-cell filtered” (R) and the other called “whole-blood filtered” (W). R also is known as the B1 or top–bottom method, whereas W is the B2 or top–top method.
In R, the processing occurs within 20 hours at ambient temperatures, and the hard spin — which packs the red cells — also drives off a buffy coat and plasma. The red cells undergo leukofiltration, while four buffy coats join with the plasma of one of the composite male donors. That pool is then soft-spun — to remove contaminating red cells — and is itself leukoreduced.
In W, the processing may occur in a wider, 72-hour window after refrigeration, with leukoreduction applied to the still-whole blood. Platelets are neither isolated nor expected to be viable as they divide in unknown ways between the two destination products, plasma and RBC.
The plasma may be frozen for direct infusion after a warm thaw, or cold-thawed to produce cryoprecipitate. The RBC by this method is left to contain up to 20 mL of plasma (vs. 2 mL in the B1 method). On the whole, W is “messier” (with residual vesicles, plasma and DNA).
The key differences between R vs. W, therefore, lie in the former being a warmer, sooner, postcentrifugation RBC filtration, vs. the latter being a cooler, more delayed (but precentrifugation) filtration.
There were six exposure groups (R vs. W methods, crossed by three storage groups: F vs. M vs. O).
Across three hospitals and 6 years (2008-2014, entering the buffy coat production era), more than 23,600 patients received more than 91,000 RBC.
Mid-storage red-cell filtered (M–R) RBC days (> 64,000) were taken as the “base,” inspired by the observation that Canada’s ABLE study had insignificantly higher mortality in recipients of fresh blood, some of which had to have been the hypothetically worrisome W–RBC.
Of the exposures, 19% were to pure M–R, 19% were a seventh group of at least two RBC types other than M–R (“mixed”), and the other groups were M–R mixed with the prespecified category of interest (the largest: similar-vintage M–W at 55%; then the “oldies” [O–R] at 3% and [O–W] at 2%; and finally the “freshies” [F–R] and [F–W] both at < 1%).
In the paper’s highlight figure, all but one subgroup’s HRs stretched through the unity line marking similarity with M–R. Tilted on the 1-or-less side were O–R, O–W, M–W and mixed exposures. This suggests older units, including those from the most controversial terminal week, might be vaguely better. Another overlap weighed on the greater-than-1 side with F–R, conversely implying that fresher units might be worse. The group that stood out as numerically distinct was F–W (HR = 2.19; 95% CI, 1.09-4.42), therein nailing the original concern.
Study implications
Although this retrospective analysis was limited by uneven group sizes and compositional overlap, the findings remain hypothesis generating.
The city-specific study reflected nationwide component production, the details of which were unapparent to blood users. Death, the endpoint, was incontrovertible. As such, this snapshot draws attention to whether buffy-coat production jurisdictions — such as Canada, Australia and Europe — might be different from American ones.
Until the American-led RECESS study in cardiac surgery, and the TOTAL study in Ugandan children, using leukofiltered RBC by American methods — both of which showed no storage-related outcome differences — Leo van de Watering, MD, PhD, of Sanguin in the Netherlands, had noted the preponderance of studies on longer-storage disadvantages emanating from the United States, whereas the absence of any differences with storage were prevailing in European data.
Why is this Canadian signal different? Are the differences between short- and long-stored outcomes eclipsed by some counterbalancing toxicity that is specific to fresh RBC made a certain way?
Is starting with a hard spin bad for the red cell? If we are leukofiltering later, or under cold and unseparated whole-blood conditions, are we permitting the formation of evil humors? Many readers who imagine a time-dependent brew of harmful mediators will be challenged by the idea that some poisons are created from the start of a manufacturing process, and that they dissipate without any extraction.
Because the R and W RBCs were leukoreduced at nearly the same median time (19 hours vs. 21 hours), are we satisfied with when we leukofilter, or do these data suggest we should complete this step sooner? Should we abandon cooling before filtration? Should we remove the buffy coat, the plasma or both before filtering?
If this analysis could be applied to larger and different production datasets, we might see if this observation can be reproduced and better specified. If we compare data on American RBCs with Canadian SAGM W vs. R products, what might we see?
We confront so many splits that some are either hopeless about seeing a strong signal, or reassured that — despite how we prep blood — there just isn’t anything consistently jumping out at us.
In the end, we look at this all and burst with the usual comments: We cut too far, and it can’t be real; the signal is too fuzzy to matter; or, this is serious and we must probe to confirm and mitigate.
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For more information:
Christine Cserti-Gazdewich, MD, FRCPC, is a hematologist and transfusion medicine specialist at University Health Network/University of Toronto. She can be reached at christine.cserti@uhn.ca.
Disclosure: Cserti-Gazdewich was a collaborator in the ABLE study and a coinvestigator in the TOTAL study.