Evaluating the survival of stored red cells

In case the reader is unaware, heated disputes have long existed a m o n g blood preservation experi- mentalists regarding the "best"methods for measuring the survival of preserved red cells. Three articles a n d three editorials in this issue of Transfusion attest to this state of affairs. After several years of painstaking arbi t ra t ion a m o n g head-strong scientists, Moroff et al. ' were successful in negotiating agreement on uniform procedures for performing single-label (5 'Cr) or double-label ("Cr a n d red cell survival studies, while Valeri a n d colleagues' concluded f rom a com- parison of the t w o methods in 397 survival studies that the double-label procedure is preferable when the 24- hour survival is less than 65 percent. As a counterpoint. Beutler a n d West3 maintain that "overestimated" single-label ("Cr) survivals (e.g., those <60%) may be properly interpreted by taking into account the magnitude of the overestimate as established in their report. Is the matter of red cell survival methodology now settled'?

Measuring the survival of preserved red cells is technically a tricky business. Strict a t tent ion must be paid t o every aspect of the manipulat ion of the fragile. compromised cells lest the test procedure itself produce artifactitiously low or high survivals. More- over, the sampling schedule and analysis of d a t a must be based upon sound principles of experimental design a n d statistical estimation in order t o assure accurate a n d precise survival results. Using these criteria, how d o the three articles in this issue of Transfusion measure up?

T h e stimulus for the efforts of Moroff et al. ' was the observed high interlaboratory differences in reported 24-hour survival of concentrated red cells stored f o r 35 d a y s in citrate-phosphate-dextrose-adenine o n e ( C P D A - I ) a m o n g six collaborating research labora- tories: laboratory means of "Cr 24-hour survivals varied 23 percent, ranging f rom 45 to 85 percent.' This extent of variability was considered to exceed the varia- bility d u e to different storage condi t ions or recipients and was. therefore, a t t r ibuted to lack of uniformity in the survival methods used. In sometimes s tormy meet- ings a n d lengthy correspondence, the Moroff Commit - tee of recognized experts dissected a n d opened for debate every minute procedural s tep in the single- and double-label red cell survival methods. T h e published document , therefore. represcnts compromise-con- se nsus-de r ived recommendations aimed a t minimizing differences in methodology a n d a t improving interlab- ora tory comparability. To the extent known o n the basis of scientific da ta a n d informed personal persua- sion, technical procedures were agreed upon that mini- mize artifact. while sampling schedules and da ta reduc- tion methods were chosen that maximizeaccuracyand

precision in the survival estimates. T h e level of detail in the Moroff Commit tee recommendat ions extends t o the procedural aspects thought t o be the most likely sources of variability: e.g., needle sizes; method, conditions, and extent of component mixing a n d washing; a n d , of critical importance, choice of sampling times a n d methods of d a t a analysis. T h e first blood sample is t o be taken 5 minutesaf ter injection of 5'Cr-labeled red cells in the single-label procedure, a time chosen a s the initial time af ter which complete mixing of injected cells is known t o occur in the majority of recipient^.^ Five blood samples d r a w n during the 5- t o 15-minute postinfusion interval a re t o be used t o establish by logarithmic regression the extrapolated zero time "Cr concentrat ion. T h e samp- ling time interval was established o n the basis of observations made o n d a t a collected f rom more than 100 survival studies of red cells preserved f o r varying periods of time in citrate-phosphate-dextrose ( C P D ) and citrate-phosphate-dextrose-adenine-one ( C P D A - 1) and two (CPDA-2),"'*' a s well a s the present report of Beutler and West? In these studies, which included 24- hour survival values that were less than 20 percent, the initial rapid "Cr-loss phase was observed t o be com- pleted within I5 minutes of infusion of labeled red cells, after which time the blood "Cr concentration stabilized. T h e choice of logarithmic regression for fitting a line for zero-time extrapolat ion may a p p e a r t o be based upon the assumption of concentration-dependent loss rate of "Cr labeled cells dur ing the initial rapid loss phase. In truth. t h e logarithmic analysis represents the least biased procedure chosen from a m o n g a number of empirical estimation techniques, none of which per- fectly model the initial loss phase of 5 ' C r dis- appearance.

This is not t o say that the Moroff Commit tee recommendat ions will g o unchallenged. Indeed, they a r e recommendat ions only, unsupported by da ta indicating that their use will achieve the desired reduction in interlaboratory variability o r will improve accuracy and precision of viability estimates. S o m e would take issue, for example, with the possible nonuniform labeling that may result dur ing addi t ion of 5 'Cr-sodium chromate directly t o red cell concen- trates or the possible hemolysis that might result f rom drawing concentrated labeled red cells th rough a n 18- gauge spinal needle. Perhaps a 50 percent suspension of red cells in 0.9 percent saline pr ior t o labeling would avert these addi t ional potential sources of variation. T h e importance of these recommendat ions, however, is the publication of procedures of sufficient detail that they can be reproduced reasonably in different labora- tories. It must be ascertained by prospective evaluation whether strict adherence t o the recommended proce-

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dures will improve reproducibility and comparability among laboratories and result in more accurate and precise survival measurements.

A convincing comparison of two general methods for estimating red cell survival entails the objective application of each method under conditions that allow maximal opportunity for each method to yield accurate and precise results. Optimally, the methods should be applied concurrently and prospectively, with careful attention paid to consistency in the performance of each minute procedural detail. Even in a carefully monitored, prospective research effort, variations in procedure can occur that may have profound effects on the experimental results. For example, during cooperative evaluations of the in vivo viability of blood preserved with adenine, low 24-hour "Cr survivals in one laboratory were traced to the inadvertant washing of "Cr-labeled red cells with distilled water instead of the recommended saline.

Valeri et al.' calculated single-label 24-hour 51Cr red cell survival values retrospectively from 397 selected survival study data sets collected during 8 years of routine application of a double-label (5'Cr and I 2 ' l ) procedure and compared the survival estimates result- ing from the two procedures. Although the limited description of procedures utilized in their report would allow for considerable variation in application in the hands of others, i t is clear that their single-label method differed in several substantive ways from that recommended by the Moroff Committee. In Valeri's method, preserved red cells were incubated with "Cr for 30 minutes at 37OC and were subsequently injected into the recipient without washing. Thus, in contrast to the Moroff Committee recommendation, a mixture of red cell-bound and -unbound "Cr was injected, the red cells having been subjected to a n additional heat stress in the presence of nutrient-depleted preservative media. Although with their procedure Valeri et al. attempt to compensate for the injection of unbound "Cr by subtracting i t out in the plasma of subsequent blood samples, this approach does not distinguish between infused unbound "Cr from "Cr in plasma which is cleaved during the rapid cell destruction phase from "Cr-labeled cells. The sampling schedule and data analysis method for estimating the zero-time "Cr concentration, however, represents the most material difference in the single-label procedures used by the Valeri group. Their use of a straight line constructed using data from only two blood samples, drawn lOand 20, or 15 and 30 minutes postinjection of "Cr- labeled red cells, violates two important principles of optimal sampling theory and statistical estimation': the choice of the number and timing of samples in a kinetic-type experiment should be based on use of an appropiate kinetic model and nominal values for the

parameters of the model in order to assure accurate and precise experimental estimates. Translated into nonstatistical vernacular, this means that the number and timing of blood samples should be derived from knowledge of the "Cr disappearance curve during the rapid disappearance phase. Based upon the usual form of this curve (see Fig. 1 i n reference I and Fig. I . 15 in reference 9), i t is obvious why the limited and ill- timed samples used by Valeri et al. resulted in under- estimates of the zero-time 51Cr concentrations. (The reader can easily verify the adverse consequences of extrapolating lines through the 10,20and 15,30minute postinfusion points.) The use of only two points to derive a line of "best fit" is an unsound practice. Starting sampling late in the rapid-loss phase and extending i t into the stable phase produces data that arise from two different kinetic processes. Although data analysis procedures are available to interpret data obtained in this manner correctly, this is more than is warranted for the simple estimation of 24-hour "Cr red cell survivals. Figure I in the paper of Moroff et al. ' illustrates how the proposed sampling scheme and data analysis method of their committee is designed to result in more accurate and precise extrapolation to zero- time than Valeri's method when the data in fact are in the expected kinetic form. However, the suggestion of Valeri et al.' cannot be ignored, namely, that the performance of a sampling scheme such as that proposed by the Moroff Committee should be investi- gated in cases where the 24-hour survival is less than 65 percent.

The report of Beutler and West' provides a scientifically sound evaluation of the performance of the single-label procedure ("Cr) across a wide spectrum of 24-hour red cell survivals (<20- 100%). Using""'1'c- labeled autologous red cells to provide "true"estimates of red cell mass, the extrapolated "Cr red cell mass estimates were shown to be accurate to within 10 percent when survivals exceeded 60 percent. The red cell mass estimation error ranged from 16 to 28 percent when 24-hour survivals were less than 20 percent. When translated into 24-hour survival estimates, the (overestimated) "Cr-derived value was 26 percent when the "true" survival was 20 percent. The authors argue that the absolute overestimation (always <lo%, usually <5%) inherent in "Cr-based survival estimates enables the valid use of the single-label ( " 0 ) tech- nique. This report, therefore, constitutes a timely re- sponse to the suggestion of Valeri et al.' by rigorously evaluating "Cr-derived survival estimates against a 9 9 m T ~ standard. However, does a study of 12 preserved red cell units in a single preservative media, only 3 units of which showed 24-hour survivals of less than 60 percent, constitute, by itself, a reliable basis for all future applications of this single-label technique?

I l<,\\\l L \lo\ I9hJ \',,I 2.4 \<, 2 EVALUATING S T O R E D R E D CEI-L SURVIVAL 99

Might not the routine application of a double-label procedure using "Cr and yy'nTc be preferable? Apart from investigation of errors inherent in the use of the

Cr-based procedure, Beutler and West demonstrated the feasibility of employing a 5 'Cr/yymTcdouble-label procedure with obvious merits.

All matters relating to the estimation of survivability of preserved red cells are far from settled. However, properly interpreted, the group of related papers in this issue constitute important contributions toward resol- ution of the differences in results and methodologies. Three reports demonstrate procedures that are either known to be inadequate or that are likely to be reproducible, accurate, and precise. The merits of carefully chosen, detailed, and uniform methodology in cooperative evaluations, as well as sound experi- mental design and statistical estimation, and insistence on experimental verification of theoretically "good" ideas, are self-evident. Finally, blood preservation experimentalists need not feel bound to employ the particular single- or double-label procedures discussed by Valeri et al. and the Moroff Committee, but may also consider alternative techniques such as double- labeling with 9y"'Tc and 5'Cr as described by Beutler and West, a technique that may render moot the weaknesses of the former methods that have been so ho t ly debated. Carl C. Peck, MD, COL MC

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References Moroff G , Sohmer PK, Button LN. Proposed standardization of methods for determining the 24-hour survival of stored red cells. Transfusion 198424: 109-14. Valeri C R , Pivacek LE. Ouellet R , Gray A. A comparison of methods of determining the 100 percent survival of preserved red cells. Transfusion 1984:24: 105-8. Beutler E, West C . Measurement of the variability of stored red cells by the single-isotope technique using "Cr: analysis of validity. Transfusion 1984:24: 100-4. Pcck CC, Moore GL, Bolin RB. Adenine in blood prcscrvation. C R C Critical Reviews in Clinical Laboratory Sciences I98 I ; 13: 173-212. Strumia M M , Colwell LS, Dugan A. l h e measure of erythro- poeisis in anemias I. the mixing time and the immediate post- transfusion disappearance of T-1824 dye and of 51-Cr-tagged erythrocytes in relation t o blood volume determination. Blood

Zuck T F , Bensinger T A , Peck CC. et al . The in vivo survival of red blood cells stored in modified C P D with adenine: report of a multi-institutional cooperative effort. Transfusion 1977;17:

Sohmcr PR, Moore GL, Bcutler E, Peck CC. In vivo viability of red blood cells stored in CPDA-2. Transfusion 1982;22:479-84. Federov VV. Theory of optimal experiments. New York: Aca- demic Press, 1972. Mollison PL . Blood transfusion in clinical medicine. 5th ed. Oxford: Blackwell. 1972.

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Carl C . Peck, M D , C O L M C , DivisionofChnical Pharmacology, Departments of Medicine and Pharmacology, Uniformed Serviccs University of the Health Sciences. Bethcsda. M 11. [Reprint requests]

On presenting balanced views

Editorials appearing in this issue of the journal comment upon articles also published in this issue. In the recent past, referees usually have recommended rejection of highly controversial manuscripts. The editors have come to realize that these rejections may d o a disservice to our readers by denying them the opportunity to evaluate for themselves the conclusions drawn from new (or perhaps old) data. In this issue, we initiate the publication of controversial articles accompanied by comments by investigators knowledgeable in the matters presented. I t is not our intent to controvert authors, but rather to present balanced views of difficult and contentious topics.

I t is appropriate to inaugurate this editorial approach with articles about posttransfusion red cell survival studies. Debates about the proper method for performing these studies have raged for many years. It is not expected that these debates will be stilled by the current published material. But, readers who need to know will have the most recent information available to them.

I t is our intent to leave readers thoughtful. For example, one might question whether a double-label red cell survival method, with its small increased risk compared to a single-label method, should be used to determine whether an anticoagulant- preservative is acceptable. Significant error in the single-label method is present only in those units that show a survival somewhat less than that acceptable by the Federal Drug Administration. Or should we strive for the greatest exactitude in all matters? Editors should not decide the merits of these issues, but rather present the merits to their readers. TFZ