Cell and Tissue Banking1: 121–127, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

Hemodilution due to blood loss and transfusion and reliability ofcadaver tissue donor infectious disease testing

Ted EastlundDepartment of Laboratory Medicine and Pathology, Medical School, University of Minnesota, Box 609,Mayo bldg., 420 Delaware Street SE, Minneapolis, MN 55455, USA (Tel.: 612-273-3821; Fax: 612-273-5410;e-mail: eastl002@tc.umn.edu)

The transplantation of human bone, cartilage, cornea,skin and heart valve has become commonplace in theU.S. Over 300,000 bone allograffs and 42,000 corneaswere transplanted in 1992 [1–3]. Since these are ofhuman origin, the potential for donor-to-recipient dis-ease transmission exists and occurrences have beendocumented [4–7]. This is minimized through donorselection steps which include blood testing for infec-tious disease markers.

In 1987 a case was reported of HIV transmissionto multiple recipients of organs derived from a donortesting negative for HIV antibodies (anti-HIV) [8]. Thefalsely-negative test was due to massive blood loss andhemodilution from rapid infusion of blood, colloid andcrystalloid solutions. Testing of the HIV-infected organdonor was negative when blood was sampled immedi-ately after receiving blood transfusions amounting totwo to three total blood volumes and an additional largevolume of crystalloid solution over an 11-h interval.When a blood sample was obtained 48 h later and testedfor anti-HIV, the test was positive due to intravascularreplenishment of immunoglobulin from extravascularsites.

Transplantable organs and tissues are commonlyderived from cadaveric donors who died traumaticdeaths and received blood, blood components and crys-talloid solutions prior to donation. At a tissue bankin Minnesota, we found that 45% of 100 consecu-tive cadaveric tissue donors received blood transfu-sion prior to death and infectious disease testing. Toavoid falsely negative results, tissue procurement agen-cies perform infectious disease tests on pre-transfusionspecimens or on post-transfusion specimens if an eval-uation indicates that the sample was not hemodilutedto the extent that test results would be altered. Thefollowing review addresses whether blood loss replace-ment by transfusion causes hemodilution sufficientto render the sample invalid for infectious diseasetesting.

Physiologic considerations – importance ofblood loss

The transfusion of blood colloids or crystalloids doesnot by itself create hemodilution sufficient to affecttest results unless the donor has experienced blood lossprior to sampling. Without loss of blood, infusions donot lower blood protein concentrations to any greatextent. There are many clinical examples wherebynon-bleeding patients receive large volumes of bloodand crystalloids intravenously without seriously reduc-ing the concentration of red cells, blood proteins orimmunoglobulins. Nearly three decades ago it wasshown that isotonic salt solutions, when administeredintravenously, equilibrate between the blood and theextravascular fluid space in an average of 23 min [9].More recently this was demonstrated in healthy volun-teers [10]. In a non-bleeding person receiving an intra-venous bolus of 0.9% sodium chloride equal to 46%of the subject’s blood volume, 60% of the saline dif-fused out of the intravascular space in less than 20 min.Immediately after the bolus, the hematocrit of nor-movolemic subjects declined 6% and returned towardbaseline despite a continuous saline infusion at ratesexceeding 5 ml/kg/h. Many cadaveric organ and tissuedonors receive some form of intravenous fluids dur-ing premortem attempts at resuscitation. Non-bleedingpatients with myocardial infarction and heart failuremay experience severe hypotension and intravenousfluids may be administered in large quantities, oftenmany liters per day. Many cadaveric brain-dead organdonors, who may also be tissue donors, have sufficientbrain damage to impede production of anti-diuretichormone. Even without blood loss they may receiveup to 10–15 l of crystalloid solutions intravenously inone day. This is matched by a similar urine output andplasma protein constituents including infectious dis-ease markers are not lowered by dilution. In contrast apatient with severe blood loss receiving a similar large

122 Ted Eastlund

volume of intravenous fluids can have a marked low-ering of plasma protein including immunoglobulins,through dilution.

Physiologic considerations –Ig re-equilibration after blood loss

Blood loss alone without intravenous fluid administra-tion poses no hemodilution threat to the reliability ofinfectious disease testing. However, during attemptsat resuscitation following blood loss, large volumesof crystalloid solutions that are protein-poor andimmunoglobulin-poor may be infused. Immediatelyafter infusion of crystalloid to replace shed blood, theprospective donor’s blood protein and immunoglob-ulin concentrations are lowered. Crystalloid solutionsrapidly leave the circulation but intravenous fluids con-taining colloids remain in the circulation longer andtherefore can potentially contribute to hemodilution ofimmunoglobulin for a longer time period. Therefore,the amount and type of infusion is important to con-sider when estimating its contribution to dilution ofthe remaining protein which is to be measured duringinfectious disease testing.

How to estimate the contribution of crystalloids,colloids, plasma (with normal concentrations ofimmunoglobulin) and red blood cells varies with eachfluid transfused. In general, one can estimate there-equilibration of immunoglobulin within the vascularspace when the amount of blood loss is known andthe interval prior to sampling is determined. However,infused crystalloids may remain within the vascularspace only briefly and infused plasma protein fractionsmay remain longer; and if plasma is used as a replace-ment fluid (with normal IgG content), it may alter therate of IgG equilibration. It becomes more complexwith the uncertainty whether HBsAg, HIV antigensand viral nucleic acid re-equilibrate after blood loss andwhat the kinetics of re-equilibration are for IgM anti-bodies which have developed recently after infectionand are in a lesser concentration in the extravascularspace.

Following blood loss, intravascular protein ispredictably replenished from extravascular sites(Figure 1). Several decades ago this was demonstratedin humans by removing 20% of the total blood vol-ume acutely [11]. Blood volume returned to normal in72 h and 95–100 percent of replenished protein camefrom a pre-labeled extravascular pool [11]. However,if crystalloids or other immunoglobulin-poor fluids are

Figure 1. A simplified model for understanding the replacement ofintravascular protein levels after blood removal.

infused to replace blood loss, the immunoglobulinlevel may be acutely depressed and the reappearanceof the prospective donor’s immunoglobulin may bedelayed. According to Chopek and McCullough [12],the gradual removal of one total blood volume andreplacing it with albumin can reduce the concentra-tion of intravascular immunoglobulin to 35% of itsinitial level if measured immediately without replen-ishment by protein in the extravascular space. Over50% of the total body immunoglobulin is extravascu-lar and intravascular restoration takes place quickly.The intravascular immunoglobulin refill rate is 3–5%of the intravascular content per hour and replenishmentshould be nearly complete by 24 h after removal or lossof a single blood volume and replacement with fluidfree of immunoglobulin [12]. Keller and Urbaniak [13]demonstrated that a total blood volume removal andreplacement with fluids free of immunoglobulin can beperformed every 2 to 3 days without depleting bloodimmunoglobulin concentrations. These studies suggestthat blood samples taken 24 h after blood loss and fluidreplacement, even if over one total blood volume islost, should not be more than moderately depleted ofthe donor’s immunoglobulin.

The effect of in vitro dilution oninfectious disease test readings

Heck et al. [14] studied the effect ofin vitro hemod-ilution on detectability of anti-HIV including somesamples with low-level reactivity. They diluted eightsera from patients with repeatedly reactive results inan enzyme immunoassay (EIA) for anti-HIV-1/HIV-2.

Hemodilution and transfusion causing infectious disease tests 123

Samples were diluted with Ringers lactate and plasmato 20% and 50% and tested with two different testkit manufacturers at two different laboratories. Theyconcluded that 50% of the blood sample volumecould safely be replaced of non-serum diluent and thesensitivity of the test is not affected.

We conducted similar studies to determine the effectof more extreme levels ofin vitro hemodilution on theEIA readings of patient serum samples positive for anti-HCV, anti-HIV and HBsAg [15]. Representative posi-tive donor sera were diluted with 0.9% sodium chlorideor negative control sera provided by the test kit manu-facturer (Abbott Laboratories, Abbott Park, IL, USA)and tested by EIA. Samples were chosen which weretypical positives. Absorbance readings were examinedat different dilutions tested. A typical HBsAg posi-tive sample with an optical density absorbance read-ing of > 1.0 required dilution at 1 : 360,000 beforeabsorbance readings became less than the cutoff read-ing that defined a reactive (positive) test (Figure 2). Aserum sample positive for anti-HCV became nonreac-tive after diluting to 1 : 16 (Figure 3). A sample positivefor anti-HIV with a typical optical density absorbancereading of> 1.5 did not have readings below the cutofflevel until diluting to 1 : 10,000 to 1 : 20,000 (Figure 4).These studies are consistent with the concept that EIAabsorbance readings are reduced with hemodilution butthere is not a straight line relationship (Figure 5). A verylarge dilution is needed before readings begin a steepdecline.

Figure 2. Hemodilution and HBsAg readings. Closed circles rep-resent absorbance (optical density) readings in the HBsAg EIA testafter dilution with 0.9% sodium chloride at the various levels indi-cated. The open circles represent absorbance readings after dilutionwith the negative control plasma provided by the manufacturer of theHBsAg test kit.

The effect of dilution by pooling negativewith positive samples

Several studies have demonstrated that diluting serumfrom individuals positive for anti-HIV with serum fromup to 10 or 15 other individuals negative for anti-HIVdoes not reduce the sensitivity of testing. Howanitzet al. [16] diluted serum 30-fold and it remained

Figure 3. Hemodilution and anti-HCV readings. Closed circles rep-resent absorbance (optical density) readings in the anti-HCV EIAtest after dilution with 0.9% sodium chloride at the various levelsindicated. The open circles represent absorbance readings after dilu-tion with the negative control plasma provided by the manufacturerof the anti-HCV test kit.

Figure 4. Hemodilution and anti-HIV readings. Closed circles rep-resent absorbance (optical density) readings in the anti-HIV EIA testafter dilution with 0.9% sodium chloride at the various levels indi-cated. The open circles represent absorbance readings after dilutionwith the negative control plasma provided by the manufacturer of theanti-HIV test kit.

124 Ted Eastlund

Figure 5. The theoretical and actual effect of dilution on enzymeimmunoassay absorbance readings.

reactive for anti-HIV by EIA. There have been severalstudies attempting to reduce reagent expenses associ-ated with screening of large numbers of the populationfor HIV by pooling sera from many individuals priorto testing. Cahoon-Younget al. [17] combined 5000samples into 500 pools. Testing was performed bothon individual samples and on the 500 pools. Therewere 104 individuals who were positive when all 5000were tested individually and when pooled there were1–3 reactive samples in each of the 500 pools. Allindividuals with HIV antibodies were detected whentesting pooled samples. There was complete agreementbetween testing in pools and testing of individual sam-ples. In addition, a false positive sample (EIA repeatreactive but negative with HIV Western blot confir-mation testing) became nonreactive with the dilution.Most positive samples were diluted 1 : 10 due to pool-ing but because there were one to three reactive samplesper pool, a few of the positive samples were diluted1 : 3 or 1 : 5. The EIA reactivity reading of the positivesamples in the pools was 30–130% of the EIA readingsfrom testing the individual samples. Most of the dilutedreactive samples had EIA readings that were 20–50%lower than the EIA readings of undiluted individualpositive samples.

Behetset al. [18] tested 8000 sera of workers andspouses in Japan. There were 2.4% of these that werepositive for anti-HIV. When tested in pools of 10 so thatthere were 800 pools, all samples which were positivewere identified. There were no false negative pools.Dilution from pooling improved specificity with fewerfalse positive results. A comprehensive study by Klineet al. [19] showed similar results. They reported three

studies. One involved 975 persons with 36 positivesamples and 65 pools (15 samples per pool). All poolswhich had 2–3 positive samples were HIV positive.Two borderline positive samples were not detected inthe pools. In another set of experiments testing 1380persons, they combined 15 samples per pool and had 92pools tested. Of the pools, 51 contained positive sera.All pools with reactive samples were positive in thetesting. Another subset of experiments had 138 poolswith 10 samples per pool. Fifty of the pools contained atleast 1 positive specimen. All pools with reactive sam-ples were positive. The preceding four studies takenin aggregate report that anti-HIV was detected in 394of 396 positive individual serum samples even afterdilution into pools with a dilution factor of 10–30. Onlytwo borderline samples were not detected. The falsepositive samples became negative with dilution. Thisgives a sensitivity of 99.4% and specificity of 100%.

Other studies have shown that even weakly reac-tive samples can be heavily diluted without loss ofdetectability. Monzonet al. [20] tested 3000 samplesin pools of 5, 10, and 20 samples per pool and foundthat even samples with very low level reactivity, suchas that observed in recent seroconversions, remaineddetectable when in a pool with 4 other equal sizednegative sera. A study by Jiet al. [21] showed thateven larger dilutions do not cause falsely negative anti-HIV test results. They diluted weakly-reactive anti-HIV samples with 20–50 other samples and the poolremained reactive. Others have addressed these issuesand pooling seems to be an effective and sensitivemethod for mass screening [22–26]. Interestingly, stud-ies using pooled sera have discovered a number of seracontaining anti-HIV-1 (Western blot positive) whichwere repeatedly reactive in the diluted EIA test poolsbut not detected when the undiluted sample was testedon its own [26,27].

The above studies clearly demonstrated that aninfected person’s blood can be heavily diluted and thesensitivity of EIA tests for infectious disease testingcan still be relied upon. However, this conclusion isbased on the characteristics of EIA testing, whereby alarge amount of dilution is necessary prior to observ-ing even a small decrease in EIA reading (Figure 5).Infectious disease testing using other methods such asviral nucleic acid testing using genomic amplificationtechniques may behave differently with hemodilution.

Recently Lefrereet al. [29] tested hepatitis B, hep-atitis C (HCV) and HIV nucleic acid in diluted poolsof sera and found only a small decrease in sensitivity.Yerly et al. [30] tested pools of 50 sera for HIV-1 andHCV RNA using polymerase chain reaction techniques

Hemodilution and transfusion causing infectious disease tests 125

and found that sensitivity was not significantly reduced.Despite a 50-fold dilution they were able to identify allsamples of persons who had recent infection with viralRNA present but EIA antibody tests were negative.Despite dilution they reported sufficient sensitivity todetect 33 copies/ml of HCV RNA and 1000 copies/mlof HIV RNA.

Management of transfused cadaveric tissueor organ donors to avoid false-negativetest results due to hemodilution

In the United States, federal governmental regulationsand national professional standards require physicianinterpretation as to suitability of blood samples priorto infectious disease testing of a prospective cadaverdonor who has had blood loss and transfusion. In1993 U.S. federal regulations [31] were first published,with subsequent modification [32], requiring quaran-tine of tissue from adult donors who had blood loss andreceived greater than 2 l of blood and colloids within48 h of blood sampling or greater than 1 l of crystalloidwithin 1 h of sampling. The donated tissue was not tobe used unless a pretransfusion sample was availablefor testing or an algorithm is used to evaluate blood andcolloid volumes administered in the 48 h prior to bloodsampling to ensure that plasma dilution sufficient toalter test results has not occurred.

American Association of Tissue Bank Standardsrequire tissue banks to follow written proceduresregarding hemodilution limits for infectious diseasetesting of post-transfusion blood samples [33]. Accept-ability limits must be part of written procedures. Stan-dards of the American Red Cross Tissue Servicesrequire that in the case of blood loss and transfusionwithin 48 h of death, a preinfusion sample must beused [34]. A post-infusion sample may be used inpatients with major clinical blood loss provided thatthe tissue bank physician has evaluated whether bloodand crystalloid infusions have compensated for bloodloss, hemodilution is 50 percent or less of the totalblood volume and the tissue bank physician gives writ-ten approval. The estimated amount of hemodilutiondepends upon the type of material infused and theamount of time elapsed since infusion.

In determining whether post-transfusion hemodilu-tion is extensive enough to possibly affect test resultsa practical but conservative approach is needed thattakes into account the necessary caution to avoid therisk of accepting a donor who is infected. Many tissuebanks have adopted this approach. Some tissue banks

have taken a different approach in testing donors withtransfusions. LeForet al. [35] have described a proce-dure of concentrating the protein in donor blood sam-ples suspected to have been hemodiluted. In their studythey found that a pretransfusion sample was not avail-able for about 20% of their donors who had receivedblood or had incomplete transfusion histories. Theyused the unambiguous quantitation of serum albuminand total protein to define hemodilution and, if present,hemoconcentration of sera by ultrafiltration to normalprotein levels was performed prior to infectious dis-ease testing. Their results showed excellent correlationbetween serum dilution and protein concentration anda quantitative recovery of 96.9% upon hemoconcentra-tion. Known positive sera (anti-CMV; anti-HTLV; anti-HIV; anti-HBs; anti-HCV and HBsAg) were added tonormal sera and diluted up to 1 : 2000, well beyonddetectable levels. A qualitative recovery of 100% anda quantitative recovery of 97.6% of antibody or anti-gen reactivity was achieved upon hemoconcentrationand retesting. Their hemoconcentration method has notbeen widely adopted.

Summary

Hemodilution as a cause of falsely-negative test resultsmust be considered in cadaver tissue and organ donorsdying with massive blood loss and transfusion. Admin-istration of blood, colloid and crystalloid does notalter EIA test results unless there is an extremelylarge amount of blood loss and hemodilution is severe.Infectious disease antibody tests are very sensitive andin vitro studies show their performance is not affectedby the amounts of hemodilution expected during mostresuscitation attempts, even with blood loss. Evaluat-ing the blood loss and transfusions can give an esti-mate whether hemodilution is significant. The amountof hemodilution varies with the amount of blood loss,the amount and type of material infused and the timecourse of blood loss, transfusion and blood sampling.An algorithm can guide and simplify making a decisionwhether to accept a diluted blood sample for testing.No one algorithm can be comprehensive enough tocover all possible variations of donor blood loss, trans-fusions and sample testing. The tissue bank physicianhas a very important role in making final interpretationsabout the acceptance of blood samples from prospec-tive donors, particularly if there has been potentialblood loss. However, determining tissue and organdonor suitability is an inexact science requiring physi-cian judgment. This is demonstrated once again in the

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assessment of hemodilution and deciding whether touse blood samples for infectious disease after bloodloss and transfusion. The decision involves making areasonable balance of risk versus benefit and the role ofthe tissue bank physician in the evaluation and decisionmaking is important. As in the practice of medicinein general, minimum and general requirements can beset regarding blood sample suitability after transfusionbut every possible medical history scenario cannot beconsidered in advance.

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