radioactive chromium 51 asdm5migu4zj3pb.cloudfront.net/manuscripts/102000/102855/jci5310… ·...

THE USE OF RADIOACTIVE CHROMIUM51 AS AN ERYTHRO-CYTE TAGGINGAGENTFORTHE DETERMINATIONOF

REDCELL SURVIVAL IN VIVO 1,2

By FRANKLIN G. EBAUGH, JR.,3 CHARLESP. EMERSON,AND JOSEPHF. ROSSWITH THE TECHNICAL ASSISTANCE oF ROSEALOIA, PEARLHALPERIN,

AND HELEN RICHARDS

(From the Robert Dawson Evans Memorial Laboratory, Massachusetts Menorial Hospitals,and the Department of Medicine, Boston University School of Medicine,

Boston, Mass.)

(Submitted for publication May 27, 1953; accepted August 18, 1953)

One of the most useful methods available for thequantitative measurement of hemolytic rates inclinical subjects and for the evaluation of red cellviability after storage is based on the survival oftransfused erythrocytes. Access to such data,however, has been restricted because of limitationsin the methods hithertofore available for measur-ing red cell survival in vivo.

The differential agglutination method of count-ing donor cells, the procedure most commonly em-ployed, has limitations in that large transfusionsare required;* the donor blood must be devoid ofall antigenic isoagglutinins which are not likewisepossessed by the recipient; the recipient's cellsmust contain agglutinogen A or B or Mor a com-bination of the three agglutinogens which is notcontained in the donor blood; and, finally, thismethod excludes the use of autotransfusion, whichwould eliminate the risk of transmission of he-mologous serum jaundice.

The labelling of donor cells with radioactive iron(Fe55) permits the conduct of survival studies on ashort term basis (1-3) but re-utilization of radioac-tive iron released from hemolyzed donor cells andthe subsequent incorporation of this label in the re-cipient's red cells preclude its use in studies ex-tending for periods longer than 24 to 48 hours.

1 This work was supported in part by a contract betweenthe Atomic Energy Commission and the MassachusettsMemorial Hospitals, and funds from the American CancerSociety of Massachusetts, Inc., Boston Blood GroupingLaboratory.

2 Presented in part before the Committee on Blood andRelated Problems, National Research Council, Washing-ton, D. C., September 17, 1952, and the American Federa-tion for Clinical Research, Eastern Section, Boston, Mas-sachusetts, April 16, 1953.

8 Part of this work was performed while a Public HealthService Research Fellow of the National Cancer Institute.

Furthermore, this method requires the accumula-tion in the prospective donor of radioactive ironin relatively high concentration, a procedure whichis not desirable. Other tagging methods that havebeen described, involving the use of sulfhemoglobin(4), N15 (5), C14 (6), p32 (7), and Zn65 (8), aresimilarly objectionable from one or another stand-point.

Gray and Sterling (9, 10) have demonstratedthat a firm union between erythrocytes and chro-mium can be established in vitro with great rapidityand with no apparent damage to the exposed cells.These observations imply that radioactive chro-mium might serve admirably, not only as a tag-ging device for measuring circulating red cell vol-ume as proposed by these authors, but as a basisfor measuring red cell survival in vivo. The po-tential advantages of its use in this manner areseveral:

1. By reinjecting chromium-tagged red cellsinto their original host, i.e., autotransfusion, itmight be possible to measure the longevity of eryth-rocytes in their native environment. Such an ap-proach would extend the application of this typeof study to hemolytic disorders based on inherentred cell defects which are not amenable to investi-gation by transfusion, and would insure the validityof survival data in all types of cases.

2. This method may provide a simple and defi-nite method of evaluating the effectiveness ofmethods and media for in vitro preservation oferythrocytes in stored blood. Relatively smallsamples of donor blood might suffice for accuratecounting after injection. A reduction in volumeof injected material might be achieved to the ex-tent that several aliquot samples from one unit ofblood might be transfused after successive intervals

1260

MEASUREMENTOF RBC SURVIVAL BY CHROMIUM51TAGGING

of storage, or after storage under a variety of con-ditions, affording a more accurate basis for study-ing the rate of deterioration of preserved red cells.

3. By substituting autotransfusion for transfu-sions in survival studies, the risk of infection, in-cluding hepatitis, and the danger of isoimmuniza-tion in the recipient are excluded.

It has been reported (9, 10) that erythrocytesbind 80 to 90 per cent of sodium chromate(Na2Cr51O4) when trace amounts of the latterare added to cell suspensions in vitro, the globinportion of the hemoglobin molecule being the siteof the combination. These observations have beenamplified and extended by a series of experimentsdesigned to determine:

(1) The effect of storage on the kinetics ofchromium uptake by erythrocytes;

(2) the influence of chromium, and the effect ofthe conditions imposed by the labelling pro-cedure, on the post-transfusion of red cellsexposed to this metal in vitro;

(3) the avidity and stability of the linkage be-tween erythrocytes and chromium; and

(4) the degree to which sodium chromate re-leased from hemolyzed red cells becomesincorporated into other circulating red cells,or into red cells subsequently produced bythe bone marrow of the recipient, the oc-currence of which would invalidate redcell survival data obtained by this taggingprocedure.

METHODS

Preparation and administration of Na2Cr'104 labelled bloodDonor blood used in these experiments was collected

and stored in collapsible bags constructed of polyvinylplastic4 containing 75 ml. of acid-citrate-dextrose (ACD)solution N. I. H. Formula A.5 Donor red cells weretagged with radioactive chromium 6 by introducing 3 to 5ml. of an aqueous solution containing Na.Cr'O4 (pH 6.0to 8.0) and with a specific activity of 0.25 to 1.0 milli-curies per mg. of chromium metal followed by approxi-mately 10 volumes of isotonic saline into the plastic donortubing connected to the bag containing whole blood and

4 "Blood Packs" distributed by Fenwal Laboratories,Inc., Framingham, Massachusetts.

6 Seventy-five ml. of this solution contains trisodium ci-trate, 1.65 Gm., citric acid, 0.60 Gm., and dextrose, 1.84Gm.

60btained from Abbott Laboratories, North Chicago,Illinois. Wewish to express our gratitude to Dr. DonaleeTabern for his kind cooperation.

ACD diluent. Na.Cr'O4 should not be autoclaved whenmixed with ACD solution, since it will decompose underthese conditions and become incapable of labelling erythro-cytes. The contents of the bag were thoroughly mixedand equilibration of the blood and chromium salt allowedto proceed for various periods of time and under a varietyof conditions which are specified in Table I. The bloodsof Experiments 1 and 2 were continually agitated on amechanical shaker during the period of equilibration; thebloods for all the other experiments were gently mixedby inversion every 10 to 20 minutes during the equilibra-tion period. Final preparation of the tagged blood forinjection included the preliminary separation of cells andplasma by rotation at 2000 rpm. in an International Cen-trifuge (Model B. P.) for 30 minutes at room tempera-ture, followed by the removal of approximately 30 to 85per cent of the plasma. In Experiments 1 and 2 the taggedred cells were washed once in their native plasma. InExperiment 3, the donor cells were suspended in saline,equilibrated with Cr51, washed and finally resuspended insaline before injection. These procedures were calcu-lated to insure the maximum degree of chromium bindingwith least exposure to damage by the erythrocytes, andreduction to a minimum of the extra-corpuscular chromiumin the injected mixture.

The first series of transfusions (Series I, Experiments1-9) involved the injection of normal donor blood whichhad been stored at 4 to 6' C. for 0 to 26 days, then taggedwith Na,Cr'104 by the method described above. Recipi-ents were healthy medical students, each of whom wassubjected to a 500 ml. phlebotomy immediately beforetransfusion. 'None of these subjects, with one exception,had been transfused previously; (Subject CN had re-ceived experimental transfusions on three occasions dur-ing the preceding two years).

Individuals receiving experimental transfusions in Se-ries II experiments were hospitalized patients withouthematologic abnormalities or diseases known to influencethe life span of erythrocytes. In preparation for theseexperiments, approximately 250 ml. of a 500 ml. ACDblood unit was tagged with Cr" prior to storage. Thetagged blood was stored at 4 to 6' C. for periods specifiedin Table I. The remainder of this unit was stored in theabsence of chromium, and the red cells were tagged im-mediately prior to injection. Fifty ml. aliquots of eachportion were transfused at weekly intervals, after the re-moval of 80 per cent of the plasma, and the resuspensionof the cells in a sterile isotonic solution of sodium chloridecontaining 1 Gm. of dextrose per 100 ml.

As a precaution against bacterial infection of the re-cipient, stored blood was cultured aerobically and anaero-bically at 5' and 37' C. on chopped meat, blood broth, andblood agar, and gram stained smears of the material wereexamined microscopically prior to injection.

Isoimmunization by transfused red cells was excludedby the observation of a negative Coomb's test, four to fivemonths after transfusion, performed on fresh cells fromthe original donor following their incubation for one hourat 37' C. with the serum of the recipient.

1261

a 00 0%

8 N t8 a0 0t-. ~014 No '0.4 ) c to00UC4 '0U)c0%000% 000000000c 0 a 00000 00 t. m0 o' 0 U) U)

_. _ _4 _4_4 _

q I- 0 00

o t'. 4 r. %o C4n0 00a 0%000 .0SO C0%0 0c 0c0000t0% 0- 0 00U)nUak 0 loo0 o, o0 Coa, 0o 00Os >r o ulouIf

o 1a so cio_0%C" U)U) t2.C* _U)U)0%¢U) *qe404.0%0000 o0 0oo oIoolooat000U__

044,010 i i aU 00 Id

Ons '0_r1 O>o boo

- oo 0i I in 't 00 No -0 ON i c \Incq n-

o 0X > >.C X X > .fi 0 0- f I=tgt|r gr =tgo*+ _ o+ O S 0 0N 0_ Os^S_ o

00 1 00 00 0 o0- Cl U) Cl

z z ~~~4~~z.eI~ ~ ~ mI0

EQU E-E E-E O~+j

EOD4Ed4-D "aD40.0 0.0 0.0 0.0 0.0 z;0 0.0

_.~~~~~~~~~~~2

0; L -; U

00 0% 0 ) U U) 0%O as 0% 00 o% 0o 00

'0 '0 '0 _0 co

cs o ooo co

.0 .0 . 0 .0 .0

q Cl 0 (4 00 U

o 0s U) 0B U) 0 U)

00 0oO 00)U U)'0 U)

o o

0m

V ~ ~ ~ ~ ~ 00 U) U) U)

_ U)e U) '0 1'

1262

.0

mgt.,_

._

'00

02

0- d0200.

-oCO

8

033CO

.2-..

A 0Q4

>

I :3

00@It~~~~~~~~~~~~~~~~~

CO

02

02

CO

02

.E

l0

.00

1¢

'.5.

0o

0

~0

b0

beD

C)

o

b_

'0

.0*0

co

00Cd

0

0

0

O

.0

02

.°

d

0

u

'0a.0

0 4i.-d 6 0* &.. D

"

U-

0

0.0.

.fi a

0 Cd

.St¢w

-,8.0

0

X4

0L)b0

40

0-

0

0

0)

I.

0)

*-

(402.-v

'0 a0038 0m X

02tu.2>2 bo

I 420012 22

,> 40-

8o000 0%0%VNoooOwv obo^

C

o%cou) in 00 00 co ) '0

00

tv~-oo.eqeq --

0v%eRr0 iu~00Or'ee0%v010co004cooqeqW

ov0u)e 000W- eqorovoo

. .4

a..t' o.-u.

0.0 ~ n f0.0

00LoW 00vv

0SE0 00 .

- 00

0. 0%

z z

CU Ccoe 1eq

1263

0.._

'0

30:

la0

V

0

'0.0

10

4-

0

00

0

.0

0

0i

a

I

I

0 0. 0 000 00 0000 00 0%Ol0% 00 %0t %0't'

U. I 0

eq0 eq0C4coon 0Nloc

.0 ou3 .E040co33o

o UcOYOuU:N:s^On O00":_ _0\0*

0 1 1

U> 0

O~~~~~O

OI U

.20~~~~~~0

'4 CUd o C'c. '

( U

(3 Cl

od co

00 000' 00

00

00 00 00

'0. 0 0

o - eq

I

0

=I

to

t010

Vi

040%

Et °

v ._E-4 CU...

cU 0 ew4

la a0 00 0

00._

beco

CU

'0.9

t.0VC_

o40.

100.0.0

0

p I

I;1

.4

I

0

._

4ica

0

ct

.2UcJ

m

C*00

00

be

V

10co

.0

'010

0Uv

.0

'00

0

00

0

0

0 *a-44 .

a 0-

.04

V04)

(.4)

(.0

.2 V.. .0'

.-a

0u8

0602

ma;.

@Q-Cit $ cyg

(z306I

I I I I

I I

ou) I.'@,o C5 fO N CE)CC) oeO )oc O '0 oN 09oNNOOonNrNr 'Ooou) ovU)U) 0bN1E)C) CONqE)t O%NrN

'OC )@O)t)v U) NtO mm I- U) t N'OU)U)0 NUbE) COU)NN I-

' I IWin INW)VVMo Oeooo_ o N

N N* CE) N+ N N* _ _N NS -" "

A ' Ia0 AU)

NNVW 4 o N N V e 4q

-~~~~

In.~~~ ~ ~ ~ ~ ~~~

ch co o

co 0 V* I

IV Is e

N E E

CE - CE - CE)

o NC! q.CK cooo

CE) N N N

CE U)

i n ) in

-

1264

I

gP..

'0

04

4040

.S'Is40

.9*SI

0

waI =o4 2m 0o

O

1m

IC

I

I

i1

la

I

A

I

tm

W

3

C

.a

t:1

I11--

,..

d

0

02A

v

0)

0

0 6

C0

V0

A.

-4

EUC04

i 4D0 ..

-0~?U41g30 I

I

-

MEASUREMENTOF RBC SURVIVAL BY CHROMIUM5" TAGGING

Radioactivity measurementThe gammaemission of chromium 51, half life 27.5 days

(11), was counted by a well-type scintillation counter,7having a gamma counting efficiency of approximately 37per cent. Counts were performed on 3 to 4 ml. aliquotsof whole blood contained in glass test tubes (0. D. 15 mm.,or in 4 ml. Gibson hematocrit tubes).8 When test tubeswere used, the contents were always adjusted to the samevolume for any series of bloods from the same recipientby the addition of saline solution; Gibson tubes were cen-trifuged at 3000 rpm. for one hour and, after removalof the plasma, placed in the scintillation counter well.Variations in geometry due to differences in the heightof the red cell column in these specimens were compen-sated for by means of a correction factor applied to theobserved counts. This factor was based on the empiricalobservation that the counting rate decreased 1.25 percent for each unit of hematocrit increase between 25 and53 per cent. The number of counts measured in thesesamples varied from 8000 to 25,600. The backgroundranged from 100 to 116 counts per minute. Sufficient sam-ple and background counts were measured to assure astandard deviation of 2 per cent or less for all but theweakest samples (Table II). A suitable chromium stand-ard was counted every third or fouth sample tested, allcounts being corrected for physical decay of Cr'1 and forvariations in the characteristics of the scintillation counter(which were negligible) by reference to this Cr'1 standard.

The percentage of Cr'1 taken up by red cells in vitrowas determined as follows:

Aliquot portions of whole blood-sodium chromate mix-tures, 0.1 to 0.2 ml. in volume, were placed in six testtubes. Three samples were diluted immediately with 10ml. of isotonic saline, then centrifuged as quickly as pos-

TABLE II

Comparison of the theoretical counting errors of the selectiveagglutination and Cr'1 methods*

Percentage donor cells surviving destruction

Days after Selectivetransfusion agglutination Crsl counting

0 100 i 12 100 i4.030 64 d 7.7 57 2.386 14 2.2 8.0 0.32

103 4.0k 1.2 3.0 4 0.24

*These figures represent errors resulting solely fromrandom distribution, their magnitude depending upon thenumber of units counted and the ratio of the numericalvalue of the sample to that of the blank or background.The standard deviation is expressed as per cent of theoriginal number of donor cells surviving destruction onthe day indicated. The standard deviation expressed asper cent of donor cells present on a given day would be12 per cent and 4 per cent for the selective agglutinationtechnic and Crl technic, respectively, on day 0 and 28per cent and 8 per cent, respectively, 103 days after trans-fusion.

7 Sodium iodine thallium activated crystal in an AtomicInstrument Company scintillation head.

8 Macalaster Bicknell Company, Boston, Massachusetts.

sible in order to prevent further Cr' uptake. The redcell sediment in these tubes was washed three times andsuspended in isotonic saline solution. The radioactivityin each tube was measured and the percentage uptake ofchromium by the red cells calculated by dividing the ra-dioactive count in each sample of washed erythrocytes bythe total number of counts in an aliquot mixture of wholeblood and chromium, and multiplying the resulting valueby 100. The concentration of Cr' in the red cells of therecipient was obtained by dividing the number of countsobserved in each sample by its volume, expressed as ml.,and by its hematocrit reading. The hematocrit readingswere not corrected for amount of "trapped" plasma forreasons detailed below.

Blood volume measurement

The plasma volume of each recipient was determined bythe T-1824 dye dilution technique (12). The total cir-culating red cell mass was calculated on the basis of theplasma volume and venous hematocrit, the latter havingbeen modified by the correction factor 0.9 to correct forplasma occluded in the packed cell mass or for inequali-ties between the hematocrit of large and small vesselsor for a combination of both.

(1) red cell volume (ml.)plasma volume (ml.)

100 - 0.9 X venous hematocrit (%)X 100 - plasma volume (ml.)

In the first series of experiments red cell volume wasdetermined in this manner two to three months followingtransfusion; in Series II, this was done at the time of in-jection of Cr' tagged cells.

Calculation of post-transfusion survival of chromium-labelled erythrocytesThe percentage survival of transfused Cr' labelled cells

in the recipient's circulating blood at any time after thetransfusion was calculated from the following formula:

(2) Percentage donor cell survivalRadioactive Cr'1 per ml. of recipient's packed red

blood cells X recipient's total red cell massTotal erythrocyte Cr'1 transfused'

The concentration of Cr'1 per ml. of the recipient'spacked red blood cells (the "specific activity of the redcells") was calculated by dividing the radioactivity perml. of whole blood by the uncorrected centrifuge venoushematocrit value as stated above. No correction factorfor "trapped" plasma in the cell mass was applied to thishematocrit value because of disagreement on the part ofthe authors as to the magnitude of this factor (13, 14).If the hematocrit value had been modified by such a fac-tor, the specific activity of the red cells and hence the per-centage survival would have been higher than the val-ues reported in this study, the difference being equal to thevalue of the correction factor applied.

9 Note: this value does not include the small amount offree Cr'1 which was present in the injected plasma.

126S

FRANKLIN G. EBAUGH, JR., CHARLESP. EMERSON,AND JOSEPH F. ROSS

This method of calculating percentage survival is basedexclusively upon measured values, i.e., the total radio-activity of the tagged donor cells, the blood volume ofthe recipient, and the actual concentration of transfusedcells per unit volume of the recipient's blood. (SeeTable I.)

Inagglutinable cell countsRed cell counts were performed on cell suspensions

prepared by diluting 0.1 ml. samples of whole blood with20 ml. of isotonic saline solution. A 0.1 to 0.2 ml. ali-quot sample of each suspension was placed in a 12 by75 mm. test tube containing approximately 10 mg. ofdried anti-A or M serum 10 (15). A second aliquotsample was placed in a hemocytometer chamber, and thetotal red cell concentration determined on the basis of acount of 1000 cells or more. The test tubes containing thecell suspension and dissolved antisera were centrifuged at900 rpm. for one minute, after which the cell sediment wasresuspended by tapping the tube vigorously 30 to 50 times.An aliquot portion of this suspension containing a mix-ture of agglutinated and inagglutinated cells was thentransferred to a counting chamber and the free, inag-glutinated cells counted. In each instance the minimumnumber of inagglutinated cells counted was 200, or thenumber present in 18 mm.' of counting area when thenumber in this area was less than 200. Prior to eachtransfusion, a blank inagglutinable cell count was per-formed on the recipient cell suspension containing an ali-quot portion of the anti-serum reserved for that particu-lar study. The ratio of the number of inagglutinable cellsminus the blank count to the total cell count multipliedby 100 was expressed as per cent donor cells. The fielderror in the selective agglutination counts reported inthis series varied from a S. D. ± 6 to S. D. ± 15 per cent(Table II). Calculations of percentage of survival ofdonor cells counted by the selective agglutination methodwere based on T-1824 blood volume data, as in the caseof the Cr5' tagged cells.

EXPERIMENTAL OBSERVATIONS

Chromation of Erythrocytes in Vitro

Influence of temperature and pH

The rate at which chromium combined with redcells when sodium chromate was added to wholeACD blood was dependent upon the tempera-ture and pH of the mixture, as demonstrated inFigure 1 and Table III. At 390 C., 90 per cent ofavailable chromium was incorporated in the eryth-rocytes within a period of five minutes, as com-pared to a 50 to 75 per cent uptake at 260 C. and10 t4 20 per cent at 1.80 C. After thirty minutes ofequilibration the proportions taken up at 26° and

10 Powdered grouping serum, Lederle Laboratories,Pearl River, New York.

390 C. were comparable, i.e., approximately 90 percent. The influence of pH on the kinetics of thisreaction is apparent from the observation that thechromium uptake at pH 6.0 was three to four timesas rapid as at pH 7.3 (Table III).

Influence of storage, concentration of Na2Cr504,and stability of the Cr51-erythrocyte union invitro

Preliminary storage of erythrocytes at 40 to60 C. for periods as long as twenty-nine days didnot appear to reduce their affinity for chromium.On the contrary, Cr5' uptake was more rapid instored than in fresh blood, due perhaps to thegreater acidity of the former. The pH of wholeACDblood immediately after collection is approxi-mately 7.2; after storage for one week at 40 to 60C. the expected pH value is approximately 7.0,falling to 6.8 after two weeks, and 6.6 to 6.7 afterthree weeks or longer. Chromium uptake, ex-pressed in terms of the percentage bound by redcells following the addition of Na,Cr 5'04, was notinfluenced by variations in concentration rangingfrom 0.25 to 9.5 micrograms of chromium salt (ex-pressed in terms of chromium metal content) perml. of whole blood (Table I, Figure 1). Theerythrocyte-chromium bond remained stable dur-ing storage in ACDplasma, inasmuch as there wasno elution of Crp5 from tagged erythrocytes storedas long as thirty-six days (Table I, Experiments10, 13, 17, and 19).

Does Cr5' released from hemolyzed cells relabel therecipient's red cells!

The extent to which Cr5l from hemolyzed redcells may combine with intact erythrocytes in vitrowas investigated as follows:

Radioactive sodium chromate was added towhole ACDblood in a concentration of 2.5 micro-grams per ml., and the mixture, divided into threealiquot samples, was allowed to stand for ninetyminutes at 1.80, 26.60, and 390 C. The red cellsin each sample, which had taken up 40, 84, and 92per cent of available chromium, respectively, werewashed three times in saline solution and thensubjected to repeated freezing and thawing untilhemolysis was complete. Each hemolysate wasmixed with an equal volume of washed, intacterythrocytes and incubated for ninety minutes at

1266

MEASUREMENTOF RBC SURVIVAL BY CHROMIUM"1TAGGING

*0

* FRESH ACD BLOODo ACD BLOODSTORED23-28 DAYS

i.erc26.60C39.C"C

0 -..

0--.~~~~~~~~~~0~~~~~~~~~~............

0

0.

0 30 60 90 120 130 180

MINUTES

FIG. 1. THE RATE OF SODIUM CHROMATEUPTAKEBY FRESH AND AGEDACDBLOODATDIFFERENT TEMPERATURES

The pH of the stored blood was between 6.7 and 6.8. The pH of the fresh blood was 7.1to 7.2. The concentration of Na,Cr'10 (expressed in terms of chromium metal content)was 2.5 gammaper ml. in all bloods except one of the two fresh bloods studied at 26° C.which was 0.25 gammaper ml.

the same temperature to which the hemolyzedcells had originally been exposed. Finally, the in-tact cells were separated, washed, and tested forCr5l uptake, which was discovered to be negligiblein all samples (i.e., less than 0.5 per cent). Thein vitro observations were substantiated by the fol-lowing in vivo experiment: Tagged erythrocyteswere washed three times in saline and lysed by theaddition of ten volumes of distilled water. Thehemolysate was then made isotomic by the addi-

TABLE III

Effect of pH on the rate of Cr51 uptake by aliquot samples offresh erythrocytes in whol ACDblood at 2° C.*

The concentration of Na2Cr61O4 was 2.5 gammaper ml. whole blood

Per cent Crsl uptake by erythrocytes

After 3 min. After 15 min.pH equilibration equilibration

7.3 8 116.9 11 126.0 28 41

* Effect of pH on the rate of Cr51 uptake by red cells at2° C. after addition of 2.5 gamma of chromium (asNa2Cr'04) per ml. of fresh whole ACDblood.

tion of NaCl and passed through a Berkefeld filterand injected back into the original dbnor of thered cells. During the first twenty-four hours 27per cent of the injected dose was excreted in theurine. None of the injected Cr5' was fixed to, nor

reappeared in, the recipient's cells during thethirty-day observation period following the in-jection.

Survival of chromated erythrocytes in vivo

The survival of Cr51-labelled donor erythrocyteshas been observed in 19 subjects based on the ra-

dioactivity of the recipient's blood after transfusion.The results of these studies, in part, are presentedin Table I, together with data pertaining to thepreliminary processing, storage, and chromationof injected cells. Nine recipients were studied bythe combined application of the chromium labellingand selective agglutination counting methods, theresults of which are likewise incjuded in Table I(Experiments 1 to 9), and represented in graphicform in Figure 2. The survival in five recipientsof transfused donor cells from the same half unit

1001 0

ILI

4i-;

a-.

z

Q0

0-

80

60

40

20

A

1267

o-. CdCimAds

Gr5l

80- 'A- .W.-13 SPSS

40 0

40 44

0 0

100______________

I I I *

100 1 "_10--om"

0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

i- - Cui*4.0~~~~~~~~~~~~~~~~~

40-ty- <

6n0 04

w~~ ~ ~ ~ ~~~R CeOUNTING

40 -04to A.B.- V0 OM4126

w %~~~~~~'

820 4.

60-

80-- , I ,.1@@S0

60 ' 0* 0~~~~~ 44

40~~~~~

20 0 444

40-.I

I00~~~~~~~~~~~~~~~~~~~40 *0C 5 -0C0

44%~~~~4

40 N~~~~~~~~

20N40 8 10 2 0 60 8 0 2

DAYS AFTER TRANSFUSIONFIG. 2. CompARISON; oFREDCEz SuRvivAL DATA OBTAINEDB3YSELECTIV AG-GLUTINATIONq AND

Ci COUNTING

1268

0-@- CINC.fts44 0~~~~~*-.* cpU5

0 ~~~JA-- mom," mOn

MEASUREMENTOF RBCSURVIVAL BY CHROMIUM"1TAGGING

of donor blood, stored with Na2Cr51O for periodsvarying from one to five weeks, is shown in TableI and Figure 5. As a control on the effect of stor-ing red cells with Na2Cr5lO4, the survival of ali-quot portions from the other half of the same unitof blood which were tagged just before their trans-fusion is shown in Table I (Experiments 10, 12,14, 16, and 18) and Figure 5.

Comparison of red cell survival as measured simul-taneously by the chromium technique and se-lective agglutinationThe survival of Cr51-labelled erythrocytes meas-

ured in the same recipient both by radioactive andselective agglutination counting is represented bythe paired curves in Figure 2, constructed fromdata obtained by both techniques on twenty ormore occasions over a period of 100 to 130 daysfollowing each transfusion of Cr51-labelled redcells. Donor cell survival as measured by the Crp5method is represented by a complex curve whichis neither logarithmic nor linear, and the end pointof which coincides with the total disappearance ofinagglutinable cells.

Comparison of data obtained by selective ag-glutination and Crp5 counting during the first twodays (Figure 3) indicates that the former valuesare consistently higher during the first forty-eight hour period following transfusion, the degreeof difference averaging 6.7 per cent. The prob-ability that this difference was due to chance isless than 0.01 per cent. The discrepancy may bedue to a systematic error in the Crp5 and in se-lective agglutination cell survival data. It is un-likely that it is due to tagging of the recipient'serythrocytes by the small amount of injected un-bound Na2Cr51O4 (average of 6.5 per cent of thetotal injected radioactivity for Experiments 1 to 9,Table I) since only about 10 per cent of freeNa2Cr51O4 when injected intravenously tags re-cipient erythrocytes as was demonstrated in threeexperiments of which the following experiment isan example:

A solution of sodium chromate containing atotal of 0.8 mg. of Na2Cr51O4 expressed in terms ofits chromium metal content was added to 100 ml.of saline and the mixture injected intravenously.Five minutes following injection 12 per cent ofthe injected Cr5l was bound to the erythrocytesand remained fixed at that level in the erythrocytes

100

80

60

-J4

5X100

80

60

v 24 HOURSAFTER TRANSFUSION* CtroO SELECTIVE AGGLUTINATION

O *

0 0

0oa

t aaf a a*9 * 42 HOUR AFTER TRANSFUSION0

8o 0 *

0

0

o *

o 10 20 30

DAYSBLOODSTORED BEFORETRANSFUSION

FIG. 3. COMPARISONOF SELECTiVE AGGLUTINATIONANDCR' COUNTSAS A MEASUREOF SURVIVAL ON THE SAMEUNIT OF BLOOD24 AND 42 HOURSAFTER TRANSFUSION

for a period of at least one week, after which agradual decline in chromium concentration oc-curred. It may be pointed out that the amount ofchromium injected in this experiment was greaterthan the largest amount of unbound chromium in-jected in the transfusion experiments described inthis report.

All Cr51 values obtained one week or more fol-lowing transfusion are consistently lower than thecorresponding values obtained by selective agglu-tination counts (Figure 2). The difference be-tween these two curves might be explained eitheron the basis of a continuous chromium elution fromthe circulating labelled cells or on the assumptionthat the amount of chromium taken up by thedonor cells is not the same for each cell, but in di-rect proportion to the age of the red cell at thetime of collection from the recipient, i.e., the senes-cent cells binding the most Cr51, and the youngcells the least. There is no experimental data foror against either hypothesis at present. However,for purposes of discussion, the progressive declinein the concentration of Cr5' in circulating donorcells will be characterized as an elution phenome-

1269


non. The rate of Crp5 loss from intact donor cells(i.e., biologic decay), as distinguished from lossof the Crp5 attributable to the hemolysis of taggedcells, may be calculated on the basis of changesin the ratio of cell survival determined by Cr5'counts and the corresponding values based on se-lective agglutination counts. A semilogarithmicplot of the per cent of original amount of Cr5' re-maining on the donor cells for the transfusion ex-periments (Figure 4), indicates that the biologichalf-life of red cell bound Crp5 may range fromfifty-one to ninety days (Table IV). ExcludingExperiment 9 from these calculations, the resultsof which vary more than two standard deviationsfrom the mean of all the data, the mean half-life ofchromium elution is 77 ± 12. Survival of trans-fused erythrocytes can be accurately measuredduring the first two days after transfusion withoutcorrections for this loss of Crp5 since less than 3per cent of transfused donor cell Crp5 is eluted fromthe red cells during this interval. Long-term sur-vival values based on Crp5 determinations are sub-ject to error unless the phenomenon of chromiumelution is taken into account and a correction fac-

UGo

ofir:

0z00z

0-Jn

I-zo

100

801

601

40

U 20

tor introduced to compensate for its effect. Sucha correction factor may be based on the observedhalf-life of chromium elution, or biological decayof Cr51. For example, 66 per cent of injected cell-count Crp5 is present in the recipient (Experiment2, Table I, and Figure 2) on the twentieth day and53 per cent on the thirtieth day following transfu-sion; the average rate of donor cell hemolysisthroughout the ten-day period, based on uncor-rected data, would be (66 - 53)/10 or 1.3 percent per day for the ten-day period. However,only 83 per cent of the original amount of Cr5'present in the transfused cells remains twenty daysafter transfusion and 76 per cent thirty days aftertransfusion. Correcting for this "elution" of Cr5'by use of the above figures, the hemolytic ratewould then equal (80 - 70)/10 or 1.0 + 0.15 percent as calculated from the Cr5l data in contrastto the observed hemolytic rate of 0.9 per cent perday as determined by selective agglutination count-ing for the same recipient during the same intervalof time. A large part of the discrepancy betweenthe two methods is due to the variation in Crp5elutions observed in donor blood thus far studied.

I

O Oaa ka0 X

a0 q

40 60 80

DAYS AFTER TRANSFUSIONFIG. 4. THE RATE OF CR" ELUTION FROMTRANSFUSEDCRe LABELLED ERYTEROCYTES

The ordinate scale is logarithmic and indicates the percentage of original Crn presentin the circulating red cells for any given day (abscissa) after transfusion. The indi-vidual experiments are identified by the following symbols: Exp. 1-X; Exp. 2-0;Exp. 3- ; Exp. 4-0; Exp. 5-+; Exp. 6-A; Exp. 8-A; Exp. 9-LI.

a

o *a

A *+ A

A. t + + +

x 8

aa

a

1270

a


0 5 10 15 20 25 30 35DAYS BLOODSTOREDBEFORETRANSFUSION

FIG. 5. THE SURVIVAL OF REDCELLS AS MEASUREDBY COUNTINGCR1 TEN MINUTES,TWENTY-FOURHOURS, AND ONEWEEKAFTER TRANSFUSION

The closed symbols represent blood stored with Na,Cr'04, the open symbols bloodstored without Na2Cr'04.

The Effect of Chromation on Red Cell Survival

The maximum red cell survival of fresh ACDblood is commonly stated to be approximately 120days. Inspection of the curves in Figure 2 basedon selective agglutination counts indicates that theextinction point of Cr5' counts following transfu-sion of blood previously stored 0 to 8 days rangefrom 108 to 115 days. The sole exception wasfound in Experiment 3 in which the end point oc-curred at ninety-four days. Inasmuch as the sttb-ject of this experiment had previously receivedthree experimental transfusions, it may be specu-

TABLE IV

Rate of chromium 51 elution from circulatingdonor erythrocytes

Preliminary Half-life ofExperi- storage of chromium inment donor blood red cells

days

1 4 hrs. 642 4 hrs. 653 7 hrs. 904 8 days 655 13 days 876 15 days 828 21 days 879 26 days 51

Mean i S.D. 74 - 33 daysMean (excluding Exp. 9) :1= S.D. 77 :1 12 days

lated that the relatively brief survival period ob-served in this study may have been attributable toisoimmunization with the production of isoanti-bodies of an obscure variety or so weak as to es-cape detection by the indirect Coomb's test. Also,the donor blood used in this experiment waswashed twice in isotonic saline before injection,a procedure which may conceivably have damagedthe erythrocytes.

Effect of erythrocyte storage with Na2Cr5lO onsurvival

The effect on red cells of prolonged exposureto chromium, as evidenced by their post-transfu-sion survival, was studied in Experiments 10 to 19(Table I), which were conducted as follows:

Two equal portions of fresh ACD blood fromthe same unit of blood were placed in separate con-tainers. To one of these was added Na2Cr51O4.Both portions were placed in storage. At weeklyintervals, 50 ml. samples from each portion wereremoved and transfused, the portion which hadbeen stored without chromium having been taggedimmediately before injection. Comparison of thesurvival of blood stored with and without chro-mium (Figure 5) indicates that the portion ex-

1271

FRANKLIN G. EBAUGH, JR., CHARLES P. EMERSON,AND JOSEPH F. ROSS

posed to chromium continuously throughout stor-age was entirely comparable to the control sam-ples labelled immediately before transfusion.

Effect of red cell exposure to high concentrationsof Na2Cr5104

Exposure of red blood cells to sodium chromatewithin the concentration range of 1.8 to 9.5 micro-grams per ml. of whole blood (expressed as ele-mental chromium) in four fresh blood samples hadno apparent influence on immediate post-transfu-sion survival (Table I, Figure 2, Figure 5).

In order to determine the effect of relativelyhigh concentrations of chromium on red cell lon-gevity, an experiment was performed involvingthe transfusion of two aliquot samples from thesame unit of donor blood which had previouslybeen stored at 4 to 60 C. for fourteen days, thenlabelled with Na2Cr5104. The proportions ofblood and sodium chromate were such that the con-centrations of chromium metal in the two samplesdiffered by a factor of 10, i.e., 3.1 micrograms ascompared to 35 micrograms of chromium metalper ml. of whole donor blood. Inspection of thecurves representing the survival of these two por-tions (Figure 6) indicates that within twenty-four

100RECIPIENT Concentration of Cr metal

90__ (emL of Donorlabod)80 -o J.H. 3.1

g70 _ , * * C.H. 35.0

|60

~50-40 -

30-

20-

10

0 20 40 60 80 100DAYS AFTER TRANSFUSION

FIG. 6. COMPARISONOF THE SURVIVAL, AS MEASUREDBY COUNTINGCR", OF Two ALIQUOTS OF THE SAMEDONORBLOOD, ONEOF WHICHWASTAGGEDWrrH A CONCEN-TRATION OF 35 GAMMA,AND THE OTHER3.1 GAMMAOFNA,CR"O, (IN TERMSOF CHROMIUMMETALPER ML. OFWHOLEBLOOD)

hours following transfusion 50 per cent of theheavily tagged blood was eliminated, in contrastto 30 per cent destruction in the case of the lightlyexposed cells. The results of this single experi-ment are by no means conclusive, in view of thefact that these observations were made in differ-ent recipients, but suggest that exposure to chro-mium in a concentration of 35 micrograms per ml.of whole blood may have resulted in cell damageevidenced by an increase in the proportion de-stroyed immediately following injection. Through-out the remainder of their life span, both the cellswhich had been exposed to the high concentrationand those which were exposed to the low concen-tration of chromium were eliminated at rates thatwere equal to those observed for unlabelled donorblood stored in ACDplasma for the same periodof time.

Excretion of Cr5l

The excretion of Cr51 in the urine at various in-tervals after transfusion is shown in Table V. Thedata indicate that for long periods of time afterthe transfusion at least a quarter of the amount ofCrp5 known to have been removed from the circu-lation is excreted in the urine. Exceptions oc-curred during the period of rapid hemolysis in thecase of Experiments 9 and 15, where only 8.5 percent and 17 per cent, respectively, of the amountdisappearing from the circulation during the firsttwenty-four and forty-eight hours appeared inthe urine. Preliminary observations in dogs showthat during the interval of 0 to 7 days after a

TABLE V

Urinary excretion of chromium 51 compared to the amountdisappearing concomitantly from the circulation

in eight transfusion recipients

Experi-mentno.

108

15984671

Daysafter

trans-fusion

0-20-10-10-2

12-1328-2930-3130-3157-58

Per cent oftotal in'iecteddose of CrI51

disappearingfrom circu-lating bloodduring this

period

11.0294664

1.070.970.700.840.72

Per centof Cr"l

disappearingfrom the

circulationexcreted

in theurine

5245

8.51793

102272465

Per cent oftotal Crt"excretedin urine

5.713.1

3.910.9

0.990.990.190.200.47

1272


transfusion of poorly preserved blood, 85 per centof which was removed from the circulation duringthe first week post-transfusion, 20 per cent of theinjected dose was excreted in the urine and 7 percent in the feces.

DISCUSSION

The experimental evidence herewith presentedindicated that radioactive, chromium-labellederythrocytes can be employed for evaluating thepost-transfusion survival of red blood cells for pe-riods of at least twenty-four to forty-eight hoursafter. transfusion and possibly for much longerperiods of time, perhaps for their entire life span.The method is ideally suited for evaluating the ef-fectiveness of in vitro erythrocyte preservationtechniques, and possibly may prove to be of greatvalue for studying the life span of erythrocytes invarious disease states.

The considerations which are of paramount im-portance in establishing the validity and applica-bility of this method are the following:

(1) The capacity of erythrocytes to bind thechromate ion under certain conditions;

(2) The "firmness" or permanency of this bind-ing;

(3) The possibility that the labelling of erythro-cytes with radioactive chromium adverselyinfluences the survival of the cells in therecipient;

(4) The possibility that radioactive chromiumliberated from transfused red cells (eitherby elution or from cellular destruction)may re-enter and tag the recipient's owncells;

(5) The question as to the safety of this methodfrom the standpoint of radiation effects inhuman subjects receiving blood labelledwith radioactive chromium.

Erythrocyte-chromium union

The union between erythrocytes and radioac-tive sodium chromate takes place in vitro regard-less of the duration of time that the erythrocyteshave been stored prior to their exposure to so-dium chromate. This fact makes possible thelabelling and subsequent post-transfusion study ofblood which has been stored for prolonged pe-

riods of time before injection. It is also apparentfrom our studies that the chromation of freshblood and storage in the chromated state is in noway different from the standpoint of eventualpost-transfusion survival than chromation of bloodjust prior to transfusion. These features make themethod eminently satisfactory for a study of bloodstorage methods.

It is quite feasible to chromate erythrocytes atrefrigeration temperatures without subjecting themto the possibly deleterious influence of incubation,since our studies show that erythrocytes will takeup chromium at 40 C. in quantities sufficient toallow transfusion studies. The rapidity andamount of chromium taken up by red cells in-creases with increase of temperature to 390 C.

Chromation proceeds slightly more rapidly andto a somewhat greater degree at a pH of 6.0 thanat a pH of 7.2, which is reflected in a greater up-take of chromium by blood stored for significantperiods of time than by fresh blood, since the pHof stored blood is lower than that of fresh blood.

The percentage of sodium chromate bound toerythrocytes is not appreciably influenced by theamount of chromate ion present within the rangeof 0.25 to 10.0 micrograms of chromium ion perml. of blood.

Our studies do not provide information as tothe nature of the chromate-erythrocyte bond-whether it be simple physical adsorption, chemicallinkage, or enzymatic binding.

The erythrocyte-chromate linkage appears tobe quite stable, at least in vitro, inasmuch as re-peated washings with saline removed negligibleamounts of the labelling material. In vivo, how-ever, there is a steady and apparently exponentialdisappearance of the radioactive chromium fromthe labelled erythrocytes, half of the chromiumdisappearing in 77 + 12 days in our experiments.Such a disappearance might be explained eitherby a process of elution of the chromate ion fromthe erythrocyte, or by preferential rapid destruc-tion of the chromated cells. The rather wide rangeof biologic half times may well be accounted for bythe fact that the bloods on which these elution rateswere determined had been processed in a variety ofways. It may be that freshly drawn and chromatedbloods reinfused into a recipient may have a morenearly constant rate of elution.

1273


Is there labelling of the recipient's erythrocytes byradioactive chromium liberated in vivo?

This is a critical consideration from the stand-point of the validity of this method for evaluatinglong-term erythrocyte survival. Thus, if chromateion which has escaped from intact erythrocytes oris released by hemolysis from senescent red cellswere to re-enter and label the erythrocytes of therecipient, the rate of the disappearance from thecirculating cell mass of the recipient could not beinterpreted in terms of the survival of donor cellpopulation.

Analysis of the cases in which as much as fiftyper cent of the transfused, labelled cells were de-stroyed during the first twenty-four hours aftertransfusion revealed that survival, as measuredsimultaneously by selective agglutination and Cr5'methods, agreed closely following this period ofrapid destruction. If retagging of the recipient'sred cells had occurred under these circumstances,the survival as measured by Crp5 would have beensignificantly higher than that as determined byselective agglutination. The above observationdoes not exclude re-utilization of the injected Cr'by the recipient's bone marrow and the gradualincorporation of Crp5 in recipient red cells. Thispossibility is excluded by the observation that afterthe injection of Cr51-tagged hemoglobin, none ap-peared'in the recipient's red cells at any time dur-ing the following month.

Since from 1 to 17 per cent of the injected chro-mate was not bound to the donor cells employedin this study, but was present in the donor plasma,the possibility existed that recipient cells couldhave been tagged by this "free" chromate. Ex-periments have shown that no more than 12 percent of the injected aqueous chromate is attachedto the recipient's red cells. Thus, no more than12 per cent of the total amount of injected Crp5contained in the donor plasma could have becomebound to the recipient's cells. This observationhas practical as well as theoretical impqrtance,since it makes unnecessary the processing of bloodwith the aim of complete removal of the portionof added sodium chromate which does not becomefixed to the red cells.

The in 7ivo survival of chromated erythrocytes

Careful determination of the survival of chro-mated erythrocytes by the Ashby differential ag-glutination method indicates that during the firsttwenty-four to forty-eight hours after transfusion,the behaviour of chromated cells is quite compara-ble to the survival which would be expected ofnon-chromated red cells. This is true of freshbloods in which survival of the transfused cells isnormal, and of bloods stored for prolonged periodsof time in which there is initial rapid destructionof the donor cells. These findings establish thevalidity of the chromated cell method of evaluatingthe effectiveness of blood preservation methods,since it is the survival of donor cells in the im-mediate post-transfusion period which is of para-mount importance in evaluating the viability oftransfused erythrocytes.

The survival values of the bloods stored forprolonged periods of time in ACD medium asindicated by the chromated cell method are lowerby some 10 per cent than the values previously re-ported as determined by the radio-iron tagged cellmethod (1-3). This discrepancy might be ex-plained by the fact that the hematocrit value em-ployed in calculating red cell survival in thesestudies was not modified by a correction factor aswas done in the studies on survival of radio-irontagged cells. It might also reflect a deleterious in-fluence of sodium chromate on the erythrocytes.The concentration of chromium which is definitelyinjurious to erythrocytes has not been determinedin this study. -Suggestive evidence was obtainedwhich indicated that a concentration of 35 micro-grams of sodium chromate (expressed as ele-mental chromium) per ml. of whole blood didshorten the immediate post-transfusion survival ofthe labelled erythrocytes. On the other hand,variations in concentrations of chromium from1.8 to 9.5 micrograms per ml. of whole blood didnot have any detectible influence on post-trans-fusion survival of labelled erythrocytes. Untilmore data are obtained on the upper limits ofchromium concentration not toxic to erythrocytes,it is suggested that future survival studies ofchromium-labelled erythrocytes be performed onblood exposed to concentrations of chromium ofone microgram or less per ml. of whole blood.The specific activity of radioactive sodium chro-

1274


mate currently available is of sufficient magnitudeto allow satisfactory red blood cell survival experi-ments to be conducted at this level of chromiumconcentration. Survival of chromated cells overprolonged periods after transfusion appears to besomewhat less than the customarily accepted meansurvival time of 120 days. It is possible that fur-ther studies may show that these particular ob-served values may be within the normal biologicrange of erythrocyte survival. If this shorteningof survival by 10 per cent is found to be con-stant, however, it should not invalidate the pos-sible value of long-term survival studies in whichrelatively large changes in rates of erythrocytedestruction are in question (e.g., in acquired he-molytic anemia, or during induced hemolyticstates). Preliminary observations indicate thatthe method yields valuable information in study-ing the survival of an individual's own red cellswithin his own body.

Amount of radiation received by the recipientfrom injected Cr5l" 11

If the Cr51 which leaves the circulation becomesevenly distributed throughout all the body tissues,the circulating blood would contain the greatestconcentration of Cr51. The amount of Cr5' givingno more than 0.3 rep during one week would be320 microcuries for a subject with a blood volumeof 5000 ml. (16). Preliminary data obtained indogs indicate that one week after the transfu-sion of poorly preserved blood,'2 the Cr5l whichleft the circulation was not evenly distributedthroughout all the tissues, but was present in high-.est concentration in the spleen, liver, and bonemarrow. The concentration of Crp5 per gram ofspleen was 10 times the concentration occurringin the liver and bone marrow so that the spleencontained one-third of the injected dose. If oneassumes a similar type of distribution in humansubjects, an individual experiencing very rapidhemolysis of donor cells (i.e., 50 per cent or morein one day), which contained 320 microcuries,would be exposed to no more than 1.7 rep perweek delivered to the spleen. In this case it would

11 The authors wish to express their appreciation toDr. Gerald J. Hine for valuable aid and criticism in calcu-lation of the radiation doses presented.

1285 per cent of injected cells were destroyed in oneweek.

be the dosage received by the spleen which wouldbe the limiting factor in the amount of Crp5 whichcould be safely injected. In experiments wherefresh ACD blood containing 320 microcuries istransfused, the spleen of the recipient would re-ceive no more than 0.8 rep per week. The abovecalculations are conservative inasmuch as it isassumed that all the Cr51 going to the spleen re-mained there and was not cleared from this tissueand that no Cr5' was excreted.

Practical application of the radioactive chromateerythrocyte survival method

In the light of our present information, the radio-active chromate method appears ideally suited forevaluation of immediate post-transfusion survivalof erythrocytes, either freshly drawn or storedblood. The method is technically simple, highlyaccurate, can be performed with small aliquots ofblood, allows study of the survival of an indi-vidual's own erythrocytes in his own vascular sys-tem. It makes possible transfusion studies with-out the hazard of transmitting virus hepatitis, andis harmless from the standpoint of the hazard ofradiation.

It is of definite value in following short-termvariations in erythrocyte survival and possibly maybe proven to be of value in studying small changesin long-term red cell survival.

SUMMARY

1. Erythrocytes in ACD whole blood mixturecombine rapidly and firmly with the radioactivechromate ion. After equilibration for three hoursat 4 to 60 C., 60 per cent was bound to the redblood cells and 90 per cent was fixed after equili-bration for one hour at 25 to 260 C. This is trueof both freshly drawn blood and blood stored at4° C. for as long as thirty-six days. The concen-tration of this chromate salt shows no markedeffect on the rate of Cr5' uptake within the limitsof 0.25 to 9.5 micrograms. The rate of erythro-cyte Cr5' uptake increased inversely with the pHwithin the observed range of 6.0 to 7.2.

2. Post-transfusion survival as determined bythe selective agglutination method of erythrocytestagged with sodium chromate in concentrations ofless than 10 micrograms per ml. of whole blood is2 to 10 per cent shorter than the post-transfusion

1275


survival of nonchromated cells. This somewhatshorter survival of the chromated cells may or may

not be within the range of the normal biologicalvariations.

3. Na,Crl04 released from destroyed trans-fused erythrocytes is not re-utilized to tag therecipient's own red blood cells.

4. The concentration of Crl in the circulatingdonor erythrocytes decreases slowly at an ex-

ponential rate with a half-life 77 + 12 days. Thismay be due to elution of Cr"5 from the circulatingdonor cells.

5. The survival of stored blood can be meas-

ured accurately by the chromium tagging tech-nique for a period of forty-eight hours after trans-fusion without correcting for the small amount ofchromium leakage. If corrections for the amountof Cr"5 elution can be made, the method wouldallow measurement of post-transfused erythrocytesfor periods of as long as three months.

REFERENCES

1. Ross, J. F., and Chapin, M. A., Effect of storage ofcitrated blood on the survival of transfused eryth-rocytes. J. A. M. A., 1943, 123, 827.

2. Ross, J. F., Finch, C. A., Peacock, W. C., and Sam-mons, M. E., The in vitro preservation and post-transfusion survival of stored blood. J. Clin. In-vest., 1947, 26, 687.

3. Gibson, J. G., Peacock, W. C., Evans, R. D., Sack, T.,and Aub, J. C., The rate of post-transfusion loss ofnon-viable stored human erythrocytes and the re-

utilization of hemoglobin-derived radioactive iron.J. Clin. Invest., 1947, 26, 739.

4. McKerns, K. W., and Denstedt, 0. F., The use ofsulfhemoglobin for labeling erythrocytes in Preser-vation of the Formed Elements and of Proteins ofthe Blood, C. J. Potthoff, ed., Washington, D. C.,American National Red Cross, 1949, p. 4.

5. London, I. M., Shemin, D., West, R., and Rittenberg,D., Heme synthesis and red blood cell dynamicsin hormal humans and in subjects with polycythemiavera, sickle-cell anemia, and pernicious anemia.J. Biol. Chem., 1949, 179, 463.

6. Moore, C. V., Red cell survival measured with C1'tagged globin in Preservation of the Formed Ele-ments and of Proteins of the Blood, C. J. Potthoff,ed., Washington, D. C., American National RedCross, 1949, p. 10.

7. Ross, J. F., A consideration of various techniquesof evaluating post-transfusion survival of eryth-rocytes in Conference on Differential Agglutina-tion of Erythrocytes, Washington, D. C.,. 17 Sept.,1952, Committee on Blood and Related Problems,National Research Council, Division of MedicalScience.

8. Ebaugh, F. G., Ross, J. F., and Emerson, C. P.,Metabolism of radioactive zinc". To be published.

9. Gray, S. J., and Sterling, K., The tagging of redcells and plasma proteins with radioactive chro-mium. J. Clin. Invest., 1950, 29, 1604.

10. Sterling, K., and Gray, S. J., Determination of thecirculating red cell volume in man by radioactivechromium. J. Clin. Invest., 1950, 29, 1614.

11. Lyon, W. S., Disintegration of Cr51. The PhysicalRev., 1952, 87, 1126.

12. Gibson, J. G., Peacock, W. C., Seligman, A. M., andSack, T., Circulating red cell volume measured si-multaneously by the radioactive iron and dye meth-ods. J. Clin. Invest., 1946, 25, 838.

13. Cliapin, M. A., and Ross, J. F., The determination ofthe true cell volume by dye dilution, by protein di-lution, and with radioactive iron. The error ofthe centrifuge hematocrit. Am. J. Physiol., 1942,137, 447.

14. Vazquez, 0. N., Newerly, K., Yalow, R. S., andBerson, S. A., Determination of trapped plasma inthe centrifuged erythrocyte volume of normal hu-man blood with radioiodinated (I') human serumalbumin and radiosodium (Na"). J. Lab. & Clin.Med., 1952, 39, 595.

15. Emerson, C. P., The methodology of experimentaltransfusions and selective agglutination countingin studies on blood preservation, Washington, D. C.,17 Sept., 1952, Committee on Blood and RelatedProblems, National Research Council, Division ofMedical Science.

16. Marinelli, L. D., Quimby, E. H., and Hine, G. J.,Dosage determination with radioactive isotopes.II. Practical considerations in therapy and pro-tection. Am. J. Roentgenol., 1948, 59, 260.

1276

radioactive chromium 51 asdm5migu4zj3pb.cloudfront.net/manuscripts/102000/102855/jci5310… ·...

Documents