SURVIVAL OF BLOODPLATELETS LABELED WITHCHROMIUM511,2

By KNUTA. AAS3 AND FRANKH. GARDNER(From the Richard C. Curtis Hematology Laboratory, Peter Bent Brigham Hospital, and the

Department of Medicine, Harvard Medical School, Boston, Mass.)

(Submitted for publication February 11, 1958; accepted May 2, 1958)

Various criteria exist for the evaluation of theintegrity, viability and physiological activity ofhuman blood platelets. Alterations in plateletmorphology, clot promoting and clot retractingactivities, oxygen consumption, clinical effect onbleeding disorders, and the life span of platelets inthrombocytopenic recipients have been used asmeasurements of platelet function. The transfu-sion studies in thrombocytopenic recipients indi-cate that platelets have a life span of three to sevendays and have been helpful in the classification ofcertain pathological disorders (2). These ob-servations have been dependent upon obtainingdonors with high platelet counts and accepting theerrors associated with platelet counting. The diffi-culty in procuring adequate donors and the de-sirability of measurements other than platelet enu-meration have initiated numerous studies of thein vivo survival of platelets labeled with radio-active isotopes.

The earlier studies concerning measurement ofthe life span of blood platelets with isotopes priorto 1955 have been reviewed by Odell, Tausche andFurth (3). In Table I the various isotope tech-niques previously reported are tabulated for com-parison. The recent report by Leeksma and Cohen(13) has demonstrated a life span of labeledplatelets of eight to nine days. These investiga-tors followed the activity of radioactive phos-phorus in platelets after the recipient hadreceived diisopropylfluorophosphonate (DFP32)parenterally. Reisner, Keating, Friesen and

1 This investigation was supported in part by a re-search grant from the Department of the Army, Officeof The Surgeon General (DA-49-007-MD-701) and theJohn A. Hartford Foundation, Inc.

2A preliminary report was presented at The SixthCongress of The International Society of Blood Trans-fusion, 1956 (1).

a Travelling Fellow, Commonwealth Fund, New York,1955 to 1956. Present address: Department of Medicine,Rikshospitalet, Oslo, Norway.

Loeffler (14) have noted that radioactive sodiumchromate could be bound to platelets in vitro.When these platelets were transfused to recipients,a survival period of five to eight days could bemeasured. Similarly, transfusions of in zivo la-beled p32 platelets from polycythemic donors havehad a life span of seven days in normal recipients(15).

The present investigation was in progress at thetime of the observations by Reisner and co-work-ers (14). A workable technique is presented forthe in vitro labeling of human blood platelets withradioactive chromium and evaluating their sur-vival after transfusion to normal recipients. Com-ments regarding details of the method will bepresented.

METHODS

Material. Whole blood donations were obtained fromvolunteer medical staff and patients. In many instancesin sitro studies were done using blood obtained fromtherapeutic phlebotomies.

Preparation of platelet suspension. The following tech-nique was derived from past experience in the prepara-tion of platelet suspensions using plastic bag equipment.Five hundred ml. of whole blood is collected by gravityinto polyvinyl chloride plastic bags (Fenwal Laborator-ies) containing 50 ml. of 1.5 per cent disodium ethylene-diaminetetracetic dihydrate (NaEDTA) in saline. Asnoted previously (16) Arquad04-coated 15 gauge male-hub needles are used. The plastic tubing leading fromthe needle to the plastic bag has a "Y" type division thatallows 10 ml. of blood to be withdrawn to exclude tissuejuice contamination and to evaluate venous blood flow.The whole blood is centrifuged at 2° C. for 15 minutesat 1,300 rpm. (325 G) in an International Centrifuge,Model PR-2. After centrifugation, the platelet-richplasma is transferred to another plastic bag containing15 ml. of 2 per cent Triton® 5 in saline using a piercing

'Arquad® is a trade-mark of Armour Chemical Di-vision, Armour and Co., Chicago, for Tris (2-hydroxy-ethyl) dodecylamine and N,N'(2,3-dihydroxypropyl)-N-alkyl-trimethylenediamine (A-15).

5Triton® is a trade-mark of Rohm & Haas Co., Phila-delphia, for oxyethylated tertiary octyl phenol formalde-

1257

KNUT A. AAS AND FRANK H. GARDNER

TABLE I

Summary of isotope platelet survival studies

Mode of IndicatedAuthor Year Isotope tagging Species life span

Julliard and associates (4) 1952 PU In vitro Human 2 hoursMueller (5) 1952 P3 In vitro Rabbit 2 hoursOdell and associates (6) 1953 C14 In vivo Rat 3-4 daysMorgan and associates (7) 1954 1151 In vitro Rabbit 1-2 hoursRobertson and associates (8) 1954 Cr5'C13 In vitro Rat 2 daysOdell and associates (9) 1954 C14 + S36 In vivo Rat 5-6 daysOdell and associates (10) 1955 C14 In vitro Rat 1-3 daysOdell and associates (10) 1955 S35 In vivo Rat 4-5 daysDesai and associates (11) 1955 pa In vitro Human Apparent ½-

survival time35-50 hours

Morgan and associates (12) 1955 Na2Cr104 In vitro Rabbit 3-4 daysLeeksma and Cohen (13) 1956 DFPU In vivo Human 8-9 daysReisner and associates (14) 1956 Na2Cr5'O4 In vitro Human 5-8 daysAdelson and associates (15) 1957 pa In vivo Human 7 days

coupler (16). A positive pressure pneumatic press ishelpful in allowing careful separation of plasma from thered cells and buffy coat. The plastic bag containing theplatelet-rich plasma is distended with air to prevent plate-let trapping in folds of the bag during centrifugation andis then centrifuged at 20 C. for 30 minutes at 2,300rpm. (1,000 G). The platelets separate out as a creamybutton at the bottom of the bag. The platelet-poor su-pernatant plasma is withdrawn under sterile conditionsand saved, leaving approximately 10 ml. of residual plasmawith the platelet button.

Labeling of platelets. Through the plastic inlet tub-ing attached to the bag, 10 ml. of 0.2 per cent Triton® insaline solution is injected by syringe. The bag is kneadedbetween the fingers to resuspend the platelet button untilno macroscopic clumps are visible. Three hundred mi-crocuries of sterile radioactive sodium chromate with aspecific activity of 20 microcuries Cr' per microgram isinjected into the bag through the plastic tubing and di-rected into the platelet suspension. The chromate solu-tion is mixed and the platelet suspension is incubated for15 minutes at room temperature (20 to 220 C.). There-after, the suspension is centrifuged again at 20 C. for 30minutes at 2,300 rpm. (1,000 G), and the plasma-Triton®solution is drained from the platelet button by gravityand saved to determine its residual Cr'1 activity. Fifteenml. of the sterile platelet-poor plasma, previously saved,and 15 ml. of 0.2 per cent Triton®-saline solution areadded to the platelet button and the platelets resuspended.One hundred milligrams of a 5 per cent ascorbic acid solu-tion is then added to the platelet suspension to prevent redcell binding in the recipient by excess Na2Cr'O4. Theplatelets are dispersed by kneading, and about 100 ml. ofair is allowed to enter the bag to facilitate complete in-fusion of the platelet suspension. A short piece of plastictubing is attached to the plastic bag using a piercingcoupler to decrease loss of platelets adhering to the tub-

hyde polymer and supplied as entsufon (WR-1339) byWinthrop Laboratories, New York.

ing. Usually a saline infusion has been started in therecipient to assure the rapid injection of the 30 to 35ml. volume of platelet suspension. From the averagedonor, the platelet concentrate in a 35 ml. volume usuallyhas a 3.5 X 10' cu. mm. platelet count. The entire pro-cedure requires about two hours from the phlebotomy tothe infusion of labeled platelets.

Comments on method

1. Anticoagulant. Plasma preparations prepared withacid citrate dextrose (ACD) solution (National Insti-tutes of Health formula) have numerous microscopic ag-gregations of platelets that do not resuspend after centri-fugation and labeling with Cr51. Earlier studies havedemonstrated that platelets are discrete and not clumpedwhen Na2EDTAwas used as the anticoagulant (16, 17).No evaluation was made in this present study to de-termine if microscopic clumping of platelets influencedviability after labeling and infusion.

2. Differential centrifugation. The speeds listed havebeen derived by arbitrary studies to achieve the maxi-mum yield of platelets in platelet-rich plasma (16).About 70 per cent of the platelets are recovered fromwhole blood by this procedure.

3. Platelet suspension. Irreversible platelet agglutina-tion has been controlled by the use of surface-activeagents, especially Triton® (WR-1339) (18), which al-lows rapid suspension of platelets and is an importantfactor in decreasing the time interval of handling plate-lets in vitro.

Platelet radioactivity in the recipient. To follow thefate of the transfused Cr' labeled platelets in the re-cipient, 20 ml. blood samples are drawn from the re-cipient at one-half, two and four hours, and daily there-after, until no further radioactivity can be detected in therecipient's platelets. The blood is collected using Ar-quad®-coated needles and silicone-coated syringes, and isplaced in 50 ml. silicone-coated tubes containing 2.0 ml.of 3 per cent Na.EDTA solution in saline. Two ml. of

1258

SURVIVAL OF BLOOD PLATELETS LABELED WITH CHROMIUM

whole blood is removed to determine radioactivity of thewhole blood sample. The tube is then centrifuged at 20C. for 15 minutes at 1,000 rpm (175 G). The platelet-rich plasma is removed from the red cells with a silicone-coated pipette and transferred to a 10 ml. silicone-coatedtest tube which is centrifuged at 3,000 rpm (1,200 G) for30 minutes at 2° C. The platelet-poor plasma is de-canted and the platelet "button" at the bottom of thetube is resuspended in 10 ml. of 0.2 per cent Triton® sa-line solution and recentrifuged. The supernatant salinesolution is decanted and saved to determine its radioac-tivity, while the platelet button remains in the test tubefor determination of its radioactivity. The red cell massand buffy coat from the initial centrifugation are elutedtwice with 10 ml. aliquots of 0.2 per cent Triton® salinesolution. The supernatant saline solutions from the twocentrifugations are pooled and saved to determine radio-activity. Platelet counts of the whole blood, platelet-rich plasma, platelet-poor plasma, red cell mass and redcell eluates ("wash solution") are determined by phasecontrast microscopy (19). Two ml. aliquots of wholeblood, platelet-poor plasma, platelet "wash solution," redcell mass and red cell "wash solution" are used to de-termine radioactivity in these respective separations.

Daily 24 hour urine collections were obtained fromfour recipients for 22 days to determine excretion of Cr`activity. These specimens are counted in a thallium-activated sodium iodide well-type scintillation counter(Tracerlab Model P-20A) with three-inch lead shielding.All samples are counted to maintain a counting errorof less than 5 per cent. Values followed for more than24 hours are corrected for physical decay.

Determination of Cr`1 labeling of platelets. One ml. ofthe labeled platelet suspension is withdrawn from theplastic bag before infusion to determine the total radio-activity of the volume transfused. One ml. of the plasma-Triton® solution is removed before suspension of thelabeled platelets in the platelet-poor plasma and is meas-ured to determine the radioactivity remaining in plasma-Triton® solution that was not bound to platelets. Thesummation of the radioactivity is considered to be thetotal Cr51 that was available to label the platelets fromthe 300 microcuries injected into the bag. Inasmuch assome platelets will adhere to the plastic bag surface, theinjected labeling dose of chromate (300 microcuries) isnot used to determine the percentage of activity boundto the platelets.

Radioactivity of final platelet suspensionRadioactivity platelet-poor plasma

+ X 100 = per centRadioactivity of final platelet suspension Cr6'_

labeledplatelets

Previous studies have indicated that one may anticipateby platelet counting 50 to 60 per cent initial recovery ofthe transfused concentrated platelet suspension in the re-

cipient's blood volume (16). These studies involved theplatelet suspension in only one rapid centrifugation

(1,000 G). The present method of labeling involves cen-trifuging the platelets twice at high speed. A singleplatelet recovery study has been done without Cr' la-beling to evaluate such a manipulation of the platelets.In this instance 46 per cent of the theoretical plateletyield was counted in a thrombocytopenic recipient. The50 per cent loss of the transfused platelets has been at-tributed to viability changes during manipulation.

In vitro studies. To determine the optimal conditionsfor labeling platelets with radioactive chromium, nu-merous in vitro observations were performed. Wholeblood was collected in 500 and 125 ml. plastic bags andcentrifuged to prepare the platelet suspensions as de-scribed previously. Platelet suspensions in 10 ml. ofplasma were incubated with radioactive sodium chro-mate at 30 C., 20 to 22° C. (room temperature), and37° C. The suspensions were centrifuged and the residualradioactivity of the platelets was expressed as the per-centage of the total radioactivity of the platelet sus-pension before centrifugation. Approximately 1 per centof the Cr51 activity remained with the platelet mass at30 C. and 370 C., while 3.6 per cent remained at 22° C.

The lower percentage at 370 C. has been attributed toelution of the radioactivity into the surrounding plasmamedia. Likewise, the Cr51 bound to the platelets was notincreased beyond an incubation period of 15 minutes.Labeled platelet preparations used for clinical studieswere incubated for 15 minutes at room temperature.

Relationship of Cr51 concentration. To determine theefficiency of binding Cr51 to platelets, a platelet-plasmasuspension was divided into 12 ml. aliquots in silicone-coated test tubes. The platelet count was 323,000 cu. mm.Varying amounts of Na2Cr104 from 3 microcuries to 25microcuries diluted to equal volumes in saline were addedto the platelet suspension, and the samples were incu-bated 15 minutes at room temperature. The tubes werethen centrifuged at 1,000 G at 3° C. and the platelet but-ton measured for radioactivity. A linear relationship be-tween the amount of Cr51 introduced into the platelet sus-pension and the radioactivity bound to the platelets wasobserved. Similarly, if the same experiment is repeatedwith the Na2Cr51O remaining constant and the plateletcount varied by plasma dilution, more radioactivity isfound in the platelet button with the highest plateletcount. In essence, the platelets will be labeled with Cryin direct relationship to the number of platelets in thesuspension.

Suspending medium. When platelet-rich plasma isutilized as the medium in which to label platelets, 1 to 3per cent of the total radioactive sodium chromate is boundto the platelets. Such suspensions usually had plateletcounts of 300,000 cu. mm. Platelet concentrates sus-pended in 10 ml. of plasma (platelet count 2 millioncu. mm.) averaged 9 per cent labeling, and when suchconcentrates were prepared from donor patients withpolycythemia vera (platelet counts 5 to 6 million cu. mm.),15 to 25 per cent of the radioactive chromate was boundto the platelets. The average platelet concentrate sus-pended in a saline-Triton® solution had 15 per cent ofthe radioactivity bound to the platelets.

1259

KNUT A. AAS AND FRANK H. GARDNER

Previous studies have emphasized the selective bind-ing of chromic chloride to plasma proteins (20). Lessthan 1 per cent of Cr' is bound to the platelets ifCr'1Cl. is used in platelet-rich plasma suspensions whileplatelet concentrates in 10 ml. of plasma bound 1 to 2per cent of Cr'Cl,. Higher residual radioactivity inthe platelet concentrate is obtained by the removal ofplasma and suspension of platelets in saline-Triton@ solu-tion. However, use of saline-Triton@ solution requiresadditional centrifugation of the platelets and a prolongedtime of preparation. Likewise, phase microscopy studiesreveal marked morphological changes in platelets sus-pended in saline. Therefore, to hasten the preparation ofplatelets for in vivo studies and to prevent effects ofsaline contact, the smaller percentage of bound Cr' ac-tivity in plasma concentrates has been preferred in thesestudies.

Cr'1 elution from platelets. The addition of ascorbicacid to the Na2Cr'O4 labeled platelet concentrate reducesthe chromium to trivalent form. In vitro incubation ofthe ascorbic acid-treated labeled platelets with red cellsdemonstrated no transfer of Cr' to the red cells, andsimilar platelet suspensions do not label buffy coatpreparations. One hour incubation at room temperatureof the Cr' labeled platelets in 40 ml. of platelet-poorplasma results in elution of 40 to 70 per cent of the

4M42a \PLATELET SURVVL

4ao T.H.

3=360

3~~~~~OI~~~~~RATWLTPORR.*SMA30XD

a-\DCLW

l-MED CL MSS

CR"' \_- ATELET MMWIMCWTS24\DPER 2 0 1 KO

M1JE

0

DAMS

FIG. 1. MEASUREMENTSOF CR" ACTIVITY IN NACR'O,LABELED PLATELETS AND SEPARATEDBLOOD COMPONENTSAFTER INFUSION IN A NORMALVOLUNTEERRECIPIENT

radioactivity into the plasma, and ascorbic acid doesnot alter this rate of elution. Cr'1 activity from theNaCr'O4 labeled platelets is eluted to a similar degreein saline-Tritong solution, but platelets labeled withCr'1Cl. have less elution (20 to 25 per cent) when incu-bated in plasma or saline. This rapid elution of Cr'from the platelets has not been observed after infusionand may reflect improvement in vivo of platelet viabilityand metabolic function.

The release of Cr' from the platelet membrane shouldnot be confused with the binding of chromium to theplastic material. When Cr'Cl, saline solution was in-troduced into a plastic bag with no platelets, 12 per centof the injected radioactivity remained on the plastic sur-face even after repeated washing. Depending upon thelength of the inlet tubing used to introduce the Cr'Cl,into the bag, as much as 21 per cent of the injected dosemay bind to the tubing and not be eluted despite repeatedwashing. In contrast NaCr'O0 is associated with negli-gible adherence of radioactivity to plastic; namely, 0.1per cent in the plastic bags and 0.04 per cent in the plasticinlet tubing. Similar observations have been noted inother applications of chromium salts (21).

It may be concluded from these in vitro observationsthat chromium' chloride and sodium chromate' can beused to label platelets. Sodium chromate offers greaterease in the preparation of the labeled platelets and lessdifficulty in the evaluation and measurement of radioac-tivity.

RESULTS

In zivo observations

Radioactive sodium chromate labeled plateletswere transfused to 30 recipients with normalplatelet counts. Heterologous or autologous la-beled platelets were used with no untoward reac-tions such as pain, fever, urticaria or phlebitis.Inasmuch as each volunteer recipient receivedonly one labeled platelet transfusion, there was noconcern that platelet sensitization would developto alter the survival period (2). In these studiesall radioactive components of the recipient's venousblood samples were followed to evaluate localiza-tion of radioactivity. The term "wash water" wasused to denote the saline washings of red cells andplatelets, and the radioactivity of these componentsis graphed in Figure 1. The peak of the Cr5' ac-tivity of the platelet "button" at 24 hours after in-fusion is about 30 to 50 per cent of the wholeblood radioactivity. The separated platelet buttonin this study indicates that the labeled plateletshad a life span of 10 to 11 days. A parallel declinein whole blood activity is associated with the lossof platelet activity. The radioactivity of theseparated platelet-poor plasma represents: 1) un-

1260

SURVIVAL OF BLOOD PLATELETS LABELED WITH CHROMIUM

COUN4TSPER 30'

MINUTE \ I

2.400,

;o.

.-t 19 4 29 12 3 6 8HOURS DANS

FIG. 2. NAOCROO.LABELED PLATELETS IN A NORMALVOLUNTEERRECIPIENT

The maximum Cr' activity was observed at 19 hoursafter infusion.

bound Cr"5 in the plasma of the platelet concen-trate that was not removed completely before thelabeled platelets were resuspended in sterile plate-let-poor plasma for infusion; and 2) Cr"5 fromnonviable platelets that constitute the 50 per centof platelets not accounted for in the recipient afterinfusion. The radioactivity of the platelet-poorplasma declines rapidly during the first 24 hoursafter infusion, although some Cr" may be foundin the plasma for a month indicating minimallabeling of plasma proteins by Na2Cr504. Thered cells are not tagged in this procedure asidefrom the few that remain in the platelet-richplasma during preparation. The radioactivity inthe red cell mass during the first 24 hours afterthe transfusion is derived from entrapped plasma.Thereafter the small residual radioactivity in thecentrifuged red cells is due to platelets. The radio-activity in the saline washings (wash water) ofthe platelet button is negligible, about 1 per centof the whole blood radioactivity.

The maximum radioactivity of the separatedlabeled platelets is always noted about 24 hoursafter the infusion. The platelet radioactivity wasfollowed at frequent time intervals in one recipientto determine the maximum radioactivity which inthis instance occurred at 19 hours (Figure 2).Probably the time intervals vary among recipientsdepending upon when the maximum numbers oflabeled platelets reappear in the circulation. Themaximum platelet activity observed 24 hoursafter infusion has been used to compare the percent of labeled platelets appearing in the circula-tion during the first few hours. This progressiveincrease in Crp activity of the platelet "button"has been graphed in Figure 3. The marked vari-ability of platelet activity half an hour after trans-fusion is startling. The delayed appearance ofcirculating platelets after transfusion of plateletconcentrates into thrombocytopenic recipients hasbeen noted previously (15, 16), (Figure 9).

CIRCULATING LABELLED PLATELETS

THE FIRST HOSAFTER TRANFUSION

70

so

/

SO

CR lyco'

FIG. 3. PLATELET RADIOACTIVITY WITH TIMEThe observed maximum Cr' activity of the separated

platelets is plotted as the 100 per cent value. The per-centage of Cr' during the initial period after infusion isrelated to the maximum value which is observed about 20hours later.

1261

KNUT A. AAS AND FRANK H. GARDNER

XI2 4i 2~\ 3 8§ 1X liii~ . .. *~ CW I20 FIG. 4. PLATELET SURVIVAL INNORMA240 4~. 3 5 7 0 - 56 DI ~ 1

A. The initial studies are shown in normal volunteer recipients by the technique described. The measurement ofthe .Cr"l labeled platelets suggests an. exponential curve of the platelet survival in vivo. B. The last seven studiesperformed with technical improvement indicate a liniear loss of the labeled platelets from the circulation. Plateletradioactivity is plotted as in Figure 3.

In all the platelet survival studies done withthe described procedure, the radioactivity of the

labeled platelets has been followed for 9 to 11

days. The Cr5' activity of the platelet button ob-served during this period suggests a linear loss ofthe labeled platelets, indicating that platelets havedisappeared from the circulation by senescence

rather than random destruction. The plottedvalues have a more linear relationship during thefirst eight days, while alterations in platelet via-bility during the labeling procedure probably en-

hanced the exponential appearance of the plottedcurves during the last three days that radioac-tivity was measured. The influence of the manipu-lations of the initial platelet concentrate may beappreciated by comparison of the survival curves

in Figure 4. The eight platelet curves to the left

represent values obtained during the early phaseof the.investigation, while the seven curves to theright represent. the last seven recipients studied.The more linear, loss of Cr5' platelet activity inthe.-later studies reflects improved handling ofthe platelets. during the labeling procedure.

Modifications of transfusion procedurePrevious studies have emphasized the value of

ascorbic acid to prevent further binding of sodiumchromate to the red cell membrane. One studywas performed to determine whether ascorbicacid could be omitted from the technique (Figure5). While the platelet button radioactivity couldbe measured, the Cr5' activity of the whole bloodwas 10 times as high as anticipated due to thelabeling of the red cells per se. In certain patho-

1262

SURVIVAL OF BLOOD PLATELETS LABELED WITH CHROMIUM

CR K)O

COUNTS 900 HOLEBLOODPER

MINUTE Soo

700-RED CELL MASS

600 \_

400 *300 -PLATELETS

100 2

0 2 5 6 7 8 Th_DAYS

FIG. 5. PLATELET SURVIVAL WITHOUTAscoRBc Acm-FEMALE, M. W.

The omission of ascorbic acid from the platelet con-centrate before infusion does not influence the inter-pretation of the labeled platelet survival period. Thelabeling of red cells by the hexavalent chromium pro-longs the period of circulating radioactivity and limitsrepeated observations in the recipient.

logical states the Cr51 bound to red cells would becumulative if repeated observations were made toevaluate therapeutic responses in thrombocytopenicrecipients.

In the present procedure the platelets are cen-trifuged after the initial labeling with Na2Cr51O4to remove residual Cr51 in the surrounding plasma.About 80 to 90 per cent of the Cr5l mixed withthe platelet concentrate remains in the plasma.Hence it is desirable to remove the 240 to 270microcuries of Cr5l from the suspension. How-ever, this additional centrifugation may lessenplatelet viability. Therefore, one study was doneto determine whether omission of the last cen-trifugation in the preparation of the platelets forinfusion would alter the observed survival period.

57000

54000

51000

48000

450a0

42000

38000

36000

3300a

CR5' 30000COUNTS

PER 2 700MINUTE2400C

21000

18000

15000

1t200

9000

6000

300C

0

PLATELET SURVIVAL

FW.WITHOUTWASHING

PLATELET

POORPLAS

PLATELETS

I POO.P_.

U2HOURS

O-x RED CELLWASHWATERS

-o REDCELL MASS

*- PLATELETWASHWATER

-WHOLEBLOD

I

;MA

DAYS

FIG. 6. INFUSION OF CR" LABELED PLATELETS WITH-OUT REMOVAL OF EXCESS NACR"04 IN PLASMA BYCENTRIFUGATION

There is no improvement or alteration of platelet val-ues by elimination of this additional centrifugation.Measurements were discontinued after seven days, al-though adequate Cr" activity was present in the plateletbutton for a longer period of observation.

In Figure 6 the survival period indicates no pro-longation of the Cr5' platelet activity, and further-more the higher dosage of Cr51 would limit theevaluation of platelets to one study and would notallow repeated observations in thrombocytopenicrecipients.

The in vitro studies with Cr51C13 indicate thatplatelet concentrates could be labeled although thelarger portion of radioactivity resides in theplasma of the final platelet concentrate. One

I

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11

It).

.1263

KNUT A. AAS AND FRANK H. GARDNER

- RED CELL WASHWERS

0-0 RED CELL MASS

PLATELET WASHWATER

BLOOD

DAYS

FIG. 7. PLATELET SURVIVAL OF CReCLS FEMALE, B. G.Cr"Cl3 labeled platelet concentrates may be followed

for nine days in this study. This chromium salt offersno advantage over Na2Cr5O4 and prolongs circulatingradioactivity by the associated binding of plasma proteins.

recipient received Cr51C13 labeled platelets, andthe results are plotted in Figure 7, showing thatthe radioactivity of the Cr5' platelet button couldbe followed for nine days. However, the highresidual plasma activity emphasizes the value ofNa2Cr5104 for in vitro labeling.

The selective separation of platelets by centrifu-gation was demonstrated in one recipient who in-advertently received a test dose of KI131 for evalu-ation of thyroid function. Despite the markedincrement of radioactivity in the whole blood on

the sixth day of study, a linear survival curve ofCr5' platelet activity could be followed for 10days (Figure 8).

Specificity of platelet labelingObservation of the plotted curves of Cr5' ac-

tivity in the recipients indicates that there is no

binding of the recipient's platelets. Measurable

radioactivity of the platelet button disappears be-tween 9 to 11 days, and no residual activity willbe found in the separated platelets thereafter.Platelet-poor plasma, separated red cells, and thewhite cell buffy coat suspended in platelet-poorplasma, were labeled with 30 microcuries ofNa2Cr51O4, and after incubation ascorbic acidwas added to each preparation. These suspensionswere then transfused into normal recipients.Blood samples from the recipients showed noradioactivity in the separated platelet buttons.

Urinary excretion of Cr5lThe Cr5' urinary excretion was measured dur-

ing the studies of Na1Cr5104 labeled platelets infour normal recipients (Table II). The urinaryradioactivity is expressed as the percentage oftotal counts of Cr5' in the infused platelet con-centrate. A large portion of the Cr51 activity in-fused into the recipient is excreted during thefirst 24 hours after the platelet transfusion. Thevariability in the initial 24 hour excretion reflectsthe efficiency of platelet labeling and is a measureof the residual Cr51 in the surrounding plasmathat was not removed completely after labeling.The excretion was followed for 22 days in eachinstance, and the percentage of Cr5' left withinthe body ranged from 19 to 35 per cent of the in-jected dose. In Table III the absolute values ofCr5l derived from the 300 microcurie labelingdose are tabulated on the basis of the per cent ofplatelets labeled. After 22 days, 6 to 18 micro-curies of Cr5' activity remained. Assuming acontinued daily excretion of 0.5 per cent of theoriginal Cr51 activity, the infused Cr5' will beexcreted in 80 to 90 days.

TABLE II

Radioactivity excreted in the urine and remaining in thebody expressed as per cent of the radioactivity

infused in the platelet concentrate

Excreted Excreted Remainingfirst first after

Recipient 24 hours 22 days 22 days

W. F. 16.8 38.6 34.5T. H. 45.0 65.0 19.7D. N. 37.6 61.0 22.0J. B. 24.0 48.0 29.0

1264

SURVIVAL OF BLOOD PLATELETS LABELED WITH CHROMIUM

DISCUSSION

The life span of Cr5 labeled human blood plate-lets is 9 to 11 days according to these studies.The manipulation of blood in plastic bag con-tainers has continued to offer the most satisfactorymethod for preparing sterile platelet concentrates.The elimination of air phase exposure in theseparation procedures assures rapid separation ofplasma components after centrifugation, and withpractice the labeled platelet concentrates may beprepared in less than two hours.

Recent studies by Campbell, Small and Dame-shek (22) have emphasized alterations of themetabolic function of human platelets during stor-age. Platelets labeled with P32 demonstratedleakage of phosphorus into plasma during storage;a lesion similar to changes noted in the red cellduring preservation studies. These metabolicchanges emphasize the difficulties associated within vitro studies, for the platelet function begins todeteriorate to some extent when the blood sampleis obtained. The rapid elution of Cr5' activityfrom the labeled platelets in vitro must be evalu-ated in relation to the time involved in making theobservation. Certainly, after the infusion of la-beled platelets into the recipient, elution cannot beobserved by the present methods.

Throughout the present technique the plateletshave been maintained in a plasma medium, sinceplatelets washed in physiologic saline show mor-phological alterations. Oxidase activity of plate-lets and the ability to promote clot retractiondiminish rapidly if platelets are suspended insaline (22). During the present investigationone transfusion was done with Cr5' labeled plate-lets suspended in physiological saline instead of

TABLE III

The radioactivity infused in the platelet concentrateand left in the body

Radioactivityof infused

platelet Radio-concentrate activityexpressed as remainingper cent of in bodythe initial Radio- 22 days

tagging dose activity afterRecipient (300 pc.) infused infusion

% Ac. Ac.W. F. 16 48 17T. H. 22 66 13D. N. 9 27 6J. B. 21 63 18

COUNTSPER

MINUTE

211 2 3 45 67 89 1011 12131415161718HWRS DAYS

FIG. 8. FAILURE OF KF' TO AFFECT PLATELET SURVIVALCURVE

On the sixth day of this study, the recipient receivedKI'31 orally to evaluate thyroid function. The separatedplatelet button radioactivity was not influenced by thecirculating P"3. Although the measurements are plottedas Cr51 activity, the whole blood and plasma reflect thepresence of Cr51 and I"5.

plasma. Although a normal life span of plateletscould be followed in the recipient's circulation, thelow radioactivity of the platelet button (15 percent of the whole blood activity) suggested thatthe majority of platelets had suffered irreversibledamage in the saline solution.

The low Cr5' activity of the separated plateletsin the recipient during the first few hours afterinfusion has not been explained completely. A re-view of the platelet counts of thrombocytopenic

1265

KNUT A. AAS AND FRANK H. GARDNER

FIG. 9. TABULATION OF PLATELET COUNTSIN THROM-BOCYTOPENICRECIPIENTS AFTER RECEIVING DONORPLATE-LETS COLLECTED IN PLASTIC BAG EQUIPMENT WITHNA2EDTA AS THE ANTICOAGULANT

The platelet counts after infusion are listed for 10 min-utes, 1, 3 and 6 hours. The infusion of whole blood,platelet-rich plasma, and platelet concentrates is as-

sociated with a delayed mixing for several hours.

recipients after transfusions from polycythemicdonors with thrombocytosis has demonstrated a

similar phenomenon. The gradual rise in cir-culating platelets in the peripheral blood has beenplotted in Figure 9. In each instance Na2EDTAwas used as an anticoagulant. Despite the rapidcollection of whole blood in plastic bags and rein-fusion without manipulation, a delayed appearance

of circulating platelets was noted. These changeswere not noted if whole blood was collected andinfused in small aliquots with silicone-coatedsyringes. The mere collection of platelets in an

anticoagulant appears to alter the membrane. Inaddition, centrifugation of the platelets for con-

centration and labeling may further effect plateletviability. In thrombocytopenic recipients onlyabout 50 per cent of the theoretical platelet yieldhas been recovered. In the present study therehas been no opportunity to compare directly theCr51 platelet activity with platelet yield, for sucha study must be done in a recipient with amega-

karyocytic thrombocytopenia.

Preliminary investigations by body surface-counting with a scintillation probe have suggestedtransient increased radioactivity over the lungsand liver areas. Possibly alterations in the plate-let membrane during the labeling procedure pro-duce a sticky surface with transient sequestrationin the reticulo-endothelial tissues. With nutri-tional and environmental equilibration, the plateletregains normal function and is released into thecirculation. Separate populations of labeled plate-lets do not appear as may be noted in the curvesof peripheral Cr51 platelet activity. The "peak-ing" of platelet activity during the first 24 hourshas been observed in all of the transfusion studies.Preliminary observations suggest that this 24 hourpeak disappears in the presence of thrombolyticplasma factors as noted in idiopathic thrombocyto-penic purpura and in platelet immunization frommultiple transfusion (2).

A review of the ABO blood groups in thesestudies indicates that incompatible transfusionsalso had 9 to 11 day survival periods. Althoughthe blood platelets appear to have grouping par-allel to the red cell ABO classification (23), in-dividual platelet transfusion studies are not af-fected by the incompatibilities. There is inade-quate evidence to demonstrate that shortenedplatelet survival periods are related to ABObloodgroup sensitization.

The measurement of labeled platelet life span inthe early transfusions of this study suggested anexponential curve when the values were plotted.Random destruction was assumed to exist follow-ing metabolic alteration of the donor plateletsduring the labeling procedure. With improvedproficiency in the preparation of the platelet con-centrates, the labeled platelet activity appears tobe linear, to suggest that the platelets have disap-peared by senescence. This linear loss of plateletradioactivity during the first nine days is helpfulevidence to exclude any pathological effect ofmetallic chromium. During the last 48 hours thatthe labeled platelets may be followed in the periph-eral circulation, the decline in Cr5' activity levelsoff. There is no adequate explanation for thisobservation, although this phenomenon may sug-gest changes in the binding of the Cr51 to theplatelet membrane and consequent elution. Thefull analysis of these changes may be evaluatedby simultaneous study of platelets labeled with

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SURVIVAL OF BLOOD PLATELETS LABELED WITH CHROMIUM

Na2Cr5104 and DFP32. In such instances theDFP32 incorporation in vivo would be the lifespan platelet reference, much as the Ashby dif-ferential agglutination survival period has beenused to compare Na2Cr5104 life span of red cells.

The present procedure with Na2Cr5104 offersa reproducible method as observed in 30 normaladult subjects. The ease of preparing labeledplatelets will allow more complete interpretationof thrombocytopenic states. At the present timethe method offers no therapeutic advantage overdirect transfusion of platelet-rich whole blood orplasma concentrates. However, labeled plateletsmay be prepared with ease from the average donorwithout seeking polycythemic donors with throm-bocytosis. The life span of platelets measuredwith DFP32 has the most reasonable physiologicapplication for the normal recipient, but largevolumes of venous blood are required to separatean adequate number of platelets. The amount ofp32 activity in the labeled platelets of thrombo-cytopenic subjects would not allow reproducibledeterminations.

SUMMARY

A standardized technique has been developedto evaluate the life span of transfused blood plate-lets in normal recipients. In vitro measurementsindicate that radioactive sodium chromate willbind to human blood platelets suspended inplasma. The factors related to time, temperatureand concentration of sodium chromate for labelingplatelets in vitro have been presented. Na2Cr5104labeled platelets have been infused into 30 normalrecipients. Determination of the radioactivity inthe recipient's platelets separated by differentialcentrifugation indicates that the transfused plate-lets have a survival period of 9 to 11 days. Norelabeling of the recipient's platelets or other bloodelements was observed. The platelet radioactivityin the recipient declines for the most part in alinear fashion to suggest that this method doesmeasure the life span of the transfused platelet.

REFERENCES

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2. Hirsch, E. O., and Gardner, F. H. The transfusionof human blood platelets. J. Lab. clin. Med. 1952,39, 556.

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7. Morgan, M. C., Keating, R. P., and Reisner, E. H.,Jr. Labeling of rabbit platelets with Iodine'.Proc. Soc. exp. Biol. (N. Y.) 1954, 85, 420.

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15. Adelson, E., Rheingold, J. J., and Crosby, W. H.Studies of platelet survival by in vivo tagging withP'. J. Lab. clin. Med. 1957, 50, 570.

16. Gardner, F. H., Howell, D., and Hirsch, E. 0. Plate-let transfusions utilizing plastic equipment. J. Lab.clin. Med. 1954, 43, 196.

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18. Stefanini, M., and Dameshek, W. Collection, pres-

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20. Frank, H., and Gray, S. J. The determination ofplasma volume in man with radioactive chromicchloride. J. clin. Invest. 1953, 32, 991.

21. Small, W. J., and Verloop, M. C. Determination ofthe blood volume using radioactive Cr': Modifica-tions of the original technique. J. Lab. clin. Med.1956, 47, 255.

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