leukocyte separation

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1965;25:1516-1520.Cancer Res

Emil J. Freireich, George Judson and Robert H. Levin

Separation and Collection of Leukocytes

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S eparation and Collection of L euk ocytes

EMIL J. FREIREICH, GEORGE JUDSON, AND ROBERT H. LEVIN'

National Cancer Institute, Bethesda, Maryland, and the International Business Machines Corporation, Endicott, New York

SUMMARY

Existing m ethods for separation and collection of leukocytes perm it collection ofonly lim ited quantities of leukocytes. A n instrum ent has been designed to processlarge volum es of blood on a continuous flow basis. The instrum ent collects venousblood, separates leukocytes in a centrifuge, and reinjects the red cells, plasm a, andplatelets. The separation process is 3060% efficient in vitro at flow rates up to 50rnl/m in. In vivo tests dem onstrated safety, sterility, lack of hem olysis, and adequateanti-coagulation. H owever, leukocyte recovery in vivo is low (under 20 % ). If thispoor in vivo recovery can be overcom e, the instrum ent should prove useful for collection of large quantities of leukocytes.

Because acute leukem ia is a disease w hich involves prim anly the leukocytes, collection of leukocytes for study isof great im portance. U nfortunately, these cells are theleast accessible component of the blood. Red cells can beobtained in pure form and in large quantity w ith ease because of their high specific gravity and their tendency toaggregation. The platelets are also relatively easy to collect because of their sm all size and low er specific gravity.Even the plasm a portion of the blood can be readily obtam ed in pure form . The leukocyte, unfortunately, is interm ediat.e and alm ost all technics of collection face theproblem s of contam ination w ith red cells and platelets.

This lim itation is aggravated by the fact that in norm alindividuals and in m ost patients w ith acute leukem ia theblood contains over 500 red cells and over 20 platelets forevery leukocyte. Finally, the leukocytes are a heterogeneous population of various myeloid leukocytes and lymphoid leukocytes. W itness the usual differential count,w hich alm ost alw ays distinguishes 5 different types of leukocytes with clearly different functions. These factspresent a m ajor obstacle to the solution of the leukem icproblem . An understanding of the physiology, biology,and biochem istry of the norm al and leukem ic leukocytew ill req uire effective tec hn ics o f leu ko cy te co llection .

For experim ental purposes there are a wide variety oftechnics for leukocyte separation (9, 19). These include

separation from a source of pure cells such as peritonealexudates (14), lym ph nodes, and thoracic duct lym ph (3,6) ; f iltration through fritted glass (4) or glass bead columns(5) to rem ove granulocytes; selective lysis of red cells (15);or flotation on density gradients (18).

By far the m ost comm only used m ethods utilize sedim entat.ion of red cells at 1 X g or at higher forces in acentrifuge. In 1948 the addition of high m olecular w eightsubstances was introduced, fibrinogen being the first substance studied (12). These m aterials cause aggregation of

1 D e ce a s ed .

red cells and greatly increase their rate of sedim entation.In addition to fibrinogen, dextran, phytohem agglutinin,p olyb ren e, a nd y -glob ulinhav e b een u sed effective ly (7 , 816, 17).

W hile m ost of these technics are useful for processingsm all quantities of blood, only the sedim entation technicshave proven practical for processing whole blood in relatively large quantities, i.e., over 500 m l. The technic oM aupin (10, 11) is am ong the best for collection of leukocytes without additives. Collection of buffy coat usuallygives yields of 3060% of the leukocytes in w hole blood.A ddition of red cell-aggregating agents usually increases

these yields to over 80 %.For supportive therapy of patients w ith leukem ia, replacem ent transfusion w ith both granulocytic and lym phocytic leukocytes offers an attractive approach. By usingas donors patients with chronic myelocytic leukemia(CM L) who have leukocyte counts 30 or m ore tim es norm al, it has been possible to show that transfusion of 10granulocytes per sq m of the body surface area of the recipient produces increm ents in circulating leukocytes of over1000/cu mm (13). For collection of the cells from suchdonors, centrifugation at slow speeds has generally produced the best separation (Table 1). Addition of dextrangreatly increases the yield of leukocytes. U nfortunately,because a plasm apheresis technic is used, som e of the dex

tran is reinjected into the donor as w ell as into the recipient. Frequent dextran injection can result in im m unization and possible anaphylaxis, and prolonged storage othis m aterial by the recipient also lim its its usefulness.The use of fibrinogen for this purpose is lim ited by thepotential contam ination w ith hepatitis virus. For thisreason we have studied 7-globulin (Table 2). The pairedstudies shown are separate units from the sam e donorstudied at the sam e tim e. Twenty m l of 7-globulin(16.5 % ) were added to each unit (500 ml) of blood.recovery w as regularly increased. U nfortunately, ad

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WBCNo.OF

UNITSCENTRIFUGAIONSPEED(rpm)TIME(mm)AVERAGELASMA

VOLUMEWBC/cumm

P L A S MATo t a l

(X1015)Yield%)23,20032001,70011,5001015017,000660030200150,0003.0021460020180170,0003.08206

DONORYI ELO(%)No

y-globulinWith-globulin1

2

Median48

32

4036262755

3387

85

8767657483

83

DonorRecovery%)B.

P.w .S.w .100,

100 ,100 ,10 0,9O, @8t @,0,80 ,6772, 69, 69, 6486,65,6472,67,64,60,55,55,48,40,3755,40,86,27,6a

V alu es in italics are m ed ian s.

1517REIREICH et al.Separation and Collection of Leukocytes

should be separated from w hole blood at a reasonablficienicy by sedim entation in a centrifuge. (b) Opeshould be conducted on a continuous flow basis toprocessing of large quantities of blood at optim alarid efficiency . (c) A vein-to-vein procedure shouused to avoid arterial puncture. (d) A n anti-coaguthat does not require anti-coagulation of the donor,its associated risks, should be employed. (e) The los

platelets, red cells, and plasm a should be m inim al toprocessing of large volum es of blood in a single donor.T he sy stem should be com pletely closed, needle-to-nw ithout any air-blood interface to obviate the dangair injection or bacterial contam ination. (g) T he esystem should contain a volum e of blood under 500all tim es. (h) T he system should be easily cleaned,disposable, and sufficiently autom ated to be operateds ingle nonprofessional operator.

A fter com pletion of 3 m odel designs, w e are curreinv estigating a centrifuge w hich subjects the blood tog of horizontal sedim ent.ing force (850 rpm ), in a vcylinder through w hich the blood passes continuousthe vertical direction. W hen the colum n of blood rthe bottom of cham ber it has separated into redbully coat , and plasm a. T here are 3 collecting portsat the outer w all w hich continuously rem oves frombottom of the packed cell layer (red cell port), oneinner w all w hich collects from the top of the plasm a(plasm a port.), and one in the center (leuk ocy te port)can collect buffy coat after enough has accum ulatedcentrifuge. Each collecting port has its outflow contrby a separate peristaltic pum p w hich perm its the opto vary the collection from each independently . Tpacity of the centrifuge is 190 m l. A ll of the desigdev elopm ental w ork w as perform ed on freshly draw nof w hole blood from norm al blood donors, w hichpooled prior to use and studied w ithin 8 hr of colleT he results of processing w hole blood at a through f5060m l/m in are show n in T able 4. Of the 4500blood run through the instrum ent, only 55 m l (1 %rem ov ed through the leuk ocy t.e port., w hich containedof the total leukocytes. T his is a 50-fold increasekocyte concentration. T his buffy coat contained onof the total red cells and 0.6 % of the platelets of theinal blood. T hese data are show n diagram m aticallyChart 1. T he red cell output contains v irtually allred cells and 5060% of the plasm a. It contains mthe leukocytes w hich w ould be lost during collection.plasm a pum p contains 30-50 % of the plasm a and 30of the platelets w ith little leukocyte contam ination.

TABLE 3R EC OV ERY O F L EU KO CY TE S F RO M S EPAR AT E D ON O

TABLE 1SE PA RATIO N O F L EU KO CY TE S F RO M D ON OR S W IT H

CHRONIC MYELOCYTIC LEUKEMIA

a M arked contam ination of buffy layer w ith red blood cells.

TABLE 2EFFECT OF 7-GLOBULIN ON RECOVERY OF LEUKOCYTES IN

PAT IE N TS W I TH C H RO N IC M Y EL O CY T IC L EU K EM I A

m in istratio n o f -y -g lo bu liri in trav eno usly c an o ccasio nallyproduce severe transitory hypotension. A lso to be emphasiz ed is the ex trem e v ariability in separation efficiencyfrom donor to donor (T able 3).

T o collect com parable quantities of leukocytes (10)from norm al donors, w ould require the cells from 20 to40 units of blood if leuk ocy te concentrations w ere 5,00010,000/cu m m . W ith the less than optim al recovery fromseparation and w ith the fact that only 5070% of thesecells are granulocy tes, 24tim es this quantity of bloodm ight have to be processed. Djerassi2 has reported thatcollection of leukocytes from a large num ber of units ofnorm al blood can increase the circulating leukocytes in arecipient. T o process quantities of blood of this m agnitude a leuk apheresis technic w ould be desirable. B ierm anet at. (1, 2) have described leukapheresis of norm al personsand leukem ic patients w ith the use of the A DL Cohn fractionator.

RESULTS AND DISCUSSION

We set out to design an instrum ent w hich w ould processlarge quantities of w hole blood from a donor, w ithout. anyadditiv e, utiliz ing a leuk apheresis technic. T he objectiv esof the instrum ent design w ere as follow s : (a) L eukocytes

2 Djerassi, A cute L eukem ia Task Force, Novem ber 18, 1963.



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PORTVOLUMEml @WBC1 0 ' %POL l S

A ND B AN DS

X lO@ %LY M PUS

A ND M ON OS

X 10' % HEM O GLO BINm % PLATELETS1O @Re d

cellPlasma

Leukocyte2960

1500

5566

33

19.2

0. 5

13.540

2

586.0

09.835

0563.5

0. 53.545

1045537

059 8

0110.6

4. 51.064

280.6Total450010023.310017.31007.810053810016.4100

SampleTotal W BCLymphs andmonos ((@@Polysand

bands (%)Hemoglob ingm/lOOl)17.79270216.29160312.545440.8413.417761.4513.122721.566.715771.473.132653.182.123752.991.732623.3

1518 Cancer Research

TABLE 4

Vol.25,October1965

SEPARATION OF WHOLE BLOOD IN THE CONTINUOUS FLOW CENTRIFUGE

free atmosphere, arid lack of hem olysis of red cells, weturned to in vivo study. This required design of a pum pfor m etering of anti-coagulant , acid-citrate-dextrose A(ACD-A), in the proper proportion during bleeding.was accom plished by an occlusive roller pum p rolling ontubes sim ultaneously. The ratio of the cross-sectionalvolum es w as selected to deliver the proper am ount of anticoagulant to blood on a continuous basis. M ixing occursnear the phlebotom y needle and further m ixing occurs during passage through the pum p. The centrifuge acceptsblood on a continuous basis, and the outflows from the redcell and plasm a pum ps are com bined and reinjected intravenously on a continuous basis through a 2nd needle.Thus m ost of the red cells, plasm a, and platelets are returned to the donor. Because the donation of blood isfrom an antecubital vein, a pressure cuff and m uscularforearm work are required to ensure adequate flow rates.Thus, the bleeding m ust be interm ittent to perm it restperiods. This collation of interm ittent bleeding with cont.inuous flow through the centrifuge was accom plished bydesign of a buffer system . It operates autom atically andis designed to lim it the am ount of blood away from thedonor at any tim e to less than 500 m l. It operates as follows : The donor bleeds at a faster rate than the flow intothe centrifuge. The excess blood is collected in a bufferbag. W hen the bag fills so that the m axim um am ount oblood is obtained, the instrum ent senses this, stops thebleeding pum p, deflates the blood pressure cuff, and initiates a needle rinse to rinse the noncitrated blood out of thetube, m aintaining a slow drip of saline to keep the donorneedle open. This is the rest period. The centrifugecontinuously draw s blood from the buffer bag, processesit, and reinjects it into the donor. W hen the bag is nearlydepleted, the instrum ent senses this and initiates the bleeding. The needle rinse is stopped, the pressure cuff is inflated, and the bleeding pum p begins. This phase of thein strum en ta tio n is efficie nt a nd effectiv e.

Initial clinical trial was begun in patients w ith chronicleukem ia. The first experim ent involved processing o250 ml of whole blood, and this quantity was progresincreased in subsequent studies to a m axim um of 11,000m l of whole blood processed at 1 tim e in a single donor.To date 44 experim ents have been perform ed in 9 patients,ranging from 15 m m to over 6 hr in duration. In 1 donorwith chronic m yelocytic leukem ia 72 liters of blood wereprocessed over a 23-day period ; 59 liters were processed in1 1 days of this period, being the m ost intensive leukapsis to date. This donor had 1 .6 X 1012 leukocytes remoin this 1 1-day period. His white count at the start of

h uffy co at. o r w hite cell pu mp has 30 6 0% o f th e leu kocy tesw ith som e red cell and platelet contam ination.

A lso of interest is that som e degree of separation oflym phocytes arid granulocytes is also observed (Table 5).If a large buffy coat is allowed to accumulate, and is thenrem oved from the plasm a side out to the packed red celllayer, the 1st. sam ples have alm ost pure lym phocytes andthe latter sam ples contain predom inantly granulocytes.In the experim ent shown, the peak of lym phocytes is seenin Tube 2, containing over 90 % lym phocytes, while thegranulocyte peak is observed in Tube 4, where 76 % of cellsare grariulocytic. These data are shown graphically inChart 2. The peak leukocyte concentration is in Sam ple2, before m easurable red cell contam ination is observed.The red cells increase in quantity whereas leukocytes decrease after Sample 6. The peak concentration of lymphocytes precedes the peak for granulocytes, the latter beingcontaminated with red cells.

A fter in vitro studies had established sterility, pyrogen

R B C%

too PLI4TPLAS

@ : I@ r:i i@ fl[T@ :

CHART 1.Separation of whole blood by continuous flow

centrifuge. Red cell port (I?) contains outflow from outer wall ofcentrifuge. Plasma port (P) is outflow from inner wall. Whitecell port (jjr) the buffy coat collection. The fraction of red cells(R B C ), leukocytes (IV B C ), platelets (P L AT ), and plasm a (P LA S)which appears in each port is shown in %.

TABLE 5S EPAR ATI ON O F L EU K OC Y TE S



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FR E IR E IC H et al.Separat ion and C ollection of L euk ocy tes

x,oe

4

S A M PLE N U M B ER

C H A RT 2.Separation of lym phocy tes and granu locy tes in the cen tr ifuge. T heabsolute num bers of total leukocytes (Total), lym phocytes (Lym ph), and granulocytes

(P oly) a re shown on the left ver tica l a xis in units of 10@cells. The hemoglobin is shownin gm on the right vertical axis. Sam ples w ere rem oved from the plasm a side of the buffycoat (Sam ple 0) out toward the packed red cell layer (Sam ple 9).

1519

18. Totc /0 Ly mp hPo /yA N b

AA I,

I

A - A @%

0 O .@

period w as 188,000 and rem ained at this level, ending w itha count of 208,000. In addition his splenom egaly increased during the procedure. Platelet count w as notsignificantly decreased by this procedure.

T hese prelim inary studies w ere designed prim arily togive inform ation on safety and operating effectiveness ofthe system . The potential toxicity of citrate injection hasbeen studied. EKG m onitoring is em ployed, and Q -T interval is observed during the procedure. A t rates of injection below 50 m l of blood per m m , no effects are ob

served. A t higher rates, paresthesia about the face andfingers has been noted even though Q Tintervals are norm al. Plasm a levels of citrate increase to 2535m g/100m l at the tim e Q Tinterval prolongation is seen. D ecreasing the rate of in j ection resu lts in p rom pt reversal of

these signs. Citrate levels have returned to under 5 m g/100 m l w ithin 1 hr after cessation of injection in all 7 studies to date.

Blood coagulation is not altered, and m easurem ent ofplasm a prothrom bin, recalification tim e, and fibrinogenlevels show s no significant change. Plasm a hem oglobinlevels have rem ained in the norm al range for all studiessave 1,w hen a hem olyt.ic episode occurred because of personnel failure. The step in question has since been auto

m ated to avoid recurrence. N o evidence of clinical pyrogen or bacterial contam ination has been seen in 39 of thestudies. Patients have been afebrile and asym ptom atic.In our early trials, 1 m ajor fever-chill reaction and 4 lowgrade febrile episodes w ere observed im m ediately following the procedure ; each lasted less than 6 hr. W e haveincorporated a w ater bath at 38 C to w arm the bloodprior to reinjection, w hich has cooled tow ard room temperature. Since then, no febrile reactions have been observed. These studies have established the safety of theins t rument .

W hile the instrum ent has proven safe for in vivo leu

kapheresis, the efficiency of leukocyte separation in vitrocould not be confirm ed in vivo. A s already em phasized,initial studies were conducted in patients with chronicleukem ia. The 2 patients w ith chronic lym phocytic leukem ia had circulating w hite counts of 17,000 and 190,000/cu m m . R ecovery ranged from 25 to 60 % of the leukocytes passing through the instrum ent and consisted alm ostentirely of sm all lym phocytes, as did the peripheral blood.Five patients w ith chronic m yelogenous leukem ia w erestudied. W hite counts ranged from 10,000 to 250,000.

Leukocyte recovery ranged betw een 2 and 22 % (m edian,12 % ). In these pat ien ts r ecovery consisted alm ost entirely of im m ature m yeloid cells. B ecause w e felt that thepoor leukocyte recovery could result from the very abnorm al leukocyte-erythrocyte ratios in these patients, w e studied 3 patients w ith norm al peripheral blood, 1 patient w ithC M L in rem ission , and 2 pat ien ts w ith m alignan t d isease.In 7 experim ents leukocyte recoveries have ranged from 1to 17 % (m edian, 9 % ), and those leukocytes collected arealm ost all lym phocytes. Thus, the in vivo experim entshave given very poor leukocyte recoveries to date.

The cause of the difference betw een in vitro and in vivoexperim ents rem ains enigm atic at present. W e have investigated the im portance of tem perature. Experim ents

w ere run w ith an ice bath, at room tem perature, and w itha 37 C bath. Blood w as equilibrated at these tem peratures before in vitro experim ents. Leukocyte recoveriesw ere low er in the cold (20 % ), but room tem perature (20 22 C )and 37 Cgave com parable separation efficiency, 58and 44 % , respectively. M oreover, both granulocytes andlym phocytes w ere collected. The effect of hem atocritw as investigated by preparing plasm a rich in w hite bloodcells and preparing sam ples from 6 to 17 gm /100 m l ofhem oglobin. A gain no m ajor difference w as observed; recoveries ranged from 30 to 80 % with m edian recovery of50 % , indep enden t of h em oglob in con cen t rat ion .



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Cancer Research52 0

T o date w e have not. been able to duplicate the low invivo recoveries in in vitro experiments. We are left withtw o possibilities : the first is that blood 48hr old differsfrom fresh blood in som e param eter; the second is thatpooling several units of blood causes som e alteration inphysical properties. Com mon to both is the possibilitythat som e red cell aggregation has tak en place in vitro andaided in separation. T hese possibilities are being inves

tigated.Even if the problem of in vivo separation is overcom e,other problem s still rem ain. T he present laboratorym odel uses hand-assem bled com ponents including 64 ft ofplastic tubing, 50 ny lon f riction f ittings, 10 three-w ay stopcock s, arid 1 1 intrav enous solution sets and bottles. T heseare all cleaned arid assem bled by hand, representing 3 hrof w ork . Cleaning arid assem bling the centrifuge requires1 h r : 3 0 m m for a ssem b ling the appa r a tu s an d 2 hou r s fortear-dow n procedure. T he seal assem bly is nondisposable,hand-tooled, and expensive. Evidently , w e have m uchdevelopm ent ahead to m ake the instrum ent easy to usean d m o stly d is po sab le .

If these problem s can be solved, w e hope that an instru

m ent w ill result w hich w ill m ake leukocyte collection inquantity a reality . T his tool could be enorm ously usefulfor overcom ing the obstacle of leukocyte collection andserv e as a biopsy technic for study of these im portant organsy stem s, th e leu ko cy tes.

REFERENCES1. Bier man , H . R ., K elly, K . H ., Byr on , R . L ., an d M ar sha ll, G .

J . L eucaph er esis in M an . I . H em atologica l O bser vat ionsFollow ing Leucocyte W ithdraw al in Patients w ith N onh em atolo gic al D iso rd ers. B rit. J. H aem ato l., 7: 5 1 6 3, 96 1.

2. B ierm an, H. R., M arshall, G. J., K elly , K . H., and B yron, R .L . L eu ka ph er esis in M an . I II . H em atologic O bser va tion s inPatients w ith Leukem ia and M yeloid M etaplasia. B lood, @1:16481,963.

3. F ar r , R . S. E xp er im en ts on th e F ate of th e L ym ph ocyte. An at .

Record, 109:515-34, 1951.

4. F lem in g, A . A Sim p le M eth od of R em ovin g L eu k ocytes fr omB lo od . B rit. J. E xp tl. Path ol., 7 : 2 81 8 6,9 26 .

5. G ar vin , J . F actor s Affect in g th e Ad h esiven ess of H u m anLeucocytes and Platelets in V itro. J. Exptl. M ed., 11961.

6. H arris, T. N ., and H arris, S . B iological and Technical Fin th e Dem on st r a t ion of An tib od y P r od u ct ion b y L ym p h a t icTissu e. J. Im m u no l., 6 4: 4 5 5 6, 95 0.

7. Lalezari, P. A N ew Technic for Separation of Hum ancy tes. B lo od, 1 9: 10 91 4, 96 2.

8. Li, J. G., and Osgood, E. E. A M ethod for the Rapid Stion of L euk ocy tes and N ucleated Ery throcy tes from BM arrow w ith a Phy tohem agglutinin f rom R ed B eans (PIn s v ulg aris). Ib id ., 4 : 6 70 7 5, 94 9.

9. M au p in , B. T ech n iq u es d e sep a r at ion d es glob u les b lan cs. R evH m atol.,14: 25065,5579,4386,959.

10. . E tu d e d e la sep a r a t ion d es leu cocytes a p a r ter d u san ghum ain. S ang, @ 1:6, 1950.

11. M aup in , B., and C ha r y, R . P r esen ta t ion d ' une nou vellem t ho ded e s ep aratio n d es le uc oc yte s e t d es p laq ue ttguines. Ibid., 28: 336, 1952.

12. Minor, A. H., and Burnett, L. A Method for Obtaining LivingL euk ocy tes f rom H um an Peripheral B lood by A cceleraE ry th ro cy te S ed im e ntatio n. B loo d, 8 : 7 99 8 1, 948 .

13. M or se, E . E ., Br on son , W ., C a r b on e, P . P ., an d F r eir eich , EJ . E ffect iven ess of G r a nu locyt e T r a nsfu sion fr om D on or s w itChronic M y elocy tic L euk em ia to Patients w ith L euk

C lin. R es., 9: 332, 1961.14. Pon d er , E ., an d M acL eod , J . L eu cocyte M or p h ology iR abbits w ith Induced Peritoneal Ex udates. J. Ex ptl.67: 83946,938.

15. Sin ger , T . P., Silb er ba ch , L ., a nd Sch war tz, S. T he P rep ar at ion of M or p h ologica lly In tact L eu k ocytes fr om P er ip h er alB lood by M eans of G ram icidin and Lysolecithin. B lood,i ssue No. 1: 8287,947.

16. S koog, W. A ., and B eck , W. S . S tudies on Fibrinogen,and Ph yto hem ag glu tin in M e tho ds o f Iso latin g L eu kIbid., 11: 436, 1956.

17. T ullis, J. L . S eparation and Purification of L euk ocy tePlatelet s.bid.,:8 9196, 952.

18. Va llee, B. L ., H u gh es, W . L ., an d G ib son , J . A . A M eth od foth e Sep a r a t ion of L euk ocytes fr om W hole Blood b y F lot a t ionon Serum Albumin. Blood, special is sue No. 1 :889947.

19. Wa lfor d , R . L . L eu kocyt e A nt igen s a nd A nt ib od ies, C h . 1. N ew

Y o rk : G ru ne an d S tratto n, 1 96 0.

Vol. 25, October 196


leukocyte separation

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