BritishJournal of Haematology, 1976,34,395.

Preparation and Storage of Platelet Concentrates I. FACTORS INFLUENCING THE HARVEST O F VIABLE PLATELETS FROM WHOLE BLOOD S. J. SLICHTER AND L. A. HA-

Puget Sound Blood Center and Department of Medicine, University of Washington School of Medicine, Seattle, Washington, U.S.A. (Received 27 December 1975 ; acceptedfor publication 8 March 1976) SUMMARY.Factors affecting the yield and viability of concentrated platelets have been investigated in a blood component programme. It was found that 86+ 1% of the platelets from a unit of whole blood can be concentrated without loss of viability by processing ACD or CPD anticoagulated blood at room temperature. The steps are initial centrifugation at 1000 g for 9 min to harvest platelet-rich plasma; centrifugation of the platelet-rich plasma at 3000 g for 20 min to pack the platelets; and resuspension of the platelet pellet in residual plasma after 14 h. Platelets are given to control thrombocytopenic bleeding. For example, the use of platelet transfusions in patients with leukaemia has been associated with a decline of haemorrhagic deaths from 22 to 14% over a 5 year period (Hersh, 1965). Initially, whole blood or plateletrich plasma were used as sources of platelets, but concentrates permit the administration of the required number of platelets without volume overload. The increasing demands for platelets require that blood centres operate as efficiently as possible in providing these and other blood components. This study was carried out to identify the optimum techniques for preparation of platelet concentrates within the practical limits imposed by a blood transfusion service. In a companion article the problem of platelet storage is discussed (Slichter & Harker, 1976).

MATERIALS AND METHODS Blood from routine blood bank donors (450 ml) was collected into the first bag of a Fenwal double or triple bag system containing either 67.5 ml of acid-citrate-dextrose (ACD; NIH formula A, PA-20 or PA-30) or 63 ml of citrate-phosphate-dextrose (CPD; GF-25N or GF-~sN).Fenwal plastic PL-130 bags were used for bleeding and PL130 or PL-146 bags for transfer packs. Platelets were separated from other components using a Sorvall RC-3 centrifuge with horizontal head and swinging buckets. The radius measured from the centre of the rotator head to the bottom of the cup was used in calculating gravitational force. Frequent calibration insured accuracy of brake time and number of revolutions per minute. Centrifuge Correspondence: Dr Sherrill J. Slichter, Puget Sound Blood Center, Terry at Madison, Seattle,Washington 98104,U.S.A.

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time was measured from onset until the rotator head had completely stopped. A maximum of 2 min was allowed for braking. All procedures were carried out at room temperature (22k 2°C). Platelet harvestfrom whole blood. A total of 4 5 units of whole blood were tested using six centrifugal forces between 175 and 1600 g for periods ranging from 3 to 14 min. After centrifugation, the platelet-rich plasma (PRP) up to the red cell interface was delivered by a Fenwal plasma extractor into the satellite bag. For some samples, varying weights of ACD solution were transferred into the satellite bag before blood collection to reduce the final pH of the PRP (Chappell, 1966). Platelet harvestfrom PRP. PRP from zg6 units of whole blood collected in ACD or CPD was recentrifuged to pack the platelets. The minimum centrifuge time required to harvest at least 95% of the platelets was measured for four centrifugal forces between 1500 and 4000 g applied for 10-30 min. Supernatant was either returned to the red cells or transferred to a second satellite bag to prepare cryoprecipitate. The platelet pellet was then resuspended in 20 ml of residual plasma by gentle manual agitation following a 14 h delay (Mourad, 1968). for acidified units (pH 6 .s), immediate resuspension was possible. Platelet counting. Platelet counts were obtained on whole blood, PRP, platelet-poor plasma (PPP), and platelet concentrates. All samples for counting were collected in 10% EDTA (0.1 ml per 5 ml sample) to promote disaggregation of platelet clumps. Concentrates were allowed to stand 2 h following addition of EDTA. Counting was done by an electronic particle counter (Bull et al, 1965; Harker & Finch, 1969). Duplicate samples were diluted in Isoton (Harleco, Santa Ana, CA) to a concentration within the counting efficiency of the machine: 13 p1 whole blood in 25 ml Isoton; 3 pl PRP in 25 ml; sop1 PPP in 25 ml; and 3 pl concentrate in so ml. Results correlated well with those obtained using phase microscopy, and the reproducibility of the technique was k 3y0. Total counts were obtained by multiplying the platelets countkpl in blood or plasma by the corresponding volume determined by weight. The platelet yield in PRP was expressed as a percentage of the original whole blood platelet content and the yield in the PC as a percentage of the PRP content. Data on ACD- and CPD-collected units were pooled since no significant difference in platelet yield was detected. Viability measurements. Viability of the concentrated platelets was determined by measuring recovery and survival of autologous "Cr labelled platelets (Aster & Jandl, 1964; Harker & Finch, 1969; Paulus, 1971). The labelling procedure required an additional centrifugation of the platelet concentrate at 1000g for 15 min to remove unbound radioactivity. Labelled concentrates wcre resuspended and injected intravenously using a syringe so that all cells were infused. Initial recovery (labelled platelets remaining in circulation after infusion) was calculated from the platelet counts/inl at time zero (determined by extrapolation) multiplied by the blood volume (75 mg/kg body weight) and divided by the 51Cr platelet activity injected. Survival was determined by measuring disappearance of radioactivity from blood at five daily intervals following the injection. When the disappearance curve was linear, survival time was determined by simple extension to zero activity; when it was curvilinear, the initial slope was extrapolated to zero. For exponential curves, survival was calculated by dividing the half-life disappearance by In 2. Because platelet viability is a function of recovery and survival, it is expressed in units of area under the survival curve (e.g., Fig 5 ) . The platelet

Preparation of Platelet Concentrates

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viability index compares the viability obtained for a given experimental procedure with that of controls. Control recovery and survival values were established in 16 normal subjects using autologous platelets prepared by the following technique to prevent platelet injury (Aster & Jandl, 1964; Harker & Finch, 1969). Whole blood collected in ACD was centrifuged at 500 g for 12 min. After transfer of PRP to the satellite bag, it was acidified to pH 6.5 by addition of 0.15 M citric acid. The PRP was then centrifuged at 1500 g for 15 min to pack the platelets, followed by immediate resuspension using gentle manual agitation. AABB procedure. For comparative purposes, platelet concentrates were prepared from I I units of whole blood collected in ACD following procedures outlined by the American Association of Blood Banks (AABB) (1970): initial centrifugation at 4470 g for 3 min followed by centrifugation of the PRP at 5000 g for 5 min.

RESULTS Counting E$iciency Formation of platelet aggregates in the counting samples would result in serious underestimation of platelet yield, particularly for the concentrates. In response to this concern, more than 200 concentrates were counted using the method described: 94-t 4% (SE) of the platelets present in PRP were detected in the concentrate, while 2 + 1% of the platelets remained in the PPP. The remaining 4% were unaccounted for. This small loss of platelets suggests that aggregation does not seriously underestimate the yield. Subsequent studies were undertaken to determine the importance of using EDTA. The preparation of 20 ACD concentrates was monitored with and without the addition of EDTA to counting samples. The following counts were obtained, respectively: PRP, 415 ooof 15 ooo/pl and 405 ooo+ 20 ooo/pl; PPP, 14 ooo* ~ooo/pland 15 ooof 1ooo/p1; and concentrate, I 436 ooo+ 57 ooo/pl and I 395 ooof 62 ooo/pl. These data indicate that addition of EDTA is not a critical factor in electronic counting of platelet concentrates.

Centrifugotiontime (min)

FIG I. Platelet yield in PRP as a function of centrifugation time at expressed as percentage of platelet content in whole blood (WB).

1000 g.

Yields

mean+^

SE)

Centrifgation Whole blood. The gravitational forces investigated gave optimum platelet yields after specific times; for example, a centrifugal force of 1000 g produced maximum yield in PRP after g min (Fig I). The time at which optimum platelet yield occurred was inversely pro-

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Slichter and L. A. Hurker

>

I

4

8

12

16

20

24

28

Centrifugation time (min)

FIG 2. Time of maximum platelet yield in PRP for six centrifugal forces between 175 g and 1600g. Time is inversely proportional to logarithm of force applied.

portional to the logarithm ofthe centrifugal force applied (Fig 2). Optimum platelet yields for the six centrifugal forces are plotted in Fig 3. A maximum (89+ I%) occurred at a force of 1000 g for g min; the corresponding PRP volume was 280f 5 ml. PRP. The minimum times required to harvest 95% of the platelets in PRP were found to be inversely related to the logarithm of the centrifugal force applied (Fig 4). The shortest time not associated with platelet injury was 20 min at 3000 g (see Table I).

Resuspension of Platelet Pellets To achieve consistent resuspension of platelet pellets from nonacidified PRP, a delay of I+ h was required prior to gentle manual mixing (Mourad, 1968). Shorter delays produced visible clumping in I S + 5% of the concentrates. Platelet pellets from acidified PRP (PH 6.5) could be resuspended immediately without macroscopic aggregate formation. Yield Based on the predicted yields from the two centrifugation steps, the final concentrate should contain approximately 85% of the platelets in the unit of whole blood (95% of 89%). To verify the expected yield, 16 concentrates were prepared following the steps outlined (centrifugation at 1000g for g min followed by centrifugation of the PRP at 3000 g for 20 min). An average of 86+ 1% of the available whole blood platelets were detected in the final concentrate. Platelet Viability Platelet recovery and survival values for the sixteen controls are shown in Fig 5 . Recovery averaged 59f 4% and survival 8.1 & 0.2 d and this was assigned a viability index of I. Using the method of platelet preparation described for controls, a significantly higher recovery would be expected only in splenectomized individuals (Harker, 1971). Since in a closed bag system acicllication by citric acid is not possible, studies were performed on ACD transfer.

Preparation

of Platelet Concentrates

I

J 15

I

5

0

399

centrifugal tme

10 ( x qavity x 10')

FIG3. Optimum platelet yields in PRP (mean* I SE) for centrifugal forces shown in Fig 2. Maximum harvest of 89 f 1% occurs at 1000g for 9 min.

10

I

I

I

I

J

10

20

33

40

Centrifugationtime (min)

FIG 4. Minimum centrifugation times to harvest 95% of PRP platelets for four centrifugal forces between 1500 g and 4000 g.

TABLE I. Effect of centrifugation on platelet viability Centrifugation

No. of observationr

Force (xg)

Time (min)

Recovery 59+4 59+5 s6* 3 55+3

I so0

IS

2025

30

15

3000

20

16

4000

I0

16* 6

Platelet

(%I

Survival

(4 8.1 If: 0.2

Viability index I.OO+O.O~ 0.05

8.2 k 0.2

0.99

8.2If:O.Z

0.97+0.07 0.88&o.o5f

7.6k0.3

Platelets prepared from ACD-acidified PRP (pH 6.5); control platelets acidified with either 0 . 1 5 ~citric acid or ACD. Results are expressed as the average+ I SE. * Control data. t Significantly different from control (P< 0.05).

S. J. Slichter and L. A. Harker

400

60

1

2

6

4 Survival ( d

8

1

FIG 5 . Recovery and survival of autologous platelets measured at five daily intervals in 16 normal controls. Points and bars represent mean+ I SE, respectively. See text for description of platelet preparation and viability measurements. Shaded portion = platelet viability.

T

6.2L

0

I

10

I

20 ACD tmnsferred (ml)

I

30

FIG6. Relationship between amount of ACD transferred to satellite bag and pH of the PRP. Points and bars represent mean& I SE for 4-8 units of blood tested, respectively. Volume of PRP = 280+ 5 Id.

At a constant plasma volume, the amount of transferred ACD was inversely proportional to the resultant pH (Fig 6). To achievc a pH of 6.5 in a plasma volume of 280 ml (volume after centrifugation at 1000g for 9 min), 25 ml of ACD was required. No visible clotting of residual red cells occurred at this level of transfer. (CPD anticoagulant cannot be used to acidify PRP because it has a pH of 6.9.) To determine the effect of centrifugation on viability of platelets, concentrates prepared from ACD-acidified PRP were centrifuged at the times and forces corresponding to a 95% harvest (Fig 4). Measurements of platelet viability in the resulting concentrates indicated that thc shortest centrifugation time not associated with platelet injury was 20 min at 3000 g (Table I). The viability of platelets prepared from ACD-acidified (pH 6.5) and nonacidified PRP was compared. There was little differencc in viability for platelets harvested after 20 min at 3000 g (Table 11).

Preparation

of Platelet Concentrates

TABLE 11. Effect of anticoagulant on platelet viability Platelet No. of' observations ~~

15 I2

I1

PRP anticoagulation ~~

Recovery

Survival

(%)

(4

Viability index

561 3 53 f 4 54+2

8.2f0.2

0.97f0.07

8.3 f 0.2 8.1f0.3

0.93 f0.07

~

g ACD transfer* Nonacidified ACD Nonacidified CPD 2s

0.92+0.05

Platelets harvested after centrifugation at 3000 g for 20 min. Results are expressed as the average+ I SE.

* pH 6.5. AABB Method For the 11 concentrates prepared using AABB procedures, the platelet yield in PRP was 43%f 3% (volume 3oof 8 ml). The concentrates contained 41 5 2% of the available platelets in the whole blood unit. When 51Cr-labelled platelet concentrates were used in autologous transfusions to four normal donors, the recovery, survival and viability index were 61+ 8%, 8.2f 0.5 d and 1.0250.05, respectively. DISCUSSION Accurate, standardized centrifugation procedures are critical for efficient preparation of platelet concentrates. Our data show that centrifugation of whole blood at a force of 1000g for g min provides the maximum yield of platelets in PRP (89+ 17~). After transfer of the PRP to the satellite bag, recentrifugation at higher g forces was required to separate more than 95% of the platelets from plasma. Centrifugation time of the PRP can be decreased by applying higher gravitational forces, but only at the expense of platelet viability (Table I). Platelet viability is reduced significantly at a force greater than 3000 g for 20 min. In a recent review of techniques for preparation of platelet concentrates (Tranum & Haut, ~ g p )the , highest reported platelet yield in the concentrate was 72% of those present in the whole blood. Concentrates prepared following our procedures yielded an average of 86rf:1% of platelets present in whole blood, while those prepared using AABB procedures contained an average of 41 f2%. 10 vim measurements of platelet recovery and survival were similar using our procedure and that of AABB. For a transfusion service, selection of the most efficient method for preparing platelet concentrates depends on the interrelationship of at least seven factors : (I) centrifugation time, (2) yield, (3) viability, (4) function, (5) resuspension time, (6) storage capability, and (7) recovery of cryoprecipitable factor VIII from the PPP. In this paper we have recorded the centrifugation procedures which correspond to maximum platelet yield and viability. Other aspects of the technique were influenced by factors of storage discussed in a companion paper (Slichter & Harker, 1976). For example, while acidification of PRP to pH 6.5 allowed immediate resuspension of platelet pellets without loss of platelet viability, platelet viability during storage was compromised and yield of factor VIII in cryoprecipitate was reduced by

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50% (Pool, 1967). Similarly, while concentrates prepared from whole blood anticoagulated with either CPD or ACD had similar viability, CPD concentrates had better storage capability at 22°C.

ACKNOWLEDGMENTS

We gratefully acknowledge the helpful suggestions of Dr Eloise Giblett and Dr E. D. Thomas, the editorial comments of Mary Weickowicz and the technical assistance of Lorna Cook, Anne Halverson, Maureen Williams, Mary Francisco, Nancy Scott, Mabel Graham and Eileen Beard. This work was supported by NIH grants (CA-12190, CA-10895) and contract (NIHNHLI-70-2214). A portion of this work was conducted through the Clinical Research Center facilities of the University of Washington (NIH grants FR-37 and RP-133). REFERENCES AMERICAN ASSOCIATION OF BLOOD BANKS(1970) Manual of Component Preparation, p 160. American Association of Blood Banks,Washington, D.C. ASTER,R.H. & JANDL,J.H. (1964) Platelet sequestration in man. I. Methods.Journal ofClinica1 Investigation, 43, 843. BULL,B.S., SCHNEIDERMAN, M.A. & BRECHBR, G. (1965) Platelet counts with the Coulter Counter. American Journal of Clinical Pathology, 4, 678. CHAPPELL,W.S. (1966) Platelet concentrates from acidified plasma: a method of preparation without the use of additives. Tranifusion, 6, 308. HARKBR, L.A. (1971) The role of the spleen in thrombokinetics. Journal of Laboratory and Clinical Medicine, 77, 247. HARKER, L.A. & FINCH,C.A. (1969) Thrombokinetics in man.Journal of Clinical Investigation, 48, 963. HBRSH, E.M., BODEY,G.P., Nm,B.A. & FREIRBICH,

E.J. (1965) Causes of death in acute leukemia. Journal ofthe American Medical Association, 193, 10s. MOURAD,N. (1968) A simple method for obtaining platelet concentrates free of aggregates. Transfusion, %48.

PAULUS,J.M. (1971) Platelet Kinetics: Radioisotopic Cytological, Mathematical and Clinical Aspects. American Elsevier Publishing Co., New York. POOL,J.G. (1967) The effect of several variables on cryoprecipitated factor VIII (AHG) concentrates. Transfusion, 7, 165. SLICHTZR, S.J. & HA=, L.A. (1976) Preparation and storage of platelet concentrates. 11. Storage variables influencing platelet viability and function. British Journal ofHaematology, 34, 401. TRANUM, B.L. & HAW,A. (1972) In vivo survival of platelets prepared in CPD anticoagulant. Transfusion, 12, 168.