Synthetic Membranes: Volume II - ACS Publications - American

8 Dialysis

Processing

of

Cryopreserved

Red

Blood

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Cells ALLEN ZELMAN, DAVID GISSER, and DEXTER SMITH Center for Biomedical Engineering, Rennsselaer Polytechnic Institute, Troy, NY 12181 ROBERT STEPHEN Division of Artificial Organs, University of Utah, Salt Lake City, UT 74112 Red c e l l s , frozen i n the presence of glycerol, can be stored for years at -80 C with excellent post-thaw recovery and i n vivo survival. Today, three approaches to red cell freezing and deglyceroli z a t i o n are i n clinical use: the agglomeration method (1), the low glycerol method developed i n the United States (2, 3) and i n Europe (4) and the high glycerol procedure (5) modified and adopted by the American Red Cross (6). Estimates of the extent to which these three procedures are used i n North America, based on sales of glycerol solutions during 1976 by United States manu facturers, t o t a l 25,000 units processed by agglomeration, 30,000 by the low glycerol method and 165,000 by the high glycerol pro cedure. A l l procedures y i e l d a product of essentially equivalent quality (7, 8). Differences i n the rate of use to some extent reflect differences i n processing costs as well as the very large impact of the Red Cross Blood Program which processes an e s t i mated 50% of all units frozen i n North America. Of a l l the uses for frozen red c e l l s probably the most im portant and far reaching would be their use for inventory control. F i r s t , i n regional blood centers, c e l l s collected during peak collection times of the year could be maintained i n storage for use i n times of d e f i c i t . Second, small isolated hospitals, be cause of irregular needs, now must either tolerate an excessive rate of outdating or engage i n an extensive exchange program with a distant regional center. Frozen red c e l l s would be a solution for both of these inventory control problems but only provided that the glycerolization procedure was rapid, simple and econom ical. Some of the problems surrounding the removal of glycerol from the high glycerol preparation have been overcome through the use of c e l l washing devices. One of these, developed by the Haemonetics Company (Braintree, MA) and based on the Cohn Fractionator, performs a continuous flow wash i n a disposable bowl. The other apparatus developed by the IBM Corporation, conducts an automated batch wash i n a disposable bag. Although effective, º

0097-6156/81/0154-0109$05.00/0 © 1981 American Chemical Society In Synthetic Membranes: Volume II; Turbak, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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SYNTHETIC MEMBRANES:

HF

AND

UF

USES

these devices add c o n s i d e r a b l e cost to the use of f r o z e n c e l l s . The c a p i t a l equipment ranges from over $5,000 to over $17,000 w i t h the d i s p o s a b l e p l a s t i c components ranging from about $8.00 to over $20.00. The washing p r o t o c o l s r e q u i r e roughly 30 minutes per u n i t and these devices w i l l wash only a s i n g l e u n i t at a time.


Advantages of Frozen Red C e l l s Using Current Processing Methods. Long term p r e s e r v a t i o n i s the most obvious asset of f r o z e n red c e l l s and one which i n e a r l i e r days was assumed to be the only novel v i r t u e . This extended s h e l f l i f e has proven i n v a l u a b l e f o r the p r e s e r v a t i o n of r a r e types and c e l l s f o r a u t o t r a n s f u s i o n . I n ventory c o n t r o l i s an important a p p l i c a t i o n as w e l l , but one whose f u l l p o t e n t i a l has been l i m i t e d by the high cost of f r o z e n c e l l s . Reduced i n c i d e n c e i n non-hemolytic t r a n s f u s i o n r e a c t i o n s , as T u l l i s , Haynes et a l . (9) c o r r e c t l y observed, i s one of the f r i n g e b e n e f i t s of d e g l y c e r o l i z i n g red c e l l s . Washing f o r d e g l y c e r o l i z a t i o n removes v i r t u a l l y a l l the plasma, the p l a t e l e t s , and most of the leukocytes. White c e l l s are l e s s permeable to g l y c e r o l than red c e l l s and the g l y c e r o l i z i n g procedure used f o r red c e l l s i s damaging to leukocytes. Most of these c e l l s are destroyed by g l y c e r o l i z a t i o n alone w i t h a d d i t i o n a l d e s t r u c t i o n o c c u r r i n g during f r e e z i n g and thawing. V a l e r i (10) has shown that the p r o p o r t i o n of i n t a c t white c e l l s remaining f o l l o w i n g d e g l y c e r o l i z a t i o n decreases w i t h 4 storage of the blood p r i o r to f r e e z i n g . A f t e r 10 days, 1% or l e s s of the leukocytes w i l l remain f o l l o w i n g deglycerolization. S e n s i t i z a t i o n to h i s t o c o m p a t i b i l i t y antigens i s presumed to be reduced i n frequency i n p a t i e n t s r e c e i v i n g leukocyte-poor deg l y c e r o l i z e d c e l l suspensions. To a considerable extent, i t i s the presumption that reducing s e n s i t i z a t i o n should be b e n e f i c i a l to t r a n s p l a n t r e c i p i e n t s that has l e d to widespread use of f r o z e n red c e l l s i n d i a l y s i s c e n t e r s . Reports have both supported and c o n t r a d i c t e d these conjectures and the immunological v i r t u e s of f r o z e n red c e l l s f o r d i a l y s i s p a t i e n t s remains u n c e r t a i n . Reduction i n the i n c i d e n c e of p o s t - t r a n s f u s i o n h e p a t i t i s has been reported as a r e s u l t of conversion to f r o z e n red c e l l s . With exception of the study by T u l l i s (11), the observations are r e t r o s p e c t i v e , u n c o n t r o l l e d and f a i l to provide unequivocal evidence that red c e l l f r e e z i n g i s a defense against t h i s disease. The study by T u l l i s , although w e l l - d e s i g n e d , i s g e n e r a l l y considered to comprise too few cases of h e p a t i t i s to be c o n c l u s i v e . Mandatory t e s t i n g has r e s u l t e d i n a s u b s t a n t i a l r e d u c t i o n of type B h e p a t i t i s as a t r a n s f u s i o n problem (12). The dominant c l i n i c a l disease now i n v o l v e s a v i r u s which i s n e i t h e r type A nor type B so that i t cannot be p r e d i c t e d w i t h confidence that the v i r u s or v i r u s e s i n v o l v e d i n present day p o s t - t r a n s f u s i o n h e p a t i t i s w i l l respond l i k e the type B v i r u s to f r e e z i n g and deglycerolization.

In Synthetic Membranes: Volume II; Turbak, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Cryopreserved Red Blood Cells

Low temperature storage accounts f o r the s i m p l i c i t y of the c u r r e n t procedure. Storage at -80 C i n mechanical f r e e z e r s i s e a s i l y o b t a i n a b l e even though the equipment i s expensive and maintenance and r e p a i r r e q u i r e s p e c i a l i z e d experience. The imminent a v a i l a b i l i t y of a procedure p e r m i t t i n g storage at -20 C (13) e s s e n t i a l l y e l i m i n a t e s storage f a c i l i t y o b s t a c l e s to the use of frozen c e l l s .


Disadvantages of Frozen Red C e l l s Using Current Methods.

Processing

P r o c e s s i n g time, from the removal of a u n i t from the f r e e z e r to the removal of d e g l y c e r o l i z e d c e l l s from the washing apparatus, r e q u i r e s an absolute minimum of 30 minutes (14). Time of proc e s s i n g i s an e f f e c t i v e o b s t a c l e to the use of f r o z e n red c e l l s f o r emergency a p p l i c a t i o n s under most circumstances. On the other hand, when f r o z e n c e l l s are r o u t i n e l y d e g l y c e r o l i z e d on a l a r g e s c a l e w i t h i n a h o s p i t a l , the d i v e r s i o n of d e g l y c e r o l i z e d c e l l s f o r emergency use appears to be e n t i r e l y f e a s i b l e , and at l e a s t two l a r g e general h o s p i t a l s have reported outdating r a t e s of 5% (15) and 1% or l e s s (16), r e s p e c t i v e l y . The 24 hour outdating p e r i o d c o n s t i t u t e s one of the major problems w i t h f r o z e n red c e l l s today. This i s p a r t i c u l a r l y t r u e when c e l l s are d e g l y c e r o l i z e d at a blood center and d e l i v e r e d t o a h o s p i t a l at some d i s t a n c e . A u n i t of c e l l s s p e c i f i c a l l y d i r e c t ed to a p a r t i c u l a r p a t i e n t , and then not used, f r e q u e n t l y cannot be crossmatched f o r some other r e c i p i e n t w i t h i n the 24 hour l i m i t . The 24 hour outdating i s imposed because of the hazard of b a c t e r i a l contamination during p r o c e s s i n g . P r o c e s s i n g of m u l t i p l e u n i t s imposes a s p e c i a l hardship where more than one u n i t has been d e g l y c e r o l i z e d i n the Haemonetics d i s posable bowl. Blood centers preparing f r o z e n red c e l l s under Federal l i c e n s e are r e q u i r e d to use a v a r i e t y of safeguards t o assure t h a t a l l u n i t s washed through the same bowl are d e l i v e r e d to a s i n g l e r e c i p i e n t . Changes i n the design of the Haemonetics apparatus could o b v i a t e the need to prepare m u l t i p l e u n i t s w i t h the same bowl. But acceptable economics and more r a p i d and s i m u l taneous d e g l y c e r o l i z a t i o n are a necessary goal of blood t a n k i n g . Cost continues to be the predominant disadvantage of f r o z e n red c e l l s , although one l a r g e general h o s p i t a l r e p o r t s t h a t , s i n c e c o n v e r t i n g almost e n t i r e l y to f r o z e n or washed red c e l l s , the cost per thousand u n i t s of matched, f r o z e n c e l l s i s only 44% more than matched whole blood. At the present time, disposable s u p p l i e s f o r g l y c e r o l i z a t i o n and d e g l y c e r o l i z a t i o n t o t a l at l e a s t $35.00 per u n i t . The present s c a l e of red c e l l f r e e z i n g i s already s u f f i c i e n t to o b t a i n cost r e d u c t i o n s from volume production and i t seems u n l i k e l y t h a t s u b s t a n t i a l f u r t h e r reductions i n the costs of d i s p o s a b l e s can be a n t i c i p a t e d . Hemolysis during d e g l y c e r o l i z a t i o n accounts f o r up to 12% l o s s of the red blood c e l l s . The v a r i o u s s t e r i l e s o l u t i o n s used


112

synthetic

membranes:

hf

and

uf

uses

i n RBC washing lowers the o s m o l a l i t y too f a s t f o r the c e l l s to accomodate and the c e l l s rupture. These l o s s e s are o b v i o u s l y a major cost f a c t o r .


A New Method f o r Adding and Removing C r y o p r o t e c t i v e Agents. We propose a new method f o r both g l y c e r o l i z a t i o n and d e g l y c e r o l i z a t i o n , based on the use of a blood bag composed ent i r e l y of semi-permeable membrane m a t e r i a l , as shown i n Figure 1. Because the membrane i t s e l f would form an absolute b a r r i e r f o r pyrogens, v i r u s e s and b a c t e r i a , c o s t l y s t e r i l e s o l u t i o n s are t o t a l l y avoided. Since the blood could be c o l l e c t e d and m a i n t a i n ed i n a s t e r i l e environment, the 24 hour outdating p e r i o d could probably be g r e a t l y increased. The process could be e a s i l y automated and c a r r i e d out without t e c h n i c i a n attendance. Processing of m u l t i p l e u n i t s simultaneously could be accomplished without f e a r of cross contamination inasmuch as each blood u n i t would be contained w i t h i n i t s own membrane system and l a s t l y , the cost would undoubtedly be g r e a t l y decreased because membrane m a t e r i a l i s not very expensive and none of the s t e r i l e s o l u t i o n s would be needed. Objections to using membranes comparable to those used i n hemodialysis f o r such a procedure would be that hemoglobin and c e l l fragments formed during the freezing-thawing process could not be washed out as i n present methods. I f hemolysis could be e l i m i n a t e d by a more g e n t l e washing procedure, the only o b j e c t i o n to t h i s membrane process would be the r e t e n t i o n of white c e l l fragments: however, a d m i n i s t r a t i o n of whole blood has s i m i l a r consequences. Methods. F i g u r e 2 i s a schematic of the t r a n s p o r t c e l l which holds the blood bag. The bag i s placed i n a " w a f f l e i r o n " l i k e c e l l which a l l o w s the c e l l to be e a s i l y opened and c l o s e d . D i a l y s a t e enters the bottom, t r a v e l s through a p l a s t i c mesh which acts both as a " d i a l y s a t e mixer" and as a membrane support. The d i a l y s a t e e x i t s and then r e - e n t e r s a t the bottom. I n t h i s manner both s i d e s of the blood bag a r e washed by the s i n g l e d i a l y s a t e stream. The bag volume i s designed t o " f i l l " the compartment; t h i s prevents osmosis, due t o plasma p r o t e i n s , from s w e l l i n g the bag and d i l u t i n g the red blood c e l l s . The bag i s formed by h e a t - s e a l i n g p o l y c a r bonate membrane (American Membrane, Covina, CA). For the i n v i t r o experiments reported here, the bag i s f i l l e d w i t h 350 ml of outdated, human blood which has been g l y c e r o l i z e d according t o standard procedures s e t by the American Red Cross. The bags have 2 960 cm area on each s i d e . The t r a n s p o r t c e l l i s mounted on a shaker (Eberbach Corp, Ann Arbor, MI) and i s shaken a t 160 r e v o l u t i o n s per minute t o enhance mixing of the blood i n the blood bag during d i a l y s i s . The primary c o n s i d e r a t i o n s i n the design of the apparatus



ZELMAN


ET AL.

1

Membrane Blood B a g Maintains Sterility During blood h a n d l i n g and storage

Figure 1.

Schematic for a membrane blood bag for maintaining sterility during glycerolization and deglycerolization

dialysate

Membrane support and dialysate mixingmesh

dialysate

Transport- CeH made like a "Waffle Iron"

Figure 2.

Schematic of the transport cell that holds the blood bag



114

SYNTHETIC MEMBRANES:

HF AND U F

USES

are: 1) t o have the d i a l y s a t e f l o w i n g as f a s t as p r a c t i c a l , w i t h out wasting s a l t , t o f a c i l i t a t e mass t r a n s f e r , and 2) to lower the d i a l y s a t e o s m o l a l i t y smoothly from - 4000 mosm to 265 mosm d u r i n g a time span s u f f i c i e n t l y f a s t to be e f f i c i e n t , but not so f a s t as to cause osmotic hemolysis. F i g u r e 3 shows s c h e m a t i c a l l y the p r i n c i p l e components of the system. Tap water i s drawn by a d i a l y s a t e d e l i v e r y system (B-D Drake-Willock, P o r t l a n d , OR) commonly used f o r h e m o d i a l y s i s ^ This system produces d i a l y s a t e a t r a t e s of up to 600 ml/min a t 38 C by d i l u t i n g a concentrate w i t h tap water. The f r e s h l y made d i a l y s a t e i s piped t o a mixing f l a s k c o n t a i n i n g - 2500 ml of 4500 mosm d i a l y s a t e . As the d i a l y s a t e i s f o r c e d i n t o the f l a s k the d i a l y sate l e a v i n g the f l a s k and e n t e r i n g the t r a n s p o r t c e l l v a r i e s i n o s m o l a l i t y w i t h time according t o Table I . TABLE I E x p o n e n t i a l Decay of D i a l y s a t e O s m o l a l i t y C ( t ) = C (o) + ( C D

D

C o n c

- C (o)) D

Tank V°>

*

Dialysate Delivery System T i s time,

C

Tank

( t )

*

Concentrate Tank

% ™ D i a l y s a t e to Transport Cell

i s the constant r a t e of volume flow to the

t r a n s p o r t c e l l , V_ . i s the volume of the concentrate tank, CL(o) lank. u and Crj (°) a r e the incoming d i a l y s a t e and i n i t i a l tank concent r a t e c o n c e n t r a t i o n s r e s p e c t i v e l y and are constant i n time. This method o f d i a l y s a t e d e l i v e r y produces a very smooth e x p o n e n t i a l decay from very high o s m o l a l i t y to that of standard d i a l y s a t e . The s a l t c o n c e n t r a t i o n s of the blood bag a f t e r d e g l y c e r o l i z a t i o n w i l l be e s s e n t i a l l y that of the d i a l y s a t e and are l i s t e d i n Table I I f o r these experiments. Other c o n c e n t r a t i o n s can be e a s i l y subs t i t u t e d f o r those chosen here. onc

TABLE I I Composition of D i a l y s a t e a t 265 mosm Na K

+

130 meq/L

C l " 102 meq/L

2 meq/L

Ac

+2 Kg 1 meq/L

Glucose

31 meq/L 200 mg% (11.1 m M)


ZELMAN ET AL.


Sink


f h- p water

Deglycerolizing

a

»265mosm

1

System

dialysate |osm(t)

d i a l y s a t e delivery system

concentrate 9IOOmosm/ circulation 4500mosm [ initial v o l u m e * 2500ml

Figure 3.

Transport Cell

Schematic of the laboratory set-up for deglycerolization

800r • * 30 minutes * « 4 5 minute* • « 60 minutes

600 ^

acceptable level of residual glycerol in blood

T3

200

400 Q

6O0

D

(

m { /

800

IOOO

1200

min)

Figure 4. Residual glycerol left in 350 mL of red blood cells after deglycerolization



116

SYNTHETIC

MEMBRANES:

HF AND U F

USES

Having passed through the t r a n s p o r t c e l l , the g l y c e r o l d i s s o l v e d i n the d i a l y s a t e i s put i n the s i n k d r a i n . The c i r c u l a t i o n pump on t h e f l a s k maintains a w e l l mixed s o l u t i o n . These experiments were aimed to determine three v a r i a b l e s f o r o p t i m i z a t i o n of t h i s process: 1) What should be the i n i t i a l d i a l y s a t e o s m o l a l i t y i n order to prevent hemolysis? 2) What i s the f a s t e s t r a t e a t which the o s m o l a l i t y may be lowered without hemolysis? 3) How long should d i a l y s i s continue before the g l y c e r o l i s a t acceptable l e v e l s (< 200 mosm)? Since we knew that i n i t i a t i n g d i a l y s i s w i t h an o s m o l a l i t y above that of the i n i t i a l g l y c e r o l would prevent hemolysis, we simply i n i t i a t e d a research program to determine an optimum value

%

T

of (—

) as given i n Table I .

Tank R e s u l t s With D i s c u s s i o n . F i g u r e 4 shows the r e s u l t s of d e g l y c e r o l i z a t i o n using our 2 f i r s t prototype t r a n s p o r t c e l l of 960 cm area f o r each s i d e of 2 the membrane bag, i . e . , 1920 cm t o t a l area. I t i s c l e a r that about 50% of the samples were p r o p e r l y d e g l y c e r o l i z e d . Dye t e s t s on the f l o w p a t t e r n s i n the t e s t c e l l i n d i c a t e d that the s c a t t e r i n data was more l i k e l y due to i r r e g u l a r d i a l y s a t e flow paths r a t h e r than to b i o l o g i c a l i r r e g u l a r i t y . But these r e s u l t s do i n d i c a t e v i a b i l i t y of the process and p o i n t d i r e c t l y toward cons t r u c t i o n of an improved d i a l y s i s system. A f t e r each experiment the e x t r a c e l l u l a r f l u i d was checked f o r plasma hemoglobin. I n our l a s t 29 experiments, 11 showed zero hemolysis w i t h an average change i n plasma hemoglobin of 183 mg % ± 203 mg % w i t h a range of (0 - 500 mg % ) . The American Red Cross s e t s the l i m i t of plasma hemoglobin f o r r e i n f u s i o n a t 500 mg %. Thus even w i t h the outdated blood, which i s very f r a g i l e , our method e x c e l s i n preventing r e d c e l l l o s s e s . The long term goals of t h i s p r o j e c t a r e to d e g l y c e r o l i z e 400 ml of red blood c e l l s i n about 30 minutes. These p r e l i m i n a r y data i n d i c a t e that t h i s g o a l can be met by proper design of the t r a n s p o r t c e l l . At the time of t h i s w r i t i n g a new prototype c e l l 2 i s being assembled w i t h a membrane area of 1419 cm (each s i d e ) and w i t h d i a l y s a t e flow p a t t e r n s designed to ensure u n i f o r m i t y of f l u i d d i s t r i b u t i o n across the surfaces of the blood bag. This new t e s t c e l l w i l l c e r t a i n l y achieve our g o a l . Our primary need now i s f o r a bag composed of a p i n h o l e f r e e membrane m a t e r i a l . The bag must be p i n h o l e f r e e to ensure s t e r i l i t y ; perhaps a double l a y e r membrane would meet t h i s g o a l . We can expect that i n the near f u t u r e membrane technology w i l l p l a y a f a r greater r o l e i n the blood banking and blood processing i n d u s t r y .


8.

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117

Acknowledgement This research could not have been conducted without the h e l p , encouragement and c r i t i c i s m of Dr. H. Meryman, ANRC, Washington, D.C., Dr. A. B r i t t e n , ANRC, Albany, N.Y., and Dr. J . Eisenmann, Baker Bros., Stroughton, MA. This research i s supported i n part by P u b l i c H e a l t h S e r v i c e s grant #NIH 3 R01 HL24466.


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Meryman, H.T. I n : Red Cell F r e e z i n g : AABB. pp. 73-86, 1973.

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Meryman, H.T. Am. J o u r n a l , of Med. Tech., 41:265-282, 1975.

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Huggins, C.E. 31-53, 1973.

I n : Red Cell F r e e z i n g .

AABB Workshop, pp. 55-71,

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Synthetic Membranes: Volume II - ACS Publications - American

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