Rapid Communications

Preparation of packed red cells suitable for intravascular transfusion in utero

BRIAN R . GALLIGAN, RONALD CAIRNS, JOSEPH V . SCHIFANO, ANDERS SELBING AND STENER S. BERNVIL

King F a i s a l S p e c i a l i s t Hospi ta l and Research Centre, Riyadh, Kingdom of Saudi Arabia

Received f o r publ ica t ion August 8 , 1988; rev is ion received November 15, 1988 and accepted November 2 2 , 1988.

ABSTRACT: In t ra -card iac t ransfus ion i n u t e ro i s a recent development i n t h e treatment o f e ry th rob la s tos i s f o e t a l i s . A s with in t ra -umbi l ica l cord t r ans fus ion , t h e procedure has been described as having a high r i s k of complication charac te r ized by bradycardia and ca rd iac -a r r e s t . Intra-coronary hyperkalemia was suspected as being a major con t r ibu t ing f a c t o r towards t h e complications experienced, p a r t i c u l a r l y by t h e in t r a -ca rd iac de l ivery . The procedure i s warranted, however, i n cases where in t ra -umbi l ica l cord t r ans fus ion i s not poss ib le . Therefore, s t u d i e s comparing var ious methods of prepar ing packed c e l l s f o r i n t r avascu la r t r ans fus ion were i n i t i a t e d . of g rav i ty sedimented packed c e l l s and s a l i n e washed red c e l l s resuspended Products of s a l i n e washed/resuspended packed red c e l l s t ransfused wi th in 2 hours of prepara t ion were found t o have low potassium l e v e l s while exoosing t h e f e t u s t o minimal r i s k of t r ans fus ion t ransmi t ted in fec t ion and were the re fo re se l ec t ed t o be the most s u i t a b l e oroduct. Using t h i s DrOdUCt, a s i g n i f i c a n t decrease i n t h e incidence of severe bradycardia r e s u l t i n g i n ca rd iac -a r r e s t , following in t r avascu la r t r ans fus ion (32 procedures) was observed.

The plasma potassium l e v e l s in e i t h e r Ringer ' s l a c t a t e , pooled donor plasma o r s a l i n e were measured.

H i s to r i ca l ly severe e ry th rob la s tos i s f o e t a l i s has been t r e a t e d by in t r a -u t e r ine in t r a -pe r i tonea l or in t ra -umbi l ica l cord t r ans fus ion . It is known t h a t f e t a l outcome is very poor a f t e r i n t r a -pe r i tonea l t ransfus ion (1 1, and the re fo re in t ra -umbi l ica l cord t r ans fus ion has become t h e p re fe r r ed route . In t r a -ca rd iac t r ans fus ion i n u t e r o has been suggested a s an a l t e r n a t i v e t o in t ra -umbi l ica l cord t r ans fus ion i n t h e treatment of e ry th rob la s tos i s f o e t a l i s (2 ) . Both procedures have been repor ted t o have a high r i s k of f e t a l d i s t r e s s and mor t a l i t y charac te r ized by bradycardia and card iac a r r e s t ( 2 , 3 ) . Previous s t u d i e s on f e tuses rece iv ing in t ra -umbi l ica l cord t r ans fus ion o r newborn i n f a n t s r equ i r ing t ransfus ion have ind ica t ed t h a t markedly e leva ted plasma potassium l e v e l s were found i n g rav i ty sedimented packed red c e l l products ( 4 , 5 ) .

A recent case involved a f e t u s which suf fered two ca rd iac a r r e s t s dur ing an in t r a -ca rd iac t r ans fus ion . It i s be l ieved t h a t four day o l d g rav i ty sedimented packed c e l l products with a high plasma potassium l e v e l ( 2 4 . 7 mmol/L) may have caused t h e ca rd iac arrests when t ransfused d i r e c t l y i n t o t h e l e f t v e n t r i c l e of t h e h e a r t . Prompted by t h i s case and t h e prev ious ly repor ted cases of bradycardia and ca rd iac a r r e s t , it was decided t o inves t iga t e a l t e r n a t i v e methods of providing packed c e l l s with a low plasma potassium l e v e l f o r i n t r avascu la r t ransfus ion .

MATERIALS AND METHODS

SUBJECTS

Routine whole blood co l l ec t ion was performed us ing twenty e igh t random blood donors. The donors were separa ted i n t o fou r groups and matched f o r age/sex between groups. I n add i t ion , a serum sample was co l l ec t ed from each donor i n order t o e s t a b l i s h b a s e l h e serum potassium va lues .

MATERIALS

Quadruple blood bags (CPD-A1 Cu t t e r Labora tor ies ) and 7 cc red top evacuated tubes (Becton Dickinson) were used f o r rou t ine whole blood co l l ec t ion . The 2991 Blood Cell Processor

(COBE) with accessor ies was used t o perform the red c e l l washing. Wash so lu t ions included 0.9% sodium chlor ide (ABBOTT), R inger ' s l a c t a t e (ABBOTTI, and pooled donor plasma (volunteer blood donors) . Analysis of serum uotassium was performed on t h e Astra-4 (Beckman). The r e s u l t i n g a l i a u o t s were i r r a d i a t e d a t 5000 cGy (Gammacell 1000 A. E . C . C . 1.

METHODS

The four study groups were divided as fo l lows:

1 ) Seven donor u n i t s were s to red inver ted i n a monitored r e f r i g e r a t o r a t 1 -6OC as g rav i ty sedimented red c e l l s . These u n i t s were a l iquoted i n t o 100 cc f r a c t i o n s on days 1 , 3 and 5 of s to rage and i r r a d i a t e d . Samples from each f r a c t i o n were cent r i fuged a t timed i n t e r v a l s with t h e r e s u l t i n g plasma analyzed immediately.

2 ) The remaining twenty one donor units were s to red i n 3 x 100 cc f r a c t i o n s i n t h e same monitored r e f r i g e r a t o r . A l l u n i t s were washed us ing t h e COBE 2991 blood c e l l processor set up f o r leukocyte poor washed c e l l s according t o manufacturer 's d i r e c t i o n s . Two washes, each with 500 cc of s a l i n e were performed and t h e f i n a l wash and resuspension i n 500 cc of t he appropr ia te so lu t ion was followed by i r r a d i a t i o n a t 5000 cGy. An average f i n a l hematocrit o f 80% was obtained. The washed products were a l l s to red a t 1-6OC dur ing the observation per iod .

A ) The Sa l ine wash cohort used seven donors. Aliquots of 100 cc were washed on days 1 , 3 and 5 a f t e r co l l ec t ion . Samples obtained from each washed a l iquo t were cent r i fuged a t t i ned i n t e r v a l s with immediate ana lys i s of t h e superna tan t .

B ) The Ringer ' s l a c t a t e wash cohort used an add i t iona l seven donors f o r ana lys i s . Again, seven 100 cc a l iquo t s were washed on days 1 , 3 and 5 a f t e r co l l ec t ion . The potassium l e v e l of t h e Ringer 's l a c t a t e (4.1 mmol/L) was determined t o i l l u s t r a t e i t s e f f e c t on t h e r e s u l t a n t l e v e l i n t h e washed product. Samples were l ikewise obtained from each washed a l iquo t a t timed i n t e r v a l s .

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C ) The Pooled Donor Plasma wash cohort used the f i n a l seven donors. Seven 100 cc a l iouo t s were washed on days 1 , 3 and 5 with nooled donor ulasma. The notassium l e v e l of t h e nooled donor nlasma (2.5-3.3 mmol/L) was determined t o i l l u s t r a t e i t s e f f e c t on the r e s u l t a n t l e v e l i n the washed nroduct. Samnles from each washed a l inuo t were obtained a t timed i n t e r v a l s .

Table 3 i l l u s t r a t e s t h e numbers of f e t a l bradycardias a t i n t r avascu la r t r ans fus ion before (Period I) and a f t e r (Period 11) t h e in t roduc t ion of t h e new blood Droduct. A s t a t i s t i c a l l y s i g n i f i c a n t decrease i n severe bradycardia r a t e was noted.

Frequency o f Bradycardia Eefore i?er lod I ) and After ( P e r i o d 11) I n t r o d u c t i o n o f Sal ine kiashed Red Cel ls

Period I ?er iod I1 N S N 5 D

RESULTS --

Table 1 demonstrates t h e e f f e c t of r e f r i g e r a t e d s to rage on uotassium l e v e l s i n inve r t ed g r a v i t y sedimented red c e l l products. The o r i g i n a l donor potassium l e v e l s were within t h e normal range (3 .5-5 .0 mmol/L). Samples obtained from the donor units a f t e r t h r e e days of s to rape had plasma potassium l e v e l s cons i s t en t ly above 9 .0 mmol/L

TABLE 1. Plasma Potassium Levels i n Inver ted Gravity Sedimented Red Cell Products

Days of Storage Day 7 Day 3 Day 5

Mean K+ (mol /L 1 5.820.8" 11 .5+2.1 13.229.4

( N = 7 )

Table 2 i l l u s t r a t e s t h e e f f e c t of washing and resumens ion of s to red red c e l l products i n var ious so lu t ions . The use of uooled donor ulasma a s a resuspension so lu t ion provided the lowest post-wash potassium l e v e l s . However, s a l i n e washed red c e l l s were s u i t a b l e f o r t ransfus ion i f used within two hours of washing.

-..

TABLE 2. Supernatant Potassium Levels i n Washed Red Ce l l Products

-.-

(mmol/L)

Hours Days of Storage Post-!$ash Day 1 Day 3 Day 5

2 8.424.7" 8.213.2 S a l i n e 4 15.818.5 12.1T4.1 ( N = 7 ) 6 16.271.9 16.777.0

12 35.6z8.4 26.0+11.1

6.123.2 70.326.2 9.623.0

22.551 1 .1

2 11.5c3.2 9 .453 .9 9.152.1 Ringer ' s 4 1 7 . 1 3 . 3 12.6+6.4 12.422.3 l a c t a t e 6 2 1 . 1 ~ 5 . 6 14.376.9 16.223.4 ( N = 7 ) 12 32.728.4 19.4x9.0 21.355.1

2 8,812.5 6.952.7

Plasma 6 l7 .0z6 .3 9.951.9 ( N = 7 ) 12 32.926.3 19,322.5

* + 1 S.D.

Pooled 4 1 1 . 7 2 . 1 8.121.8

-

4.4+0.3 6.8xO.8 9.051.4

22.927.5

S i i g h t L.0 Xoderace Bradycardia 15 ( 1 9 ) 5 ( 1 6 ) :I.S.**

Severe Bradpa rd ia 1 3 ( 1 7 ) 0 (0) 0.95"' _ _ , . - -. . -. . . - -. -. - - -- ._ -. . . . . . - - -. . . - -. - - - . . -. -. -. . . -. _. . -. .

32 i o c a l Intraucerme Transfusions 77

DISCUSSION

In the hiiddle E a s t , e ry th rob la s tos i s f o e t a l i s due t o Rh iso-immunization i s not uncommon. In o u r h o s p i t a l , we see aDvoxirnatelv 25 new cases a yea r , many of whom a r e t r e a t e d seve ra l times during the pregnancy with i n t r a - u t e r i n e t r ans fus ion . Althouph t h e umbi l ica l cord is t h e n re fe r r ed route f o r venous acess , occasionaly due t o d i f f i c u l t y i n loca t inp the umbi l ica l ve in , i n t r a -ca rd iac t ransfus ion may be necessary ( 2 ) . Severa l Daners have described t h e technica l DreDaration of blood f o r exchange or i n t r a - u t e r i n e t r ans fus ions , some authors recommendinq " f r e sh" oacked c e l l s and o the r p r e f e r r i n g Dacked c e l l s r econs t i t u t ed with f r e sh frozen ulasma ( 4 , 6 , 7 , 8 ) . However, t h e subjec t of how t o nrenare blood s n e c i f i c a l l y for i n t r avascu la r deliver,v i n u t e ro has not prev ious ly been addressed. The bradycardia o re sen t ly observed during in t r a -ca rd iac t r ans fus ion may be due t o the d i r e c t ?uncture of t he h e a r t , although the same comnlication has been repor ted with umbi l ica l cord t r ans fus ion . It has been renor ted t h a t premature i n f a n t s a r e not adversely a f f ec t ed by a uotassium concent ra t ion of less than 12 mmol/L of ulasma i n blood used for exchange t ransfus ion ( 4 ) and i t may be assumed t h a t i n t r a -ca rd iac hynerkalemia is avoided due t o mixinp of t h e t ransfused olasma with the r e c i p i e n t s own nlasma Dr ior t o a r r i v a l i n t h e h e a r t . In f e tuses undergoing umbi l ica l cord t ransfus ion and in t r a -ca rd iac t r ans fus ion , we sue res t t h a t high uotassium is a maJor cont r ibu tory cause of bradycardia and ca rd iac a r r e s t , e s u e c i a l l y if the f e t u s is a l ready ac ido t i c .

In t h i s s tudy , various methods of urenar inp blood f o r i n t r avascu la r t r ans fus ion were comoared. The ob jec t ive was t o f i n d a nroduct which exuosed t h e f e t u s t o t h e l e a s t r i s k both i n terms of potassium l e v e l s and number of donor exnosures. Our nre -ex i s t ing rnethod was t o i s s u e an a l i a u o t of packed c e l l s from a u n i t of HgbS negat ive , g rav i ty sedimented, f r e s h (less than 5 days o l d ) 0 Neqative blood and i r r a d i a t e i t u r i o r t o t r ans fus ion . As can be seen i n Table 1 , a comuarison of a l i a u o t s taken from u n i t s 1 , 3 and 5 davs o l d y ie lded a mean plasma uotassium l e v e l of 5.8 mmol/L i n 1 day o ld u n i t s . 11.5 mmol/L i n 3 day o ld u n i t s and 13.2

PRBC FOR INTRA-UTERO TRANSFUSION 181 I'RANSFUSION 19X9-Vol. 29. N o 2

mmol/L i n 5 day o ld u n i t s . Following discussion i t was t e n t a t i v e l y decided between t h e blood bank and the o b s t e t r i c i a n s t h a t a plasma potassium l e v e l o f no g rea t e r than 9.0 mmol/L should be considered t h e t a r g e t value f o r blood intended f o r i n t r avascu la r t ransfusion. Clear ly , t h i s indicated t h a t g rav i ty sedimented packed c e l l s were not a s u i t a b l e product unless t he blood was extremely f r e sh . The range of values found on g rav i ty sedimented blood was a l s o of g rea t concern, one u n i t having a plasma potassium of 15.5 mmol/L a t 3 days and 32.5 mmol/L a t 5 days.

Three o the r methods were proposed f o r preparing the packed cells, a l l involved washing the u n i t s i n 0.9% Sal ine twice followed by a f i n a l wash/resuspension with e i t h e r 0.9% Sal ine, Ringer's l a c t a t e o r pooled donor plasma. Again, t he u n i t s s e l ec t ed were WS negative and the a l iquo t s were i r r a d i a t e d a f t e r washing. Ringer 's l a c t a t e was discounted as a p o t e n t i a l f i n a l wash so lu t ion because the lowest mean supernatant potassium l e v e l (2 hours a f t e r wash), was cons i s t en t ly over 9.0 mmol/L and rose r ap id ly over t h e ensuing s torage period. Pooled donor plasma gave improved r e s u l t s , with acceptable mean potassium l eve l s up t o 6 hours. However, i n o rde r t o provide t h i s product, i t was necessary t o pool 2 or 3 f r e sh frozen plasma u n i t s t o obtain s u f f i c i e n t plasma volume f o r t h e f i n a l wash, thereby increasing the r i s k of disease transmission. Washing and resuspending packed c e l l s i n 0.9% Sal ine, and using the product within 2 hours of production r e su l t ed i n a blood product with an acceptable mean value of less than 9.0 mmol/L, when using blood 5 days o ld o r less. Sa l ine washing offered a d i s t i n c t advantage over f r e sh frozen plasma, i n t h a t i t exposed t h e f e t u s t o only t h e red c e l l donor. Furthermore, it was possible t o coordinate t h e washing of t he packed cells within 2 hours of t ransfusion.

It was noted t h a t regardless of t he resuspension solut ion used, lower potassium l e v e l s were obtained when washing blood t h a t was 3 or ' 5 days o ld as opposed t o the f r e she r 1 day o ld blood. The authors suggest t h a t t h i s may be due t o s t a b i l i z a t i o n of t he red c e l l membrane by p l a s t i c i z e r re leased from the blood packs and incorporated i n t o the red c e l l membrane ( 9 ) .

Following the introduct ion of s a l i n e washed packed red blood cells followed by t r ans fus ion within 2 hours of preparat ion, we had an i n i t i a l impressive decrease in the rate of f e t a l bradycardia t h a t could later be s t a t i s t i c a l l y

confirmed. We the re fo re recommend t h a t s a l i n e washing t o a f i n a l hematocrit of 80%, followed by i r r a d i a t i o n should be considered t h e method of choice t o prepare blood f o r f e t a l i n t r avascu la r t ransfusion.

REFERENCES

(1 Holt EM, Boyd I E , Dewhurst CJ, Murray J , Naylor CJ, and Smithan J H . I n t r au te r ine t r ans fus ion : 101 consecutive cases t r e a t e d a t Queen Char lo t t e ' s Maternity Hospital . B r Med J 1973; 3: 39-43.

( 2 ) Westp-en M . , Selbing A . , Stangenberg X. F e t a l i n t r aca rd iac t ransfusions i n p a t i e n t s with severe rhesus isoimmunisation. B r Med J 1988; 296: 885-886.

( 3 ) McKenzie I Z , Bowel1 P J , Ferguson J , Cast le BM, Entwist le C C . In-utero in t r avascu la r t ransfusion of t he f e tus f o r t h e management of severe rhesus isoimmunisation - a reappraisal . B r J Obstet Gynaecol 1987; 94: 1068-1073.

( 4 ) Barnard D.R., Chapman R.G. , Simmons M.A., Hathaway W.E. Blood f o r use in Exchange Transfusion i n t h e Newborn. Transfusion 1980; 20: 401-408.

( 5 ) Batton D . G . , Maisels M.J., Shulman G. Serum potassium changes following packed red c e l l t ransfusions i n newborn i n f a n t s . Transfusion 1983; 23: 163-164.

( 6 ) Rodeck C . H . , Holman C . A . , Karnicki J . , Kemp J . R . , Whitmore D . N . , Austin M.A. Direct i n t r avascu la r f e t a l blood t ransfusion by fetoscopy i n severe rhesus i so imun i sa t ion . Lancet 1981 ; 1 : 625-627.

( 7 ) Thomson S . , Inwood M.J., Ferries P., Baker N. Hyperkalemia i n the Neonate. Canad J Med Tech 1982; 4 4 : 31-32.

(8) Grannum P . , Copel J . , Plaxe S., Scioscia A . , Hobbins J. In u t e ro exchange t ransfusion by d i r e c t i n t r avascu la r i n j ec t ion i n severe e ry th rob la s tos i s f e t a l i s . N Engl J Med 1986; 314: 1431-1434.

( 9 ) Rock G. , Toechi M . , Ganz P.R. , Tackaberry E.S. Incomorat ion of p l a s t i c i z e r i n t o red c e l l s during s torage. Transfusion 1984; 24: 493-498.