Life Sciences, Vol. 46, pp. 647-652 Printed in the U.S.A.
Pergamon Press
D E T E R M I N A T I O N OF C I R C U L A T I N G BLOOD V O L U M E BY M E A S U R E M E N T OF I N D O C Y A N I N E G R E E N DYE IN HEMOLYSATE: A P R E L I M I N A R Y STUDY
M a r t i n W. Busse, Henschen; Bernhard
Departments
MD; Stefan Zisowsky; S t e p h a n Panning, MD; Lotte Reilmann,
M e d i c a l School H a n n o v e r of Physiology, A n a e s t h e s i o l o g y D-3000 H a n n o v e r West-Germany
MD
and R a d i o l o g y
(Received in final form January 3, 1990) Summary In 8 e m e r g e n c y c a r e _ p a t i e n t s blood v o l u m e w a s d e t e r m i n e d u s i n g Cr ~I labelled e r y t h r o c y t e s and i n d o c y a n i n e g r e e n (ICG). Prior to m e a s u r e m e n t of ICG in blood with a spectrophotometer, the b l o o d w a s h e m o l y z e d w i t h Triton-X. A close c o r r e l a t i o n of r=0.97 b e t w e e n the Cr 51 and the ICG.. ~ e s t i m a t e s was found; the ICG ~1 TR_ v o l u m e was about 3~ Tower t h a n the Cr 5 volume. In ~ i v e a d d i t i o n a l in vitro e x p e r i m e n t s the ICG__ _ m e t h o d w a s found to r e f l e c t real v o l u m e s w i t h an i n s l g n l f l c a n t e r r o r of less t h a n 1%. Blood v o l u m e d e t e r m i n a t i o n w i t h ICGTR_~ c a n n o t be a p p l i e d in cases of c i r c u l a t o r y failu@e. ICG should be a d m i n i s t e r e d in a d o s e of 0.5 m g / k g of b o d y weight. For c a l i b r a t i o n purposes, a two p o i n t c a l i b r a t i o n curve (point i: p o i n t of i n t e r s e c t i o n of x and y axis; point 2 : 5 mg ICG/I of blood) is sufficient. From these p r e l i m i n a r y e x p e r i m e n t s it is c o n c l u d e d that the ICG__. m e t h o d is a r a p i d and simple t e c h n i q u e of bloo~ v o l u m e d e t e r m l n a t i o n w i t h m u l t i p l e r e p r o d u c i b i l i t y w h i c h can be c a r r i e d out in any c l i n i c a l laboratory.
In p a t i e n t s w i t h a m i s m a t c h of blood loss and s i m u l t a n e o u s t h e r a p e u t i c a l a d m i n i s t r a t i o n of fluids, r a p i d b l o o d v o l u m e m e a s u r e m e n t m a y be an i m p o r t a n t d i a g n o s t i c aid. R e p e t i t i v e d e t e r m i n a t i o n s m a y be of p a r t i c u l a r i m p o r t a n c e d u r i n g h e m o r r h a g e n i c s u r g e r y after a l t e r n a t i n g i n f u s i o n s of c o l l o i d a l or m i n e r a l s o l u t i o n s or of blood. The c i r c u l a t i n g p l a s m a and b l o o d v o l u m e or the r e s p e c t i v e changes can t h e n not be q u a n t i f i e d by r e l a t i v e c h a n g e s of h e m o g l o b i n / h e m a t o c r i t or total p l a s m a p r o t e i n c o n c e n t r a t i o n . These c o n d i t i o n s m a y be even m o r e c o m p l i c a t e d d u r i n g n e u r o s u r g e r y and n e u r o s u r g i c a l i n t e n s i v e care, w h e n in a d d i t i o n h y p e r o s m o l a l s o l u t i o n s are administered. C u s t o m a r y m e t h o d s such as m e a s u r e m e n t of red cell v o l u m e w i t h Cr 51 l a b e l l e d c e l l s o f t e n fail the d e m a n d for simple, r a p i d and r e p e t i t i v e m o n i t o r i n g . The latter also holds for Evan's blue. A g e n e r a l 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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d r a w b a c k of all tracer m e t h o d s is the need for s p e c i a l e q u i p m e n t and the u n w e l c o m e a p p l i c a t i o n of r a d i o a c t i v e substances. T h o u g h B r a d l e y and Barr in 1968 p r o p o s e d a rapid and r e p r o d u c i b l e m e t h o d of b l o o d v o l u m e d e t e r m i n a t i o n with indocyanine g r e e n in this j o u r n a l I, this m e t h o d has not g a i n e d general acceptance. T h i s m a y be m a i n l y due to the fact that the used t e c h n i c a l device, w h i c h w a s d e s i g n e d for cardiac output measurement, was q u i t e a w k w a r d to o p e r a t e and, in addition, no routine d i a g o s t i c equipment. F u r t h e r m o r e the a p p a r a t u s has become obsolete t h e s e days and is u s e d only for special a p p l i c a t i o n s such as lung w a t e r d e t e r m i n a t i o n 2, d i a g n o s i s of cardiac shunts 3 or liver f u n c t i o n 4. As in p r i n c i p l e the f a v o u r a b l e q u a l i t i e s of ICG are well documented, a m o r e simple m e t h o d of m e a s u r e m e n t w o u l d allow fast and r e l i a b l e b l o o d v o l u m e monitoring. ICG may be easily m e a s u r e d in plasma. A g e n e r a l d r a w b a c k is the need of a special c a l i b r a t i o n c u r v e for each p a t i e n t and also for i n t r a i n d i v i d u a l l y v a r y i n g c o n d i t i o n s (own u n p u b l i s h e d results). As the c a l i b r a t i o n curve s h o u l d r e p r e s e n t the in vivo conditions, ICG should be p i p e t t e d into w h o l e b l o o d w h i c h w o u l d be c e n t r i f u g e d a f t e r w a r d s for ICG m e a s u r e m e n t . The ICG plasma c o n c e n t r a t i o n of s t a n d a r d and in vivo s a m p l e s w o u l d t h e n be c o r r e c t e d for p a c k e d cell volume. W h e n c a l c u l a t i n g the in vivo blood volume, a p o s s i b l e s y s t e m a t i c error of the h e m a t o c r i t (Hk) d e t e r m i n a t i o n would thus be avoided. On the other h a n d the m u l t i p l e Hk m e a s u r e m e n t s may be an i m p o r t a n t t i m e c o n s u m i n g factor in e m e r g e n c y diagnosis, w h e n a d i r e c t b l o o d v o l u m e d e t e r m i n a t i o n w o u l d be m u c h m o r e comfortable. U n f o r t u n a t e l y ICG m e a s u r e m e n t in whole blood w i t h a s p e c t r o p h o t o m e t e r , an e q u i p m e n t w h i c h is nearly anywhere available, is impossible. O b j e c t of this study was to introduce an u n c o m p l i c a t e d w a y of b l o o d v o l u m e m e a s u r e m e n t using ICG in h e m o l y z e d b l o o d w h i c h can be c a r r i e d out in any clinical laboratory.
Methods In 5 e x p e r i m e n t s the in vitro v a l i d i t y of blood v o l u m e (BV) m e a s u r e m e n t in h e m o l y s a t e was checked by c o m p a r i s o n of real and m e a s u r e d blood volumes. About 0.17 mg ICG w e r e m i x e d in about 85 ml of w h o l e blood (i.e. about 2 mg/l; in each s i n g l e case the b l o o d v o l u m e s and ICG amounts were m e a s u r e d and w e i g h e d w i t h h i g h p r e c i s i o n equipment). The ICG c o n c e n t r a t i o n s of a i0 p o i n t s t a n d a r d curve w e r e in the range of about 0.3 m g / l to 7 m g / l of blood. 200 ~i of the blood w e r e added to 400 ~i of T r i t o n - X (TR-X, 1%) for hemolysis. S t a n d a r d values and the in v i t r o s a m p l e s w e r e then measured with a spectrophotometer (Hitachi 150-20) at 805 nm. The w a t e r v o l u m e in ml o c c u p i e d by stroma p r o t e i n s per liter of w h o l e b l o o d was c a l c u l a t e d as 0.75 * 0.08 * [Hb] 5, w h e r e [Hb] is g i v e n in gLl (0.08 is the p o r t i o n of stroma p r o t e i n s per g r a m hemoglobinS). In 8 p a t i e n t s of the n e u r o s u r g i c a l e m e r g e n c y care u n i t the in vivo v a l i d i t y of the m e t h o d was tested. C o m p a r a t i v e blood v o l u m e m e a s u r e m e n t s w e r e c a r r i e d out in these p a t i e n t s w i t h Cr sl l a b e l l e d red cells and w i t h indocyanine green (ICG, as C a r d i o - G r e e n R, Hynsen, W e s t c o t t & Dunning, Baltimore, USA). 20 ml of a r t e r i a l b l o o d w e r e c o l l e c t e d and labelled with CR 51. One hour later w h e n this p r o c e d u r e was finished 16 mg ICG in 7 ml of w a t e r w e r e i n j e c t e d t h r o u g h a central venous catheter in about 7 sec. Prior
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to the i n j e c t i o n a 20 ml arterial sample was w i t h d r a w n for a 5 p o i n t c a l i b r a t i o n curve, w h e r e ICG in c o n c e n t r a t i o n s f r o m 0.3 mg/l to 1.9 m g / l was used. From the 2nd until the 7th m i n a f t e r i n j e c t i o n a r t e r i a l samples (2 ml) w e r e w i t h d r a w n in h a l f m i n u t e or m i n u t e t i m e intervalls. The d e a d s p a c e of the s a m p l i n g s y s t e m was not d i s c a r d e d but a r i t h m e t i c a l l y c o m p e n s a t e d for. A f t e r a d m i s s i o n of 200 ~i b l o o d to 400 ~i Triton X (1%) the s a m p l e s w e r e m e a s u r e d in a s p e c t r o p h o t o m e t e r and the c o n c e n t r a t i o n was p l o t t e d on a s e m i l o g a r i t h m i c g r a p h a g a i n s t the time (Fig. I). A r e g r e s s i o n line t h r o u g h t h e s e p o i n t s was e x t r a p o l a t e d to the time p o i n t "0" at the end of i n f u s i o n (Fig. i). The blood volume was c a l c u l a t e d f r o m the i n j e c t e d ICG a m o u n t d i v i d e d by the ICG c o n c e n t r a t i o n at the time p o i n t "0" m u l t i p l i c a t e d by 0.91 (this factor d e s c r i b e s the r e l a t i o n of w h o l e body h e m a t o c r i t to central hematocrit6). I m m e d i a t e l y after the last ICG sample was taken, the l a b e l l e d red cells w e r e r e i n j e c t e d and arterial samples w e r e c o l l e c t e d a f t e r 15, 30, 45 and 60 min. Blood volume was c a l c u l a t e d f r o m the r e l a t i o n of i n j e c t e d r a d i o a c t i v i t y to the r a d i o a c t i v i t y of the s a m p l e s m u l t i p l i e d by 0.91. In four patients, the r e p r o d u c i b i l i t y of the ICGTR_X m e t h o d was checked by a second b l o o d v o l u m e determination (BV2) 15 min after the first (BVl). The s t u d y w a s a p p r o v e d by, and c a r r i e d out a c c o r d i n g to the i n s t r u c t i o n s of the E t h i c ' s C o m m i t t e e of the H a n n o v e r M e d i c a l School. Statistics: L i n e a r r e g r e s s i o n a n a l y s i s was c a l c u l a t e d for t i m e / l o g c o n c e n t r a t i o n v a l u e s and for the c o m p a r i s o n b e t w e e n b l o o d v o l u m e s e s t i m a t e d w i t h the Cr 51 and ICGTR x methods.
IICG] ling/l)
1
05
Time
120
2/.0
360
FIG.
/.80
660
Isec)
1
ICG d i l u t i o n curve p l o t t e d on a s e m i l o g a r i t h m i c graph. The p o i n t of i n t e r s e c t i o n w i t h the y-axis is c o r r e s p o n d i n g to the ICG c o n c e n t r a t i o n at time "0". Results In v i t r o Triton-X
estimates: m e a n blood volume as d e t e r m i n e d from the ICG(ICGTR_X) m e t h o d was 84 ml +/- 7 ml in r e l a t i o n to the
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r e a l in v i t r o v o l u m e of 85 ml +/- 5 ml w i t h a m e a n d i f f e r e n c e 1.4 ml +/- 1.2 ml, i.e. an u n d e r e s t i m a t i o n of t h e r e a l v o l u m e a b o u t 1.6%.
of by
In v i v o e s t i m a t e s : m e a n b l o o d v o l u m e s of t h e p a t i e n t s m e a s u r e d w i t h t h e Cr ~ a n d ICGTR_X w e r e 4977 ml +/- 1344 ml a n d 4794 ml +/1122 ml, r e s p e c t i v e l y (Fig. 2). T h e m e a n d i f f e r e n c e w a s 184 ml +/138 ml (n.s.). T h e c o r r e l a t i o n c o e f f i c i e n t w a s 0.97. T h e r e s u l t s of t h e c o m p a r a t i v e b l o o d v o l u m e m e a s u r e m e n t s w i t h ICGTR_X in f o u r p a t i e n t s a r e d i s p l a y e d in T a b l e i.
TABLE Blood
volumes
in t w o
Patient
successive BV
BV
1
measurements Difference
1
363
371
-86
2 3 4
5226 3708 2450
5165 3596 2385
61 112 65
using
ICGTR_X
(ml)
(BVI-BV2)
BVICBTR. x
(I) 7
6 5 L 3 2 I 0
1
2
3
4
FIG. Comparison between r e d c e l l s (x-axis)
5
6
7
8VCR 5! {I)
2
blood volume measurements or ICGTR_X (y-axis).
using
Cr 51 l a b e l l e d
D~spussion T h o u g h in v i v o b l o o d v o l u m e s m e a s u r e d w i t h ICG_~ ~ or Cr 51 d e m o n s t r a t e a c l o s e r e l a t l o n s h l p in our e x p e r i m e n t s , t h e c o n v i n c i n g r e s u l t s of B r a d l e y and B a r r I c o u l d n o t c o m p l e t e l y be c o n f i r m e d . In our o p i n i o n t h i s is not a p r i n c i p a l d r a w b a c k of t h e s u g g e s t e d m e t h o d . In a g e n e r a l a t t e m p t to a d m i n i s t e r an as low d o s e as p o s s i b l e a m i n i m a l ICG a m o u n t w a s u s e d w h i c h m a d e t h e e v a l u a t i o n m o r e s u s c e p t i b e l to i n t e r f e r e n c e . A d o s e in t h e r a n g e
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of 0.5 m g / k g of b o d y w e i g h t m i g h t i m p r o v e t h i s as w a s i n d i c a t e d f r o m o w n m e a s u r e m e n t s in p l a s m a (to be p u b l i s h e d ) . T h e s a m e h o l d s for t h e c a l i b r a t i o n c u r v e s w h i c h s h o u l d h a v e b e e n p l o t t e d w i t h m a r k e d l y h i g h e r ICG c o n c e n t r a t i o n s . T h i s is d e m o n s t r a t e d in t h e in v i t r o e x p e r i m e n t s w h i c h w e r e c a r r i e d out a f t e r t h e in v i v o series, w i t h a v e r y c l o s e r e l a t i o n s h i p b e t w e e n real and m e a s u r e d volumes. T h e i n s i g n i f i c a n t u n d e r e s t i m a t i o n of the real in v i t r o v o l u m e s by 1.6% c a n p a r t l y be e x p l a i n e d by the fact t h a t the ICG_• R - - A_ m e t h, o d . m e a s u r e s w h o l e b l o o d v o l u m e m l n u s the v o l u m e of s t r o m a p r o t e l n s w h i c h is in the r a n g e of 0.8-1% 5 . The c a l i b r a t i o n c u r v e s w e r e d e m o n s t r a t e d to be linear. No c o n s i d e r a b l e d i f f e r e n c e w a s o b t a i n e d w h e n b l o o d v o l u m e s w e r e c a l c u l a t e d from a t w o - p o i n t c a l i b r a t i o n c u r v e or a five and t e n - p o i n t c a l i b r a t i o n curve, r e s p e c t i v e l y . W h e n t h e h e m o l y z e d b l o o d w a s c e n t r i f u g e d in o r d e r to o b t a i n a c l e a r solution, free of cell m e m b r a n e fragments, b l o o d v o l u m e d e t e r m i n a t i o n w a s d o n e w i t h i n about 15 m i n (with the h e l p of a p r o g r a m m a b l e p o c k e t c a l c u l a t o r ) , if T R - X t u b e s and the ICG s o l u t i o n h a d b e e n p r e p a r e d before. M e a s u r e m e n t of t h e h e m o l y s a t e w i t h o u t p r e c e d i n g c e n t r i f u g a t i o n of c a l i b r a t i o n b l o o d and samples m a y e v e n a c c e l e r a t e the p r o c e d u r e but m a y also r e d u c e t h e a c c u r a c y of b l o o d v o l u m e estimates. The m e t h o d a f f o r d s an a r t e r i a l and c e n t r a l v e n o u s c a t h e t e r . If a central v e n o u s c a t h e t e r is used, o n l y a s a m p l i n g site in the a. p u l m o n a l i s is a c c e p t a b l e . F r o m a s a m p l i n g s i t e in the v. cava sup., b l o o d w i t h a h i g h e r ICG c o n c e n t r a t i o n t h a n in the v. cava inf. or in the a r t e r i e s would be w i t h d r a w n . T h u s for any g i v e n time, i n c l u d i n g t i m e "0", a s m a l l e r d i s t r i b u t i o n v o l u m e w o u l d be c a l c u l a t e d . The u s e of p e r i p h e r a l v e i n s for ICG a d m i n i s t r a t i o n or b l o o d s a m p l i n g w o u l d g e n e r a l l y r e s u l t in w r o n g b l o o d v o l u m e m e a s u r e m e n t s (own u n p u b l i s h e d results). A p o s s i b l e s y s t e m a t i c e r r o r m a y a r i s e f r o m t h e fact t h a t t h e linear s e g m e n t of the s e m i l o g a r t h m i c p l o t w a s e x t r a p o l a t e d to the t i m e "0" i m m e d i a t e l y a f t e r the end of i n j e c t i o n . T h e l i n e a r s e g m e n t is s u p p o s e d to r e p r e s e n t ICG e x t r a c t i o n f r o m the b l o o d by the liver. T h u s if t h e t i m e f r o m the e n d of i n j e c t i o n u n t i l the dye r e a c h e s the liver is n o t t a k e n into account, an u n d e r e s t i m a t i o n of b l o o d v o l u m e in the r a n g e of 4% m a y r e s u l t 7. T h i s is in c o r r e s p o n d e n c e w i t h our f i n d i n g s of a b o u t 3% l o w e r ICGTR_X v o l u m e s (after c o r r e c t i o n for a b o u t 1% of s t r o m a p r o t e i n s ) c o m p a r e d w i t h Cr 51 v o l u m e s in this series. O n t h e w h o l e the ICGTR_X m e t h o d a p p e a r s to be an a p p r o p r i a t e w a y of fast b l o o d v o l u m e m e a s u r e m e n t as long as no c i r c u l a t o r y f a i l u r e occurs. In t h i s case a c o n s i d e r a b l y d e l a y e d m i x i n g t i m e in the b l o o d w o u l d m a k e a r e l i a b l e b l o o d v o l u m e d e t e r m i n a t i o n i m p o s s i b e l 8. T h o u g h w e r e c o g n i z e the i n c o m p l e t e state of this p r e l i m i n a r y r e s e a r c h , w e felt j u s t i f i e d to p u b l i s h the g i v e n d a t a at t h i s e a r l y s t a t e d u e to t h e i r g e n e r a l s i g n i f i c a n c e , all the more, as e a c h e x a m i n e r can w o r k out an i n d i v i d u a l p r o c e e d i n g on t h e b a s i s of t h e g i v e n values.
Acknowledgements We t h a n k M r s H. K o n r a d
for her e x c e l l e n t
technical
assistance.
References i. E.C.
BRADLEY
(1968).
and J.W.
BARR,
Life S c i e n c e s
7 1001-1007
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2. F.R. LEWIS, V . B . E L I N G S , S.L. H I L L and J.M. C H R I S T E N S E N , A n n of N.Y. Acad. Sci. 384 393-410 (1982) 3. M. SOKOLOW, M.B. MCILROY, K a r d i o l o g i e , 70-73, S p r i n g e r H e i d e l b e r g - N e w Y o r k (1985) 4. G.R. CHERRICK, S.W. STEIN, C.M. LEEVY and C.S. D A V I D S O N , J. Clin. Invest. 39 592-600 (1960) 5. D. B O E N I N G and N. MAASSEN, J . A p p l . P h y s i o l . 54 118-122 (1983) 6. S O L O M O N N. ALBERT, B l o o d Volume, J o h n A d r i a n i (ed), 147, C h a r l e s C. Thomas, S p r i n g f i e l d ' I l l i n o i s ' U S A (1963) 7. K. H A N E D A and T. HORIUCHI, T o h u k u J. exp. Med. 148 49-56 (1986) 8. G.J. KUHN, B.C. WHITE, R.E.SWETNAM, J.F. MUMEY, M.F. RYDESKY, J.E. T I N T I N A L L Y , R.L. K R O M E and P.J. HOEHNER, Ann. Emerg. Med. i0 417-419 (1981)
Pergamon Press
D E T E R M I N A T I O N OF C I R C U L A T I N G BLOOD V O L U M E BY M E A S U R E M E N T OF I N D O C Y A N I N E G R E E N DYE IN HEMOLYSATE: A P R E L I M I N A R Y STUDY
M a r t i n W. Busse, Henschen; Bernhard
Departments
MD; Stefan Zisowsky; S t e p h a n Panning, MD; Lotte Reilmann,
M e d i c a l School H a n n o v e r of Physiology, A n a e s t h e s i o l o g y D-3000 H a n n o v e r West-Germany
MD
and R a d i o l o g y
(Received in final form January 3, 1990) Summary In 8 e m e r g e n c y c a r e _ p a t i e n t s blood v o l u m e w a s d e t e r m i n e d u s i n g Cr ~I labelled e r y t h r o c y t e s and i n d o c y a n i n e g r e e n (ICG). Prior to m e a s u r e m e n t of ICG in blood with a spectrophotometer, the b l o o d w a s h e m o l y z e d w i t h Triton-X. A close c o r r e l a t i o n of r=0.97 b e t w e e n the Cr 51 and the ICG.. ~ e s t i m a t e s was found; the ICG ~1 TR_ v o l u m e was about 3~ Tower t h a n the Cr 5 volume. In ~ i v e a d d i t i o n a l in vitro e x p e r i m e n t s the ICG__ _ m e t h o d w a s found to r e f l e c t real v o l u m e s w i t h an i n s l g n l f l c a n t e r r o r of less t h a n 1%. Blood v o l u m e d e t e r m i n a t i o n w i t h ICGTR_~ c a n n o t be a p p l i e d in cases of c i r c u l a t o r y failu@e. ICG should be a d m i n i s t e r e d in a d o s e of 0.5 m g / k g of b o d y weight. For c a l i b r a t i o n purposes, a two p o i n t c a l i b r a t i o n curve (point i: p o i n t of i n t e r s e c t i o n of x and y axis; point 2 : 5 mg ICG/I of blood) is sufficient. From these p r e l i m i n a r y e x p e r i m e n t s it is c o n c l u d e d that the ICG__. m e t h o d is a r a p i d and simple t e c h n i q u e of bloo~ v o l u m e d e t e r m l n a t i o n w i t h m u l t i p l e r e p r o d u c i b i l i t y w h i c h can be c a r r i e d out in any c l i n i c a l laboratory.
In p a t i e n t s w i t h a m i s m a t c h of blood loss and s i m u l t a n e o u s t h e r a p e u t i c a l a d m i n i s t r a t i o n of fluids, r a p i d b l o o d v o l u m e m e a s u r e m e n t m a y be an i m p o r t a n t d i a g n o s t i c aid. R e p e t i t i v e d e t e r m i n a t i o n s m a y be of p a r t i c u l a r i m p o r t a n c e d u r i n g h e m o r r h a g e n i c s u r g e r y after a l t e r n a t i n g i n f u s i o n s of c o l l o i d a l or m i n e r a l s o l u t i o n s or of blood. The c i r c u l a t i n g p l a s m a and b l o o d v o l u m e or the r e s p e c t i v e changes can t h e n not be q u a n t i f i e d by r e l a t i v e c h a n g e s of h e m o g l o b i n / h e m a t o c r i t or total p l a s m a p r o t e i n c o n c e n t r a t i o n . These c o n d i t i o n s m a y be even m o r e c o m p l i c a t e d d u r i n g n e u r o s u r g e r y and n e u r o s u r g i c a l i n t e n s i v e care, w h e n in a d d i t i o n h y p e r o s m o l a l s o l u t i o n s are administered. C u s t o m a r y m e t h o d s such as m e a s u r e m e n t of red cell v o l u m e w i t h Cr 51 l a b e l l e d c e l l s o f t e n fail the d e m a n d for simple, r a p i d and r e p e t i t i v e m o n i t o r i n g . The latter also holds for Evan's blue. A g e n e r a l 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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d r a w b a c k of all tracer m e t h o d s is the need for s p e c i a l e q u i p m e n t and the u n w e l c o m e a p p l i c a t i o n of r a d i o a c t i v e substances. T h o u g h B r a d l e y and Barr in 1968 p r o p o s e d a rapid and r e p r o d u c i b l e m e t h o d of b l o o d v o l u m e d e t e r m i n a t i o n with indocyanine g r e e n in this j o u r n a l I, this m e t h o d has not g a i n e d general acceptance. T h i s m a y be m a i n l y due to the fact that the used t e c h n i c a l device, w h i c h w a s d e s i g n e d for cardiac output measurement, was q u i t e a w k w a r d to o p e r a t e and, in addition, no routine d i a g o s t i c equipment. F u r t h e r m o r e the a p p a r a t u s has become obsolete t h e s e days and is u s e d only for special a p p l i c a t i o n s such as lung w a t e r d e t e r m i n a t i o n 2, d i a g n o s i s of cardiac shunts 3 or liver f u n c t i o n 4. As in p r i n c i p l e the f a v o u r a b l e q u a l i t i e s of ICG are well documented, a m o r e simple m e t h o d of m e a s u r e m e n t w o u l d allow fast and r e l i a b l e b l o o d v o l u m e monitoring. ICG may be easily m e a s u r e d in plasma. A g e n e r a l d r a w b a c k is the need of a special c a l i b r a t i o n c u r v e for each p a t i e n t and also for i n t r a i n d i v i d u a l l y v a r y i n g c o n d i t i o n s (own u n p u b l i s h e d results). As the c a l i b r a t i o n curve s h o u l d r e p r e s e n t the in vivo conditions, ICG should be p i p e t t e d into w h o l e b l o o d w h i c h w o u l d be c e n t r i f u g e d a f t e r w a r d s for ICG m e a s u r e m e n t . The ICG plasma c o n c e n t r a t i o n of s t a n d a r d and in vivo s a m p l e s w o u l d t h e n be c o r r e c t e d for p a c k e d cell volume. W h e n c a l c u l a t i n g the in vivo blood volume, a p o s s i b l e s y s t e m a t i c error of the h e m a t o c r i t (Hk) d e t e r m i n a t i o n would thus be avoided. On the other h a n d the m u l t i p l e Hk m e a s u r e m e n t s may be an i m p o r t a n t t i m e c o n s u m i n g factor in e m e r g e n c y diagnosis, w h e n a d i r e c t b l o o d v o l u m e d e t e r m i n a t i o n w o u l d be m u c h m o r e comfortable. U n f o r t u n a t e l y ICG m e a s u r e m e n t in whole blood w i t h a s p e c t r o p h o t o m e t e r , an e q u i p m e n t w h i c h is nearly anywhere available, is impossible. O b j e c t of this study was to introduce an u n c o m p l i c a t e d w a y of b l o o d v o l u m e m e a s u r e m e n t using ICG in h e m o l y z e d b l o o d w h i c h can be c a r r i e d out in any clinical laboratory.
Methods In 5 e x p e r i m e n t s the in vitro v a l i d i t y of blood v o l u m e (BV) m e a s u r e m e n t in h e m o l y s a t e was checked by c o m p a r i s o n of real and m e a s u r e d blood volumes. About 0.17 mg ICG w e r e m i x e d in about 85 ml of w h o l e blood (i.e. about 2 mg/l; in each s i n g l e case the b l o o d v o l u m e s and ICG amounts were m e a s u r e d and w e i g h e d w i t h h i g h p r e c i s i o n equipment). The ICG c o n c e n t r a t i o n s of a i0 p o i n t s t a n d a r d curve w e r e in the range of about 0.3 m g / l to 7 m g / l of blood. 200 ~i of the blood w e r e added to 400 ~i of T r i t o n - X (TR-X, 1%) for hemolysis. S t a n d a r d values and the in v i t r o s a m p l e s w e r e then measured with a spectrophotometer (Hitachi 150-20) at 805 nm. The w a t e r v o l u m e in ml o c c u p i e d by stroma p r o t e i n s per liter of w h o l e b l o o d was c a l c u l a t e d as 0.75 * 0.08 * [Hb] 5, w h e r e [Hb] is g i v e n in gLl (0.08 is the p o r t i o n of stroma p r o t e i n s per g r a m hemoglobinS). In 8 p a t i e n t s of the n e u r o s u r g i c a l e m e r g e n c y care u n i t the in vivo v a l i d i t y of the m e t h o d was tested. C o m p a r a t i v e blood v o l u m e m e a s u r e m e n t s w e r e c a r r i e d out in these p a t i e n t s w i t h Cr sl l a b e l l e d red cells and w i t h indocyanine green (ICG, as C a r d i o - G r e e n R, Hynsen, W e s t c o t t & Dunning, Baltimore, USA). 20 ml of a r t e r i a l b l o o d w e r e c o l l e c t e d and labelled with CR 51. One hour later w h e n this p r o c e d u r e was finished 16 mg ICG in 7 ml of w a t e r w e r e i n j e c t e d t h r o u g h a central venous catheter in about 7 sec. Prior
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to the i n j e c t i o n a 20 ml arterial sample was w i t h d r a w n for a 5 p o i n t c a l i b r a t i o n curve, w h e r e ICG in c o n c e n t r a t i o n s f r o m 0.3 mg/l to 1.9 m g / l was used. From the 2nd until the 7th m i n a f t e r i n j e c t i o n a r t e r i a l samples (2 ml) w e r e w i t h d r a w n in h a l f m i n u t e or m i n u t e t i m e intervalls. The d e a d s p a c e of the s a m p l i n g s y s t e m was not d i s c a r d e d but a r i t h m e t i c a l l y c o m p e n s a t e d for. A f t e r a d m i s s i o n of 200 ~i b l o o d to 400 ~i Triton X (1%) the s a m p l e s w e r e m e a s u r e d in a s p e c t r o p h o t o m e t e r and the c o n c e n t r a t i o n was p l o t t e d on a s e m i l o g a r i t h m i c g r a p h a g a i n s t the time (Fig. I). A r e g r e s s i o n line t h r o u g h t h e s e p o i n t s was e x t r a p o l a t e d to the time p o i n t "0" at the end of i n f u s i o n (Fig. i). The blood volume was c a l c u l a t e d f r o m the i n j e c t e d ICG a m o u n t d i v i d e d by the ICG c o n c e n t r a t i o n at the time p o i n t "0" m u l t i p l i c a t e d by 0.91 (this factor d e s c r i b e s the r e l a t i o n of w h o l e body h e m a t o c r i t to central hematocrit6). I m m e d i a t e l y after the last ICG sample was taken, the l a b e l l e d red cells w e r e r e i n j e c t e d and arterial samples w e r e c o l l e c t e d a f t e r 15, 30, 45 and 60 min. Blood volume was c a l c u l a t e d f r o m the r e l a t i o n of i n j e c t e d r a d i o a c t i v i t y to the r a d i o a c t i v i t y of the s a m p l e s m u l t i p l i e d by 0.91. In four patients, the r e p r o d u c i b i l i t y of the ICGTR_X m e t h o d was checked by a second b l o o d v o l u m e determination (BV2) 15 min after the first (BVl). The s t u d y w a s a p p r o v e d by, and c a r r i e d out a c c o r d i n g to the i n s t r u c t i o n s of the E t h i c ' s C o m m i t t e e of the H a n n o v e r M e d i c a l School. Statistics: L i n e a r r e g r e s s i o n a n a l y s i s was c a l c u l a t e d for t i m e / l o g c o n c e n t r a t i o n v a l u e s and for the c o m p a r i s o n b e t w e e n b l o o d v o l u m e s e s t i m a t e d w i t h the Cr 51 and ICGTR x methods.
IICG] ling/l)
1
05
Time
120
2/.0
360
FIG.
/.80
660
Isec)
1
ICG d i l u t i o n curve p l o t t e d on a s e m i l o g a r i t h m i c graph. The p o i n t of i n t e r s e c t i o n w i t h the y-axis is c o r r e s p o n d i n g to the ICG c o n c e n t r a t i o n at time "0". Results In v i t r o Triton-X
estimates: m e a n blood volume as d e t e r m i n e d from the ICG(ICGTR_X) m e t h o d was 84 ml +/- 7 ml in r e l a t i o n to the
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r e a l in v i t r o v o l u m e of 85 ml +/- 5 ml w i t h a m e a n d i f f e r e n c e 1.4 ml +/- 1.2 ml, i.e. an u n d e r e s t i m a t i o n of t h e r e a l v o l u m e a b o u t 1.6%.
of by
In v i v o e s t i m a t e s : m e a n b l o o d v o l u m e s of t h e p a t i e n t s m e a s u r e d w i t h t h e Cr ~ a n d ICGTR_X w e r e 4977 ml +/- 1344 ml a n d 4794 ml +/1122 ml, r e s p e c t i v e l y (Fig. 2). T h e m e a n d i f f e r e n c e w a s 184 ml +/138 ml (n.s.). T h e c o r r e l a t i o n c o e f f i c i e n t w a s 0.97. T h e r e s u l t s of t h e c o m p a r a t i v e b l o o d v o l u m e m e a s u r e m e n t s w i t h ICGTR_X in f o u r p a t i e n t s a r e d i s p l a y e d in T a b l e i.
TABLE Blood
volumes
in t w o
Patient
successive BV
BV
1
measurements Difference
1
363
371
-86
2 3 4
5226 3708 2450
5165 3596 2385
61 112 65
using
ICGTR_X
(ml)
(BVI-BV2)
BVICBTR. x
(I) 7
6 5 L 3 2 I 0
1
2
3
4
FIG. Comparison between r e d c e l l s (x-axis)
5
6
7
8VCR 5! {I)
2
blood volume measurements or ICGTR_X (y-axis).
using
Cr 51 l a b e l l e d
D~spussion T h o u g h in v i v o b l o o d v o l u m e s m e a s u r e d w i t h ICG_~ ~ or Cr 51 d e m o n s t r a t e a c l o s e r e l a t l o n s h l p in our e x p e r i m e n t s , t h e c o n v i n c i n g r e s u l t s of B r a d l e y and B a r r I c o u l d n o t c o m p l e t e l y be c o n f i r m e d . In our o p i n i o n t h i s is not a p r i n c i p a l d r a w b a c k of t h e s u g g e s t e d m e t h o d . In a g e n e r a l a t t e m p t to a d m i n i s t e r an as low d o s e as p o s s i b l e a m i n i m a l ICG a m o u n t w a s u s e d w h i c h m a d e t h e e v a l u a t i o n m o r e s u s c e p t i b e l to i n t e r f e r e n c e . A d o s e in t h e r a n g e
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of 0.5 m g / k g of b o d y w e i g h t m i g h t i m p r o v e t h i s as w a s i n d i c a t e d f r o m o w n m e a s u r e m e n t s in p l a s m a (to be p u b l i s h e d ) . T h e s a m e h o l d s for t h e c a l i b r a t i o n c u r v e s w h i c h s h o u l d h a v e b e e n p l o t t e d w i t h m a r k e d l y h i g h e r ICG c o n c e n t r a t i o n s . T h i s is d e m o n s t r a t e d in t h e in v i t r o e x p e r i m e n t s w h i c h w e r e c a r r i e d out a f t e r t h e in v i v o series, w i t h a v e r y c l o s e r e l a t i o n s h i p b e t w e e n real and m e a s u r e d volumes. T h e i n s i g n i f i c a n t u n d e r e s t i m a t i o n of the real in v i t r o v o l u m e s by 1.6% c a n p a r t l y be e x p l a i n e d by the fact t h a t the ICG_• R - - A_ m e t h, o d . m e a s u r e s w h o l e b l o o d v o l u m e m l n u s the v o l u m e of s t r o m a p r o t e l n s w h i c h is in the r a n g e of 0.8-1% 5 . The c a l i b r a t i o n c u r v e s w e r e d e m o n s t r a t e d to be linear. No c o n s i d e r a b l e d i f f e r e n c e w a s o b t a i n e d w h e n b l o o d v o l u m e s w e r e c a l c u l a t e d from a t w o - p o i n t c a l i b r a t i o n c u r v e or a five and t e n - p o i n t c a l i b r a t i o n curve, r e s p e c t i v e l y . W h e n t h e h e m o l y z e d b l o o d w a s c e n t r i f u g e d in o r d e r to o b t a i n a c l e a r solution, free of cell m e m b r a n e fragments, b l o o d v o l u m e d e t e r m i n a t i o n w a s d o n e w i t h i n about 15 m i n (with the h e l p of a p r o g r a m m a b l e p o c k e t c a l c u l a t o r ) , if T R - X t u b e s and the ICG s o l u t i o n h a d b e e n p r e p a r e d before. M e a s u r e m e n t of t h e h e m o l y s a t e w i t h o u t p r e c e d i n g c e n t r i f u g a t i o n of c a l i b r a t i o n b l o o d and samples m a y e v e n a c c e l e r a t e the p r o c e d u r e but m a y also r e d u c e t h e a c c u r a c y of b l o o d v o l u m e estimates. The m e t h o d a f f o r d s an a r t e r i a l and c e n t r a l v e n o u s c a t h e t e r . If a central v e n o u s c a t h e t e r is used, o n l y a s a m p l i n g site in the a. p u l m o n a l i s is a c c e p t a b l e . F r o m a s a m p l i n g s i t e in the v. cava sup., b l o o d w i t h a h i g h e r ICG c o n c e n t r a t i o n t h a n in the v. cava inf. or in the a r t e r i e s would be w i t h d r a w n . T h u s for any g i v e n time, i n c l u d i n g t i m e "0", a s m a l l e r d i s t r i b u t i o n v o l u m e w o u l d be c a l c u l a t e d . The u s e of p e r i p h e r a l v e i n s for ICG a d m i n i s t r a t i o n or b l o o d s a m p l i n g w o u l d g e n e r a l l y r e s u l t in w r o n g b l o o d v o l u m e m e a s u r e m e n t s (own u n p u b l i s h e d results). A p o s s i b l e s y s t e m a t i c e r r o r m a y a r i s e f r o m t h e fact t h a t t h e linear s e g m e n t of the s e m i l o g a r t h m i c p l o t w a s e x t r a p o l a t e d to the t i m e "0" i m m e d i a t e l y a f t e r the end of i n j e c t i o n . T h e l i n e a r s e g m e n t is s u p p o s e d to r e p r e s e n t ICG e x t r a c t i o n f r o m the b l o o d by the liver. T h u s if t h e t i m e f r o m the e n d of i n j e c t i o n u n t i l the dye r e a c h e s the liver is n o t t a k e n into account, an u n d e r e s t i m a t i o n of b l o o d v o l u m e in the r a n g e of 4% m a y r e s u l t 7. T h i s is in c o r r e s p o n d e n c e w i t h our f i n d i n g s of a b o u t 3% l o w e r ICGTR_X v o l u m e s (after c o r r e c t i o n for a b o u t 1% of s t r o m a p r o t e i n s ) c o m p a r e d w i t h Cr 51 v o l u m e s in this series. O n t h e w h o l e the ICGTR_X m e t h o d a p p e a r s to be an a p p r o p r i a t e w a y of fast b l o o d v o l u m e m e a s u r e m e n t as long as no c i r c u l a t o r y f a i l u r e occurs. In t h i s case a c o n s i d e r a b l y d e l a y e d m i x i n g t i m e in the b l o o d w o u l d m a k e a r e l i a b l e b l o o d v o l u m e d e t e r m i n a t i o n i m p o s s i b e l 8. T h o u g h w e r e c o g n i z e the i n c o m p l e t e state of this p r e l i m i n a r y r e s e a r c h , w e felt j u s t i f i e d to p u b l i s h the g i v e n d a t a at t h i s e a r l y s t a t e d u e to t h e i r g e n e r a l s i g n i f i c a n c e , all the more, as e a c h e x a m i n e r can w o r k out an i n d i v i d u a l p r o c e e d i n g on t h e b a s i s of t h e g i v e n values.
Acknowledgements We t h a n k M r s H. K o n r a d
for her e x c e l l e n t
technical
assistance.
References i. E.C.
BRADLEY
(1968).
and J.W.
BARR,
Life S c i e n c e s
7 1001-1007
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