Journal of Immunological Methods, 7 (1975) 283--290 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
REFINEMENTS IN THE AUTOMATED FLUOROMETRIC HISTAMINE ANALYSIS SYSTEM
REUBEN P. SIRAGANIAN Clinical Immunology Section, Laboratory of Microbiology and Immunology, NIDR, National Institutes of Health, Bethesda, Md. 20014, U.S.A. (Received 10 August 1974, accepted 18 December 1974) The automated continuous flow system for the extraction and fluorometric analysis of histamine has been further improved. Samples with or without protein can be analyzed. The use of a single pump, added nitrogen, and lower concentration of o-phthalaldehyde increase the sensitivity by 5--10 fold and allow the analysis of samples at the rate of 30 per hour. Samples which are less than 0.5 ml in volume and contain 0.1 to 10 ng/ml histamine can routinely be analyzed. The sensitivity of the system is now 0.05 ng and compares favorably with isotopic techniques.
INTRODUCTION An a u t o m a t e d c o n t i n u o u s f l o w s y s t e m f o r t h e e x t r a c t i o n and f l u o r o m e t ric analysis o f h i s t a m i n e has b e e n described (Siraganian, 1974). The s y s t e m d e p e n d s o n t h e ability to separate o r g a n i c - a q u e o u s phases in the c o n t i n u o u s f l o w s y s t e m b y the use o f special e x t r a c t o r s . However, the s y s t e m as previo u s l y described required t w o p u m p s and its o p e r a t i o n was o p t i m a l at 20 samples per h o u r . When a n a l y z i n g samples at a faster rate there was considerable c a r r y - o v e r and c o n t a m i n a t i o n f r o m o n e sample to t h e next. The present p a p e r describes several m o d i f i c a t i o n s o f t h e s y s t e m including the use o f a single, greater c a p a c i t y p u m p , a m o r e sensitive f l u o r o m e t e r , and changes in t h e chemical reaction. The r e a c t i o n o f t h e f l u o r o p h o r e with h i s t a m i n e is p e r f o r m e d u n d e r n i t r o g e n w h i c h increases t h e sensitivity o f the r e a c t i o n (Hfikanson et al., 1972). The c o n c e n t r a t i o n o f o - p h t h a l a l d e h y d e (OPT) is decreased w h i c h i m p r o v e s the r e a g e n t b l a n k fluorescence. With the m o d i f i e d system, samples are a n a l y z e d at the rate o f 30 per h o u r with a very high degree o f sensitivity and a c c u r a c y . The p r e s e n t m o d i f i c a t i o n allows a 5-fold increase in sensitivity with 50% increase in t h e rate o f sample analysis. MATERIALS AND METHODS A p p a r a tus The f o l l o w i n g T e c h n i c o n m o d u l e s ( T e c h n i c o n Corp., T a r r y t o w n , N.Y.) were used: s a m p l e r t y p e II, p r o p o r t i o n i n g p u m p II, basic s y s t e m dialyzer
284 (37°C) a f l u o r o n e p h l o m e t e r and a single pen recorder. The f l u o r o m e t e r was equipped with the 013-B008-01 flow cell. The primary filter was the narrow pass 7-60 (peak at 350 nm). A sharp cut filter 2A plus a 4-72 (Corning) were used t o g e t h e r as the secondary filter.
Reagents The following reagents were used: saline (0.9% NaC1, 0.016% Brij-35, 1.5 X 10 -3 M EDTA), 30% NaC1 solution, 5 N NaOH, 1 N NaOH (containing 1 × 10 -3 M EDTA), 0.1 N HC1, 0.73 M phosphoric acid, n-butanol and heptane. The details of the preparation of these reagents have been described previously (Siraganian, 1974). The 30% NaC1 solution was filtered before use. A 0.5 M pH 12.3 borate buffer was prepared by dissolving 30.95 gm of boric acid in 900 ml water, adjusting the pH with 50% NaOH, and bringing the solution up to 1 liter. The o-phthalaldehyde (OPT) solution was changed f r o m the c o n c e n t r a t i o n previously used to 0.5 mg per ml. For a day's use, 50 mg OPT were dissolved in 2 ml methanol ( s p e c t r o p h o t o m e t r i c grade), and this solution was brought up to 100 ml with 0.5 M borate buffer, pH 12.3. It was kept at 0°C in a dark bottle in an ice bucket. The use of fresh OPT reduced the reagent black fluorescence and keeping it in ice resulted in a more stable baseline. Histamine standards were prepared in 2% perchloric acid. The c o n c e n t r a t i o n of histamine is stated for the base.
Procedure The flow diagram for the system, including the size of the different manifold tubes, the connectors, and the coils used on the manifold, is shown in fig. 1. The sample is diluted with saline and dialyzed against the same solution. To the dialyzed stream 30% NaC1 is added. After mixing, butanol and NaOH are added and the histamine is extracted by mixing into the butanol. The organic and aqueous phases are separated in the first extractor; to the organic phase 0.1 N HC1 and n-heptane are added. After mixing, the phases are separated in the second extractor. Both extractors are placed close to the inlet side of the manifold tubes on the right side of the pump. It is crucial t h a t the second e x t r a c t o r be very close to the inlet of the manifold tubes as this part o f the stream is not segmented by air bubbles. It is also i m p o r t a n t t h a t the end o f the waste tubes f r om bot h extractors are kept at the level of the middle o f the extractors, otherwise a negative pressure will be applied to this part o f the manifold with uneven flow characteristics. In the second extractor, the lower aqueous phase is removed and resegmented with bubbles. It is then alkalinized, OPT added, and after the optimal reaction time the histamine-o-phthalaldehyde reaction stopped with phosphoric acid. The stream then passes through the fluorometer. As indicated in fig. 1 Solvaflex tubing is used for those portions of the manifold which come in co n tact with organic solvents.
285
MANIFOLD TUBES POSITION SIZE INCHES (ml/min) 0045 (080) AIR WASTE ~
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Fig. 1. Flow diagram for histamine analysis. Manifold is shown for analysis with dialysis; for operation without dialysis points A and A p are connected and pump tubes marked (+) are removed. Extra sample tubes can be left on the pump at the unoccupied positions 7 (0.23 ml/min) and 10 (0.32 ml/min) for rapid changeover to analyze samples at other sensitivities. The first 3 coils have a 1.5 cm diameter but whereas coil No. 116-0246 is 10 turns with a 2.0 mm I.D. (internal diameter) both coils 105-0089 and 105-0087 are 28 turns each and have an I.D. of 2.4 mm. The last 2 coils (190-0051-10 is 40 turns and 190-0051-04 is 16 turns) are 3.0 cm in diameter with a 2 mm internal diameter. Part numbers are listed in the Technicon catalogue.
T h e t w o e x t r a c t o r s u s e d in t h e m a n i f o l d are c o n s t r u c t e d as p r e v i o u s l y d e s c r i b e d f r o m T e c h n i c o n m a n i f o l d part N - 6 and D - 3 (Siraganian, 1 9 7 4 ) . T h e t w o n i p p l e s ( N - 6 ) are p l a c e d at t h e e n d s o f t h e w i d e - b o r e c o n n e c t o r s ( D - 3 ) a n d n o t i n s i d e t h e s i d e - a r m s . T h e f i n e s t e e l w i r e l o o p is u s e d o n l y in t h e s e c o n d e x t r a c t o r and it f u n c t i o n s e f f e c t i v e l y t o b r e a k u p s m a l l b u b b l e s . T h e u s e o f n i t r o g e n i n s t e a d o f air t o s e g m e n t t h e s t r e a m i n c r e a s e s t h e s e n s i t i v i t y o f t h e s y s t e m b y 3 0 - - 5 0 % . N i t r o g e n is p u m p e d i n t o t h e e n d o f t h e 'air' t u b e s f r o m a t a n k u s i n g s e v e r a l p r e s s u r e r e d u c t i o n v a l v e s t o a l l o w gas t o e s c a p e at a b o u t 1 0 m l / m i n . A n o v e r f l o w f r o m t h e n i t r o g e n t u b e a l l o w s t h e e x c e s s gas t o e s c a p e . A l t h o u g h t h e e f f e c t o f n i t r o g e n is o n l y o n t h e o - p h t h a l a l d e h y d e - - h i s t a m i n e r e a c t i o n , t h e m a n i f o l d in r o u t i n e u s e has n i t r o g e n rep l a c i n g air at all t h e p u m p p o i n t s . The flow system can be modified to increase the sensitivity when analyzing h i s t a m i n e in s a m p l e s w h i c h c o n t a i n l i t t l e p r o t e i n . T h e s e s a m p l e s are
286 a n a l y z e d w i t h o u t d i a l y s i s . T h i s m o d i f i c a t i o n is d e s c r i b e d in fig. 1. T h e t o t a l t i m e r e q u i r e d f o r a s a m p l e t o r u n t h r o u g h t h e m a n i f o l d is 1 6 m i n w i t h d i a l y s i s a n d 11 m i n w i t h o u t d i a l y s i s .
Manifold changes The basic manifold can easily be modified to analyze different volume samples. In general, increasing the sample uptake volume will increase the s e n s i t i v i t y o f t h e a s s a y . A s u m m a r y o f t h e p o s s i b l e c h a n g e s is s h o w n in t a b l e 1. T h e m a n i f o l d is u s e d w i t h o u t d i a l y s i s w h e n t h e p r o t e i n c o n c e n t r a t i o n in t h e s a m p l e s is less t h a n 3 . 0 m g / m l , e.g., as in T r i s A o r T r i s A C M buffers (Levy and Widra, 1973). The total volume of samples required for
analysis in this system is 0.3--0.6 ml; and histamine concentrations of 7--25 ng/ml result in full scale deflection. The amount of histamine that can be quantitated is usually 0.5 to 1% of the concentration that results in full scale deflection and therefore about I00 pg/ml. For routine use in assays of histamine release from washed human leukocytes the manifold with the 0.23 ml/min sample tube is used. W h e n s a m p l e s a r e a n a l y z e d u s i n g t h e d i a l y z e r t h e s e n s i t i v i t y is d e c r e a s e d
TABLE 1 Manifold parameters for analyzing samples with different volumes and varying histamine content. Manifold
Sample uptake tube
Sample volume required a
I.D. Flow rate (inches) (ml/min)
(ml)
Histamine % of Plateau c concentration for full scale deflection b (ng/ml)
I. Without dialysis (protein less than 3 mg/ml)
0.020 0.025* 0.030
0.16 0.23 0.32
0.3 0.4 0.6
25 10 7
93 94 92
II. With dialysis
0.020 0.025 0.030*
0.16 0.23 0.32
0.3 0.4 0.6
75 30 20
91 91 90
Samples analyzed at the rate of 30/hr; nitrogen used instead of air. Sensitivity will decrease by 35% if air is used in place of N2. * Manifold used most extensively with these two sample uptake tubes. a Total volume required in the sample cup. Actual volume analyzed is about 25% less. b Concentration of histamine standard that will give full scale deflection in the recorder; the readability of the graph is 0.5--1% of this value. The amount of histamine that can be quantitated is therefore in the 70--200 pg range. c Height of peak as percentage of plateau obtained by sampling the same standard for 5 min.
287
by about 70% (table 1). When the histamine content of the samples is low, a larger sample is analyzed to compensate for the decreased sensitivity. With dialysis, histamine concentrations of 20--75 ng/ml result in full scale deflection. The manifold with the 0.32 ml/min sample uptake tube is used extensively with samples containing large amounts of plasma and little histamine. Extra sample tubes are left on the pump for rapid changeover to analyze samples with different volumes. The manifold pump tube from extractor I is changed every 20 hr of machine operation time. The o-phthalaldehyde and sample uptake pump tubes are changed every 40 hr and all the manifold pump tubes after 160 hr of operation. These changes are important when analyzing samples at high sensitivity settings. RESULTS AND DISCUSSION
A summary of the sensitivity of the system when used under different conditions is shown in table 1. The system is slightly more accurate when
I0.0 ng/ml 80
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Fig. 2. C h a r t r e c o r d i n g o f h i s t a m i n e s t a n d a r d s a n a l y z e d w i t h o u t dialysis. S t a n d a r d s were 0 - - 1 0 n g / m l in 2% p e r c h l o r i c acid. Analysis was at 30 samples p e r h o u r using a s a m p l e u p t a k e t u b e o f 0.23 m l / m i n . T o t a l s a m p l e v o l u m e r e q u i r e d for this assay is 0.4 ml.
288 s a m p l e s are a n a l y z e d w i t h o u t dialysis. At 30 s a m p l e s / h r t h e p e a k heights are 9 2 - - 9 4 % o f t h e p l a t e a u values o b t a i n e d b y c o n t i n u o u s s a m p l e s o f t h e s a m e s t a n d a r d . This is o n e i m p o r t a n t criterion o f t h e p e r f o r m a n c e of an autom a t e d s y s t e m . When a n a l y z i n g t h e samples with dialysis t h e p e a k values are 9 0 - - 9 1 % o f t h e p l a t e a u . With b o t h m a n i f o l d s y s t e m s the c a r r y - o v e r is 1.5 to 2.5% w h e n a n a l y z i n g 30 s a m p l e s / h r . These p e r f o r m a n c e values o f t h e s y s t e m are a m a r k e d i m p r o v e m e n t o v e r t h o s e o b t a i n e d using t h e m a n i f o l d w i t h t w o p u m p s p u b l i s h e d earlier (Siraganian, 1974). In t h e s y s t e m with 2 p u m p s t h e r e is 4 - - 5 % c a r r y - o v e r at 30 s a m p l e s / h r w h i c h d r o p s to a b o u t 1.0% at 20 s a m p l e s / h r . T h e p r e s e n t s y s t e m can be used to a n a l y z e s a m p l e s at t h e rate of 3 0 / h r w i t h t h e s a m e a c c u r a c y and precision as was possible at 2 0 / h r w i t h the previous system. Fig. 2 illustrates t h e d a t a o b t a i n e d on a n a l y z i n g h i s t a m i n e s t a n d a r d s witho u t dialysis in the 0 - - 1 0 n g / m l range. T h e r e is little v a r i a t i o n in the duplicates a n d t h e c a l i b r a t i o n curve is linear. T h e r e f o r e , in a n a l y z i n g e x p e r i m e n t a l s a m p l e s o n l y o n e s t a n d a r d is used. As t h e t o t a l v o l u m e a n a l y z e d is less t h a n 0.4 ml, t h e a m o u n t o f h i s t a m i n e a c t u a l l y a n a l y z e d is 0 . 0 5 - - 4 ng. The rec o r d e r readings o f h i s t a m i n e samples a n a l y z e d with dialysis is s h o w n in fig. 3. T h e h i s t a m i n e c o n t e n t o f t h e s t a n d a r d is 0 - - 2 5 n g / m l (total h i s t a m i n e 0 - - 1 5 rig). T h e c a l i b r a t i o n curve is again linear.
DOt ,~
25.0 ng/ml
::1 /
, Ii 11II l .,oo, ,AAI![ X] J
t
Fig. 3. Chart recording of histamine standards analyzed with dialysis. Standards were 0 25 ng/ml in 2% perehlorie acid. Analysis was at 30 samples per hour using a sample uptake of 0.32 ml/min. Total sample volume required for this assay is 0.6 ml.
289 T h e m e t h o d described in the p r e s e n t p a p e r is at least 100-fold m o r e sensitive t h a n t h e a u t o m a t e d t e c h n i q u e described b y Evans et al. ( 1 9 7 3 ) and has t h e f u r t h e r advantage o f handling samples with p r o t e i n . It is a b o u t 5 times as sensitive as the m e t h o d previously described b y Siraganian ( 1 9 7 4 ) and t h e rate o f analysis is increased to 30 samples per hour. The a u t o m a t e d f l u o r o m e t r i c t e c h n i q u e c o m p a r e s very f a v o r a b l y with t h e e n z y m e - i s o t o p i c assay f o r histamine. The isotopic t e c h n i q u e s described by S n y d e r et al. ( 1 9 6 6 ) and K o b a y a s h i and Maudsley ( 1 9 7 2 ) have a sensitivity of a b o u t 1 ng. The m e t h o d has been f u r t h e r refined b y T a y l o r and S n y d e r ( 1 9 7 2 ) and Levy and Widra ( 1 9 7 3 ) to measure h i s t a m i n e in t h e 0.1 to 2.0 ng range. This is a b o u t the same range as the f l u o r o m e t r i c t e c h n i q u e for analysis o f samples w i t h o u t dialysis. The a u t o m a t e d s y s t e m has the f u r t h e r advantages o f reproducibility, a u t o m a t i o n , ease o f o p e r a t i o n , and rapid analysis o f a large n u m ber o f samples.
REFERENCES Evans, D.P., J.A. Lewis and D.C. Thomason, 1973, Life Sci. 12, 327. I-~kanson, R., A.L. Ronnberg and K. Sjolund, 1972, Anal. Biochem. 47, 356. Kobayashi, Y. and D.V. Maudsley, 1972, Analyt. Biochem. 46, 85. Levy, D.A. and M. Widra, 1973, J. Lab. Clin. Med. 81, 291. Siraganian, R.P., 1974, Analyt. Biochem. 57, 383. Snyder, S.H., R.J. Baldessarini and J. Axelrod, 1966, J. Pharmacol. Exptl. Ther. 153, 544. Taylor, K.M. and S.H. Snyder, 1972, J. Neurochem. 19, 1343.
REFINEMENTS IN THE AUTOMATED FLUOROMETRIC HISTAMINE ANALYSIS SYSTEM
REUBEN P. SIRAGANIAN Clinical Immunology Section, Laboratory of Microbiology and Immunology, NIDR, National Institutes of Health, Bethesda, Md. 20014, U.S.A. (Received 10 August 1974, accepted 18 December 1974) The automated continuous flow system for the extraction and fluorometric analysis of histamine has been further improved. Samples with or without protein can be analyzed. The use of a single pump, added nitrogen, and lower concentration of o-phthalaldehyde increase the sensitivity by 5--10 fold and allow the analysis of samples at the rate of 30 per hour. Samples which are less than 0.5 ml in volume and contain 0.1 to 10 ng/ml histamine can routinely be analyzed. The sensitivity of the system is now 0.05 ng and compares favorably with isotopic techniques.
INTRODUCTION An a u t o m a t e d c o n t i n u o u s f l o w s y s t e m f o r t h e e x t r a c t i o n and f l u o r o m e t ric analysis o f h i s t a m i n e has b e e n described (Siraganian, 1974). The s y s t e m d e p e n d s o n t h e ability to separate o r g a n i c - a q u e o u s phases in the c o n t i n u o u s f l o w s y s t e m b y the use o f special e x t r a c t o r s . However, the s y s t e m as previo u s l y described required t w o p u m p s and its o p e r a t i o n was o p t i m a l at 20 samples per h o u r . When a n a l y z i n g samples at a faster rate there was considerable c a r r y - o v e r and c o n t a m i n a t i o n f r o m o n e sample to t h e next. The present p a p e r describes several m o d i f i c a t i o n s o f t h e s y s t e m including the use o f a single, greater c a p a c i t y p u m p , a m o r e sensitive f l u o r o m e t e r , and changes in t h e chemical reaction. The r e a c t i o n o f t h e f l u o r o p h o r e with h i s t a m i n e is p e r f o r m e d u n d e r n i t r o g e n w h i c h increases t h e sensitivity o f the r e a c t i o n (Hfikanson et al., 1972). The c o n c e n t r a t i o n o f o - p h t h a l a l d e h y d e (OPT) is decreased w h i c h i m p r o v e s the r e a g e n t b l a n k fluorescence. With the m o d i f i e d system, samples are a n a l y z e d at the rate o f 30 per h o u r with a very high degree o f sensitivity and a c c u r a c y . The p r e s e n t m o d i f i c a t i o n allows a 5-fold increase in sensitivity with 50% increase in t h e rate o f sample analysis. MATERIALS AND METHODS A p p a r a tus The f o l l o w i n g T e c h n i c o n m o d u l e s ( T e c h n i c o n Corp., T a r r y t o w n , N.Y.) were used: s a m p l e r t y p e II, p r o p o r t i o n i n g p u m p II, basic s y s t e m dialyzer
284 (37°C) a f l u o r o n e p h l o m e t e r and a single pen recorder. The f l u o r o m e t e r was equipped with the 013-B008-01 flow cell. The primary filter was the narrow pass 7-60 (peak at 350 nm). A sharp cut filter 2A plus a 4-72 (Corning) were used t o g e t h e r as the secondary filter.
Reagents The following reagents were used: saline (0.9% NaC1, 0.016% Brij-35, 1.5 X 10 -3 M EDTA), 30% NaC1 solution, 5 N NaOH, 1 N NaOH (containing 1 × 10 -3 M EDTA), 0.1 N HC1, 0.73 M phosphoric acid, n-butanol and heptane. The details of the preparation of these reagents have been described previously (Siraganian, 1974). The 30% NaC1 solution was filtered before use. A 0.5 M pH 12.3 borate buffer was prepared by dissolving 30.95 gm of boric acid in 900 ml water, adjusting the pH with 50% NaOH, and bringing the solution up to 1 liter. The o-phthalaldehyde (OPT) solution was changed f r o m the c o n c e n t r a t i o n previously used to 0.5 mg per ml. For a day's use, 50 mg OPT were dissolved in 2 ml methanol ( s p e c t r o p h o t o m e t r i c grade), and this solution was brought up to 100 ml with 0.5 M borate buffer, pH 12.3. It was kept at 0°C in a dark bottle in an ice bucket. The use of fresh OPT reduced the reagent black fluorescence and keeping it in ice resulted in a more stable baseline. Histamine standards were prepared in 2% perchloric acid. The c o n c e n t r a t i o n of histamine is stated for the base.
Procedure The flow diagram for the system, including the size of the different manifold tubes, the connectors, and the coils used on the manifold, is shown in fig. 1. The sample is diluted with saline and dialyzed against the same solution. To the dialyzed stream 30% NaC1 is added. After mixing, butanol and NaOH are added and the histamine is extracted by mixing into the butanol. The organic and aqueous phases are separated in the first extractor; to the organic phase 0.1 N HC1 and n-heptane are added. After mixing, the phases are separated in the second extractor. Both extractors are placed close to the inlet side of the manifold tubes on the right side of the pump. It is crucial t h a t the second e x t r a c t o r be very close to the inlet of the manifold tubes as this part o f the stream is not segmented by air bubbles. It is also i m p o r t a n t t h a t the end o f the waste tubes f r om bot h extractors are kept at the level of the middle o f the extractors, otherwise a negative pressure will be applied to this part o f the manifold with uneven flow characteristics. In the second extractor, the lower aqueous phase is removed and resegmented with bubbles. It is then alkalinized, OPT added, and after the optimal reaction time the histamine-o-phthalaldehyde reaction stopped with phosphoric acid. The stream then passes through the fluorometer. As indicated in fig. 1 Solvaflex tubing is used for those portions of the manifold which come in co n tact with organic solvents.
285
MANIFOLD TUBES POSITION SIZE INCHES (ml/min) 0045 (080) AIR WASTE ~
".'.'.'.-.'.'.','.,.'4~ ~L
Hi3
"
!
DIALYZER
A'
i~
(BASIC)
=
COIL ID_o D-O
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I
WASTE A
EXTRACTOR ~
(O-3)
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WASTE
I°
7-60
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2A ÷ 4-72
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2
2
0045
{080)
SALINE
0020
(016)
SAMPLE
0045 0051
(080) (I 00)
AIR + ~N SALINE +
0056
(I 20)
30% NaCI
0100
(289} W BUTANOL
0020
(016)
SAMPLER]I
5N NoOH
.......... {~ OlO0
7 (289~J EXTRACTOR I
D-O
(~
0075
(200)
0-0
(~
0090
(242) W HEPTANE
D-I ( ~ D-I~.~
0073 0.056
(l 69}*J EXTRACTOR (I 20) AIR ~N
0030
(0.32)
(016) W OPT
v
MIXING COIL
OIN HCL
105-0087
D-I
.
MIXING COIL
FLUOROMETER FLOW CELL 013- BO08-OI
D-I (~)
~ 4 6
EXTg~C~OR ~ [~ -
(~
~N
A-6'
WASTE ~
TO SAMPLER WASH RECEPTACLE : D
z
ION NeOH
(~
0020
,~
0.040
(060)
@.
0081
~,
- - 0051
(202) W FROM FLUOROMETER ] --(120) SALINE /
PHOSPHORIC
26 TUBE END BLOCKS
J
~SOLVAFLEX TUBING
Fig. 1. Flow diagram for histamine analysis. Manifold is shown for analysis with dialysis; for operation without dialysis points A and A p are connected and pump tubes marked (+) are removed. Extra sample tubes can be left on the pump at the unoccupied positions 7 (0.23 ml/min) and 10 (0.32 ml/min) for rapid changeover to analyze samples at other sensitivities. The first 3 coils have a 1.5 cm diameter but whereas coil No. 116-0246 is 10 turns with a 2.0 mm I.D. (internal diameter) both coils 105-0089 and 105-0087 are 28 turns each and have an I.D. of 2.4 mm. The last 2 coils (190-0051-10 is 40 turns and 190-0051-04 is 16 turns) are 3.0 cm in diameter with a 2 mm internal diameter. Part numbers are listed in the Technicon catalogue.
T h e t w o e x t r a c t o r s u s e d in t h e m a n i f o l d are c o n s t r u c t e d as p r e v i o u s l y d e s c r i b e d f r o m T e c h n i c o n m a n i f o l d part N - 6 and D - 3 (Siraganian, 1 9 7 4 ) . T h e t w o n i p p l e s ( N - 6 ) are p l a c e d at t h e e n d s o f t h e w i d e - b o r e c o n n e c t o r s ( D - 3 ) a n d n o t i n s i d e t h e s i d e - a r m s . T h e f i n e s t e e l w i r e l o o p is u s e d o n l y in t h e s e c o n d e x t r a c t o r and it f u n c t i o n s e f f e c t i v e l y t o b r e a k u p s m a l l b u b b l e s . T h e u s e o f n i t r o g e n i n s t e a d o f air t o s e g m e n t t h e s t r e a m i n c r e a s e s t h e s e n s i t i v i t y o f t h e s y s t e m b y 3 0 - - 5 0 % . N i t r o g e n is p u m p e d i n t o t h e e n d o f t h e 'air' t u b e s f r o m a t a n k u s i n g s e v e r a l p r e s s u r e r e d u c t i o n v a l v e s t o a l l o w gas t o e s c a p e at a b o u t 1 0 m l / m i n . A n o v e r f l o w f r o m t h e n i t r o g e n t u b e a l l o w s t h e e x c e s s gas t o e s c a p e . A l t h o u g h t h e e f f e c t o f n i t r o g e n is o n l y o n t h e o - p h t h a l a l d e h y d e - - h i s t a m i n e r e a c t i o n , t h e m a n i f o l d in r o u t i n e u s e has n i t r o g e n rep l a c i n g air at all t h e p u m p p o i n t s . The flow system can be modified to increase the sensitivity when analyzing h i s t a m i n e in s a m p l e s w h i c h c o n t a i n l i t t l e p r o t e i n . T h e s e s a m p l e s are
286 a n a l y z e d w i t h o u t d i a l y s i s . T h i s m o d i f i c a t i o n is d e s c r i b e d in fig. 1. T h e t o t a l t i m e r e q u i r e d f o r a s a m p l e t o r u n t h r o u g h t h e m a n i f o l d is 1 6 m i n w i t h d i a l y s i s a n d 11 m i n w i t h o u t d i a l y s i s .
Manifold changes The basic manifold can easily be modified to analyze different volume samples. In general, increasing the sample uptake volume will increase the s e n s i t i v i t y o f t h e a s s a y . A s u m m a r y o f t h e p o s s i b l e c h a n g e s is s h o w n in t a b l e 1. T h e m a n i f o l d is u s e d w i t h o u t d i a l y s i s w h e n t h e p r o t e i n c o n c e n t r a t i o n in t h e s a m p l e s is less t h a n 3 . 0 m g / m l , e.g., as in T r i s A o r T r i s A C M buffers (Levy and Widra, 1973). The total volume of samples required for
analysis in this system is 0.3--0.6 ml; and histamine concentrations of 7--25 ng/ml result in full scale deflection. The amount of histamine that can be quantitated is usually 0.5 to 1% of the concentration that results in full scale deflection and therefore about I00 pg/ml. For routine use in assays of histamine release from washed human leukocytes the manifold with the 0.23 ml/min sample tube is used. W h e n s a m p l e s a r e a n a l y z e d u s i n g t h e d i a l y z e r t h e s e n s i t i v i t y is d e c r e a s e d
TABLE 1 Manifold parameters for analyzing samples with different volumes and varying histamine content. Manifold
Sample uptake tube
Sample volume required a
I.D. Flow rate (inches) (ml/min)
(ml)
Histamine % of Plateau c concentration for full scale deflection b (ng/ml)
I. Without dialysis (protein less than 3 mg/ml)
0.020 0.025* 0.030
0.16 0.23 0.32
0.3 0.4 0.6
25 10 7
93 94 92
II. With dialysis
0.020 0.025 0.030*
0.16 0.23 0.32
0.3 0.4 0.6
75 30 20
91 91 90
Samples analyzed at the rate of 30/hr; nitrogen used instead of air. Sensitivity will decrease by 35% if air is used in place of N2. * Manifold used most extensively with these two sample uptake tubes. a Total volume required in the sample cup. Actual volume analyzed is about 25% less. b Concentration of histamine standard that will give full scale deflection in the recorder; the readability of the graph is 0.5--1% of this value. The amount of histamine that can be quantitated is therefore in the 70--200 pg range. c Height of peak as percentage of plateau obtained by sampling the same standard for 5 min.
287
by about 70% (table 1). When the histamine content of the samples is low, a larger sample is analyzed to compensate for the decreased sensitivity. With dialysis, histamine concentrations of 20--75 ng/ml result in full scale deflection. The manifold with the 0.32 ml/min sample uptake tube is used extensively with samples containing large amounts of plasma and little histamine. Extra sample tubes are left on the pump for rapid changeover to analyze samples with different volumes. The manifold pump tube from extractor I is changed every 20 hr of machine operation time. The o-phthalaldehyde and sample uptake pump tubes are changed every 40 hr and all the manifold pump tubes after 160 hr of operation. These changes are important when analyzing samples at high sensitivity settings. RESULTS AND DISCUSSION
A summary of the sensitivity of the system when used under different conditions is shown in table 1. The system is slightly more accurate when
I0.0 ng/ml 80
~g: 4 U
2
7.5 ng/ml
o~"
,I!
_..L._.~,
2 4 6 8 !0 HISTAMINE ng/rnl 5.0 ng/ml
V
Fig. 2. C h a r t r e c o r d i n g o f h i s t a m i n e s t a n d a r d s a n a l y z e d w i t h o u t dialysis. S t a n d a r d s were 0 - - 1 0 n g / m l in 2% p e r c h l o r i c acid. Analysis was at 30 samples p e r h o u r using a s a m p l e u p t a k e t u b e o f 0.23 m l / m i n . T o t a l s a m p l e v o l u m e r e q u i r e d for this assay is 0.4 ml.
288 s a m p l e s are a n a l y z e d w i t h o u t dialysis. At 30 s a m p l e s / h r t h e p e a k heights are 9 2 - - 9 4 % o f t h e p l a t e a u values o b t a i n e d b y c o n t i n u o u s s a m p l e s o f t h e s a m e s t a n d a r d . This is o n e i m p o r t a n t criterion o f t h e p e r f o r m a n c e of an autom a t e d s y s t e m . When a n a l y z i n g t h e samples with dialysis t h e p e a k values are 9 0 - - 9 1 % o f t h e p l a t e a u . With b o t h m a n i f o l d s y s t e m s the c a r r y - o v e r is 1.5 to 2.5% w h e n a n a l y z i n g 30 s a m p l e s / h r . These p e r f o r m a n c e values o f t h e s y s t e m are a m a r k e d i m p r o v e m e n t o v e r t h o s e o b t a i n e d using t h e m a n i f o l d w i t h t w o p u m p s p u b l i s h e d earlier (Siraganian, 1974). In t h e s y s t e m with 2 p u m p s t h e r e is 4 - - 5 % c a r r y - o v e r at 30 s a m p l e s / h r w h i c h d r o p s to a b o u t 1.0% at 20 s a m p l e s / h r . T h e p r e s e n t s y s t e m can be used to a n a l y z e s a m p l e s at t h e rate of 3 0 / h r w i t h t h e s a m e a c c u r a c y and precision as was possible at 2 0 / h r w i t h the previous system. Fig. 2 illustrates t h e d a t a o b t a i n e d on a n a l y z i n g h i s t a m i n e s t a n d a r d s witho u t dialysis in the 0 - - 1 0 n g / m l range. T h e r e is little v a r i a t i o n in the duplicates a n d t h e c a l i b r a t i o n curve is linear. T h e r e f o r e , in a n a l y z i n g e x p e r i m e n t a l s a m p l e s o n l y o n e s t a n d a r d is used. As t h e t o t a l v o l u m e a n a l y z e d is less t h a n 0.4 ml, t h e a m o u n t o f h i s t a m i n e a c t u a l l y a n a l y z e d is 0 . 0 5 - - 4 ng. The rec o r d e r readings o f h i s t a m i n e samples a n a l y z e d with dialysis is s h o w n in fig. 3. T h e h i s t a m i n e c o n t e n t o f t h e s t a n d a r d is 0 - - 2 5 n g / m l (total h i s t a m i n e 0 - - 1 5 rig). T h e c a l i b r a t i o n curve is again linear.
DOt ,~
25.0 ng/ml
::1 /
, Ii 11II l .,oo, ,AAI![ X] J
t
Fig. 3. Chart recording of histamine standards analyzed with dialysis. Standards were 0 25 ng/ml in 2% perehlorie acid. Analysis was at 30 samples per hour using a sample uptake of 0.32 ml/min. Total sample volume required for this assay is 0.6 ml.
289 T h e m e t h o d described in the p r e s e n t p a p e r is at least 100-fold m o r e sensitive t h a n t h e a u t o m a t e d t e c h n i q u e described b y Evans et al. ( 1 9 7 3 ) and has t h e f u r t h e r advantage o f handling samples with p r o t e i n . It is a b o u t 5 times as sensitive as the m e t h o d previously described b y Siraganian ( 1 9 7 4 ) and t h e rate o f analysis is increased to 30 samples per hour. The a u t o m a t e d f l u o r o m e t r i c t e c h n i q u e c o m p a r e s very f a v o r a b l y with t h e e n z y m e - i s o t o p i c assay f o r histamine. The isotopic t e c h n i q u e s described by S n y d e r et al. ( 1 9 6 6 ) and K o b a y a s h i and Maudsley ( 1 9 7 2 ) have a sensitivity of a b o u t 1 ng. The m e t h o d has been f u r t h e r refined b y T a y l o r and S n y d e r ( 1 9 7 2 ) and Levy and Widra ( 1 9 7 3 ) to measure h i s t a m i n e in t h e 0.1 to 2.0 ng range. This is a b o u t the same range as the f l u o r o m e t r i c t e c h n i q u e for analysis o f samples w i t h o u t dialysis. The a u t o m a t e d s y s t e m has the f u r t h e r advantages o f reproducibility, a u t o m a t i o n , ease o f o p e r a t i o n , and rapid analysis o f a large n u m ber o f samples.
REFERENCES Evans, D.P., J.A. Lewis and D.C. Thomason, 1973, Life Sci. 12, 327. I-~kanson, R., A.L. Ronnberg and K. Sjolund, 1972, Anal. Biochem. 47, 356. Kobayashi, Y. and D.V. Maudsley, 1972, Analyt. Biochem. 46, 85. Levy, D.A. and M. Widra, 1973, J. Lab. Clin. Med. 81, 291. Siraganian, R.P., 1974, Analyt. Biochem. 57, 383. Snyder, S.H., R.J. Baldessarini and J. Axelrod, 1966, J. Pharmacol. Exptl. Ther. 153, 544. Taylor, K.M. and S.H. Snyder, 1972, J. Neurochem. 19, 1343.
