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Repeated skin tests in guinea pigs with fluorescein visualization
Journal oflmmunologicalMethods,
63 (1983) 221-228
221
Elsevier JIM02763
Repeated Skin Tests in Guinea Pigs with Fluorescein Visualization James H. Wells, Megan R. Lerner, Richard A. Strecker and William A. Cain Department of Medicine and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, U.S.A.
(Received 24 September 1982, accepted 18 April 1983)
A method is presented for immediate hypersensitivityskin testing of guinea pigs using fluorescein as a contrast medium. The method allows repeated skin tests of individual animals. Results are compared with those obtained using Evans blue dye, which leaves animals unsuitable for later skin testing. The two methods give very similar direct skin test results. Key words: skin
test - - fluorescein - - guinea pig - - Evans blue dye - - immediate hypersensitivity
Introduction Skin testing for cytotropic antibodies in guinea pigs, both directly and by the indirect method of passive cutaneous anaphylaxis (PCA), is difficult to assess without a contrast medium. The most frequently employed contrast medium is Evans blue dye administered by intravenous injection at or shortly before the time of injection of antigen (Ovary, 1958). We have been engaged in studies of immediate respiratory hypersensitivity in guinea pigs, attempting to correlate both physiological and immunological variables (Cox et al., 1979; Pennock et al. 1979, 1980; Cain et al., 1980). Direct skin tests are a relatively sensitive index of early sensitization. Positive direct skin tests often occur even before reaction to antigen aerosol challenge can be detected using body plethysmography and before histamine released by antigen is detectable in the chopped lung assay as we perform it (Cain et al., 1980). In many of our studies, for example during active sensitization with more than one antigen, sequential direct skin testing is desirable. However, skin testing with Evans blue dye can be carried out only once. Thereafter, the dye, which initially attaches mainly to serum protein, stains other tissues so that subsequent Evans blue dye injections no longer provide adequate contrast. Fluorescein was first administered intravenously by Erlich (1881). More than 50 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
222 years ago, it was shown that intravenous fluorescein would cause skin luminescence when viewed in ultraviolet light (Wollheim and Lang, 1931). While studying human peripheral vascular disease, Neller and Schmidt (1945) reported that following intravenous fluorescein injection, a histamine skin test site would fluoresce brightly unless the circulation to the area was severely compromised. Fluorescein has also been used for opthalmological research (Maurice, 1967). The dye is generally considered to be safe, although exceptions to this have been reported by Stein and Parker (1971). This report describes a method for skin testing guinea pigs with fluorescein. It has proven to be safe, inexpensive, simple, and can be repeated several times per week if desired. In guinea pigs, when the dye is injected immediately after the skin tests are applied, the wheal (positive reaction) is non-luminescent under ultraviolet light and is measured against a bright background of luminescent normal skin. Quantitative direct skin tests with histamine and antigen, comparing fluorescein with Evans blue dye, indicate that similar reaction measurements are obtained with these 2 contrast media.
Materials andMethods
Experimental design Ten guinea pigs were randomly assigned to 2 groups of 5. One group received 2-weekly sensitizing exposures by inhalation of ovalbumin. On the third week this group received quantitative direct skin tests on flanks with ovalbumin on each of 2 successive days. The other (unsensitized) group was similarly tested with histamine. Fluorescein and one side of each animal were used on the first skin test day, while Evans blue dye and the contralateral side were employed on the second day.
Animals Adult, female Hartley strain guinea pigs (Charles Rivers) initially weighing 350-400 g were used in this study.
Sensitization Five guinea pigs were sensitized by 2-weekly exposures to aerosols of 1 % ovalbumin (fraction V; Sigma Chemical Company) in a Model A4 Airbone Infection Apparatus (Tri-R Instruments, Jamaica, NY) as previously described (Cain et al., 1980). Briefly, 3 ml were nebulized for 10 min, the apparatus turned off for 30 min, and the vacuum then run for 10 min to flush the chamber prior to removing the animals.
Reagents Fluorescein sodium salt (Sigma Chemical Company) and Evans blue dye (Fisher Scientific Company) were prepared as 5% solutions in 0.85% physiological saline and were filter-sterilized through 0.20 p m Nalgene filter units. The final pH of each solution was 8.5.
223 Fraction V ovalbumin and histamine dihydrochloride (Sigma Chemical Company) were prepared as stock solutions (100 /~g/ml and 1 m g / m l respectively in 0.85% saline) and filter-sterilized through 0.20/~m Nalgene filter units. Quantitative direct skin tests Prior to all skin tests, animals were shaved and residual hair removed by use of a depilatory. Animals were anesthetized with ketamine and Rompun as previously described (Cain et al., 1980). For the fluorescein dye direct skin test, 7 sites on the animal's back were injected intradermally along one side with 0.05 ml of 5-fold dilutions of ovalbumin (at a starting concentration of 100/~g/ml) or 5-fold dilutions of histamine dihydrochloride (at a starting concentration of 1000/~g/ml), and an eighth site received a saline control. One animal was tested for photographic purposes only (Fig. 1), with histamine (1000/~g/ml, 40 # g / m l , 1.6/xg/ml) and saline. Following completion of test site injections, the animal received an intracardiac injection of 1.0 ml of 5% fluorescein dye in saline. Skin test sites were measured with calipers immediately after completion of dye injection. Reactions were measured in a darkened room,
Fig. 1. Skin tests with fluorescein in ultraviolet light. Histamine skin test doses decrease from left to
(1000 f,g/ml, 40 pg/ml, 1.6/~g/ml) followedby saline.
right
224 using a light source in the ultraviolet range of 360 nm. Positive reactions were non-luminescent compared to luminescent surrounding skin. For the Evans blue dye skin tests animals received intracardiac administration of 1.0 ml of 5% Evans blue dye in saline prior to application of the skin tests. Thereafter skin tests were applied as with fluorescein testing as described above. The contralateral side was used with each dilution placed in the same relative location as with the previous fluorescein testing. Blue reaction diameters (mm) were measured in white light 20-30 min after completion of test site injections. Saline injections were used with both methods as a negative control. Diameters were subtracted from the Evans blue measurements as background. Positive reactions (non-luminescent) were never detected at saline injection sites by the fluorescein skin test. Indeed, sites injected with saline were usually hyperfluorescent.
Histopathology Animals were anesthetized as above. Biopsies of skin test sites were taken under sterile conditions at 6 h and 24 h intervals. Tissues were processed and embedded in paraffin. Sections were stained with hematoxylin and eosin or May-Griinwald giemsa.
Plasma fluorescence To demonstrate the time course of elimination of fluorescein, plasma samples were obtained. Blood was collected in heparinized tubes by cardiac puncture before fluorescein injection, 15 min later, and 1,4 and 24 h after dye injection. Plasma was separated by centrifugation and fluorescence determined with an Aminco-Bowman spectrophotofluorometer (excitation 332 nm; emission 540 nm).
Statistical methods Linear regressions of wheal diameters vs. log concentrations of both antigen and histamine reactions were computed using a Data General Eclypse M / 6 0 0 with I / O devices connected to an IBM 370/158. Programs were from BMDP (Biomedical computer programs, P series (Chasen, 1979)). The t statistic for comparison of slopes, intercepts, and correlation coefficients were those suggested by Glantz (1981).
Results
A photograph of histamine tests performed with the fluorescein method is shown in Fig. 1. Fig. 2 compares histamine dose-response curves, and Fig. 3 does the same for the ovalbumin data. Coefficients of determination were 0.85 (Evans blue data) and 0.86 (fluorescein data) for log histamine concentration versus reaction diameter. By paired t-tests neither the slopes nor the intercepts of these relationships were significantly different ( P > 0.05).
225 10
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6
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;/
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,
2.'" -
0
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/
~
c.
!
0.084
oh2
I
!
• 1.6 ' 8.0 40.0 HISTAMINE CONCENTRATION (~g/ml)
2~0
l&O
Fig. 2. Comparisons of wheal sizes obtained with different histamine concentrations using either Evans blue dye (O. . . . . . O) or fluorescein (O ©) to visualize the reactions. The linear regression lines fitted by the method of least squares are: for fluorescein, y = 1.9 log(x)+3.1, the coefficient of determination (r 2) is 0.86; for Evans blue dye, y = 1.7 log(x)+ 2.4, the coefficient of determination (r 2) is 0.85.
10 9
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0
2_
~•
0
F
~
~
,
•
•O
1 0
~ ~.00164
~ 0.~32
' ' 0.16 0.80 410 ANTIGEN CONCENTRATION ~g/ml
, 20.0
I 100
Fig. 3. Comparisons of wheal sizes obtained with different antigen concentrations using either Evans blue dye (O. . . . . . O) or fluorescein ( O O) to visualize the reactions. The linear regression lines fitted by method of least squares are: for fluorescein, y = 1.7 log(x)+4.5, the coefficient of determination (r 2) is 0.79; Evans blue dye, y = 1.2 log(x)+3.5, the coefficient of determination (r 2) is 0.71.
226 10
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;
8
,
10
,
11
,
12
EVANS BLUE WHEAL DIAMETER (ram)
Fig. 4. C o r r e l a t i o n of fluorescein visualized a n d E v a n s blue dye wheal sizes o b t a i n e d with given h i s t a m i n e c o n c e n t r a t i o n s on the same a n i m a l s (y = 0.95 ( x ) + 1.12; r = 0.85).
With the antigen dose versus reaction size data (Fig. 3), coefficients of determination were 0.71 (Evans blue method) and 0.79 (fluorescein method). These data showed that the slope of the curve obtained by the fluorescein method was significantly greater than that obtained by the Evans blue method (P < 0.05, paired t-test). Similarly, the intercept of the curve obtained with the fluorescein method was 10.
v
8.
~6-
,~.
•
o
• ~/o,/o
~:4_ z
g2. u. 1
2
4
5
7
8
9
1
11
12
EVANS BLUE WHEAL DIAMETER( r am)
Fig. 5. C o r r e l a t i o n of visualized fluorescein a n d E v a n s blue dye wheal sizes o b t a i n e d with given antigen c o n c e n t r a t i o n s o n the s a m e a n i m a l s (y = 1.2 (x)+0.0004; r = 0.92),
227 30.
-- 2 0
== P O
_= u. 10 ==
,
I
I
0 0.25 1 4 24 T i m e A f t e r Fluorescein Injection ( h )
Fig. 6. Clearance of fluorescence from plasma with time.
significantly less than that obtained with Evans blue ( P < 0.05, paired t-test). Skin test measurements made from tests employing the same doses of histamine in the same animal, comparing fluorescein vs. Evans blue dye are shown in Fig. 4. Similar data for ovalbumin are displayed in Fig. 5. Note that by this method of comparison, also, the fluorescein method appears to be slightly more sensitive (slope 1.2) with antigen but not with histamine (slope 0.95). Correlation coefficients of these comparisons indicate a high strength of association. Biopsies taken from positive skin test reactions to ovalbumin show edema and a sparse non-specific cellular reaction 6 h after antigen injection. After 24 h, a predominantly eosinophil-marked cellular reaction is found. The time course of plasma fluorescence clearance after intravenous fluorescein injection appears in Fig. 6. Fluorescein had disappeared from plasma after 24 h. Skin fluorescence clears within 4 h. Discussion These findings seem to indicate that fluorescein is a reasonable alternative to Evans blue dye for visualization of skin reactions in guinea pigs. The fluorescein assay was slightly more sensitive with antigen but not with histamine. However, results obtained with fluorescein and Evans blue dyes were essentially very similar, and animals could be retested soon after a previous test with fluorescein. The difference in mechanisms by which Evans blue dye and fluorescein appear to operate is of interest. Evans blue dye, initially bound to serum albumin, stains skin in areas of increased local vascular permeability (i.e., positive test sites). Fluorescein, injected immediately after application of the skin test, is apparently excluded from positive skin test sites. The dose of fluorescein routinely employed by us has been 0.5-1.0 ml of a 5% solution with no toxicity noted. As much as 10 ml has been injected without altering
228 test results. O n the other hand, such high doses of fluorescein serve n o useful p u r p o s e a n d can cause toxic reactions in guinea pigs due to renal t u b u l a r necrosis. While the use of somewhat larger t h a n needed a m o u n t s of fluorescein does n o t cause e x p e r i m e n t a l problems, excessive Evans blue dye doses render skin tests difficult to measure. I n our protocols we have repeated fluorescein injections at weekly intervals for up to 6 weeks i n over 100 a n i m a l s with n o a p p a r e n t harm. Fluorescein appears potentially a d a p t a b l e to a variety of skin test procedures. G o o d PCA results in guinea pigs have b e e n obtained, although we have n o t rigorously c o m p a r e d Evans blue dye a n d fluorescein P C A assays.
Acknowledgement The authors gratefully acknowledge the s u p p o r t of Dr. G,L. W i n n . This work was also supported i n part b y the Veterans A d m i n i s t r a t i o n a n d by O k l a h o m a Allergy Clinic, Inc.
References Cain, W.A., C.P. Cox, B.E. Pennock and J.H. Wells, 1980, Int. Arch. Allergy Appl. Immunol. 63, 361. Chasen, S., 1979, in: BMDP-79: BiomedicalComputer Programs P Series, eds. W.S. Dixon, M.B. Brown, L. Engelman, J.W. Franc and R.I. Jennrick (University of California Press, Berkeley, CA) pp. 230-244. Cox, C.P., W.A. Cain, B.E. Pennock and J.H. Wells, 1979, Fed. Proc. 38, 931. Erlich, P., 1881, Dtsch. Med. Wochenschr. 2, 21. Glantz, S.A., 1981, in: Primer of Biostatistics (McGraw-Hill, New York) pp. 180-228. Maurice, D.M., 1967, Invest. Ophthalmol. Visual Sci. 6, 464. Neller, J.L. and E.R. Schmidt, 1945, Ann. Surg. 121,328. Ovary, Z., 1958, Progr. Allergy 5, 459. Pennock, B.E., W.A. Cain, R.M. Rogers and J.H. Wells, 1980, Am. Rev. Resp. Dis. 121, 88. Pennock, B.E., C.P. Cox, R.M. Rogers, W.A. Cain and J.H. Wells, 1979, J. Appl. Physiol. 46, 399. Stein, M.R. and C.W. Parker, 1971, Am. J. Ophthalmol. 72, 861. Wollheim, E. and K. Lang, 1931, Verh. Dtsch. Ges. Inn. Med. 43, 134.
63 (1983) 221-228
221
Elsevier JIM02763
Repeated Skin Tests in Guinea Pigs with Fluorescein Visualization James H. Wells, Megan R. Lerner, Richard A. Strecker and William A. Cain Department of Medicine and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, U.S.A.
(Received 24 September 1982, accepted 18 April 1983)
A method is presented for immediate hypersensitivityskin testing of guinea pigs using fluorescein as a contrast medium. The method allows repeated skin tests of individual animals. Results are compared with those obtained using Evans blue dye, which leaves animals unsuitable for later skin testing. The two methods give very similar direct skin test results. Key words: skin
test - - fluorescein - - guinea pig - - Evans blue dye - - immediate hypersensitivity
Introduction Skin testing for cytotropic antibodies in guinea pigs, both directly and by the indirect method of passive cutaneous anaphylaxis (PCA), is difficult to assess without a contrast medium. The most frequently employed contrast medium is Evans blue dye administered by intravenous injection at or shortly before the time of injection of antigen (Ovary, 1958). We have been engaged in studies of immediate respiratory hypersensitivity in guinea pigs, attempting to correlate both physiological and immunological variables (Cox et al., 1979; Pennock et al. 1979, 1980; Cain et al., 1980). Direct skin tests are a relatively sensitive index of early sensitization. Positive direct skin tests often occur even before reaction to antigen aerosol challenge can be detected using body plethysmography and before histamine released by antigen is detectable in the chopped lung assay as we perform it (Cain et al., 1980). In many of our studies, for example during active sensitization with more than one antigen, sequential direct skin testing is desirable. However, skin testing with Evans blue dye can be carried out only once. Thereafter, the dye, which initially attaches mainly to serum protein, stains other tissues so that subsequent Evans blue dye injections no longer provide adequate contrast. Fluorescein was first administered intravenously by Erlich (1881). More than 50 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
222 years ago, it was shown that intravenous fluorescein would cause skin luminescence when viewed in ultraviolet light (Wollheim and Lang, 1931). While studying human peripheral vascular disease, Neller and Schmidt (1945) reported that following intravenous fluorescein injection, a histamine skin test site would fluoresce brightly unless the circulation to the area was severely compromised. Fluorescein has also been used for opthalmological research (Maurice, 1967). The dye is generally considered to be safe, although exceptions to this have been reported by Stein and Parker (1971). This report describes a method for skin testing guinea pigs with fluorescein. It has proven to be safe, inexpensive, simple, and can be repeated several times per week if desired. In guinea pigs, when the dye is injected immediately after the skin tests are applied, the wheal (positive reaction) is non-luminescent under ultraviolet light and is measured against a bright background of luminescent normal skin. Quantitative direct skin tests with histamine and antigen, comparing fluorescein with Evans blue dye, indicate that similar reaction measurements are obtained with these 2 contrast media.
Materials andMethods
Experimental design Ten guinea pigs were randomly assigned to 2 groups of 5. One group received 2-weekly sensitizing exposures by inhalation of ovalbumin. On the third week this group received quantitative direct skin tests on flanks with ovalbumin on each of 2 successive days. The other (unsensitized) group was similarly tested with histamine. Fluorescein and one side of each animal were used on the first skin test day, while Evans blue dye and the contralateral side were employed on the second day.
Animals Adult, female Hartley strain guinea pigs (Charles Rivers) initially weighing 350-400 g were used in this study.
Sensitization Five guinea pigs were sensitized by 2-weekly exposures to aerosols of 1 % ovalbumin (fraction V; Sigma Chemical Company) in a Model A4 Airbone Infection Apparatus (Tri-R Instruments, Jamaica, NY) as previously described (Cain et al., 1980). Briefly, 3 ml were nebulized for 10 min, the apparatus turned off for 30 min, and the vacuum then run for 10 min to flush the chamber prior to removing the animals.
Reagents Fluorescein sodium salt (Sigma Chemical Company) and Evans blue dye (Fisher Scientific Company) were prepared as 5% solutions in 0.85% physiological saline and were filter-sterilized through 0.20 p m Nalgene filter units. The final pH of each solution was 8.5.
223 Fraction V ovalbumin and histamine dihydrochloride (Sigma Chemical Company) were prepared as stock solutions (100 /~g/ml and 1 m g / m l respectively in 0.85% saline) and filter-sterilized through 0.20/~m Nalgene filter units. Quantitative direct skin tests Prior to all skin tests, animals were shaved and residual hair removed by use of a depilatory. Animals were anesthetized with ketamine and Rompun as previously described (Cain et al., 1980). For the fluorescein dye direct skin test, 7 sites on the animal's back were injected intradermally along one side with 0.05 ml of 5-fold dilutions of ovalbumin (at a starting concentration of 100/~g/ml) or 5-fold dilutions of histamine dihydrochloride (at a starting concentration of 1000/~g/ml), and an eighth site received a saline control. One animal was tested for photographic purposes only (Fig. 1), with histamine (1000/~g/ml, 40 # g / m l , 1.6/xg/ml) and saline. Following completion of test site injections, the animal received an intracardiac injection of 1.0 ml of 5% fluorescein dye in saline. Skin test sites were measured with calipers immediately after completion of dye injection. Reactions were measured in a darkened room,
Fig. 1. Skin tests with fluorescein in ultraviolet light. Histamine skin test doses decrease from left to
(1000 f,g/ml, 40 pg/ml, 1.6/~g/ml) followedby saline.
right
224 using a light source in the ultraviolet range of 360 nm. Positive reactions were non-luminescent compared to luminescent surrounding skin. For the Evans blue dye skin tests animals received intracardiac administration of 1.0 ml of 5% Evans blue dye in saline prior to application of the skin tests. Thereafter skin tests were applied as with fluorescein testing as described above. The contralateral side was used with each dilution placed in the same relative location as with the previous fluorescein testing. Blue reaction diameters (mm) were measured in white light 20-30 min after completion of test site injections. Saline injections were used with both methods as a negative control. Diameters were subtracted from the Evans blue measurements as background. Positive reactions (non-luminescent) were never detected at saline injection sites by the fluorescein skin test. Indeed, sites injected with saline were usually hyperfluorescent.
Histopathology Animals were anesthetized as above. Biopsies of skin test sites were taken under sterile conditions at 6 h and 24 h intervals. Tissues were processed and embedded in paraffin. Sections were stained with hematoxylin and eosin or May-Griinwald giemsa.
Plasma fluorescence To demonstrate the time course of elimination of fluorescein, plasma samples were obtained. Blood was collected in heparinized tubes by cardiac puncture before fluorescein injection, 15 min later, and 1,4 and 24 h after dye injection. Plasma was separated by centrifugation and fluorescence determined with an Aminco-Bowman spectrophotofluorometer (excitation 332 nm; emission 540 nm).
Statistical methods Linear regressions of wheal diameters vs. log concentrations of both antigen and histamine reactions were computed using a Data General Eclypse M / 6 0 0 with I / O devices connected to an IBM 370/158. Programs were from BMDP (Biomedical computer programs, P series (Chasen, 1979)). The t statistic for comparison of slopes, intercepts, and correlation coefficients were those suggested by Glantz (1981).
Results
A photograph of histamine tests performed with the fluorescein method is shown in Fig. 1. Fig. 2 compares histamine dose-response curves, and Fig. 3 does the same for the ovalbumin data. Coefficients of determination were 0.85 (Evans blue data) and 0.86 (fluorescein data) for log histamine concentration versus reaction diameter. By paired t-tests neither the slopes nor the intercepts of these relationships were significantly different ( P > 0.05).
225 10
O 9 8
O 0 0 ~
E 0
~
~
•
i. ~
0" 0/
6
D-
~< s •
4
~ "
;/
/ "
qmO
3
,
2.'" -
0
!
/
~
c.
!
0.084
oh2
I
!
• 1.6 ' 8.0 40.0 HISTAMINE CONCENTRATION (~g/ml)
2~0
l&O
Fig. 2. Comparisons of wheal sizes obtained with different histamine concentrations using either Evans blue dye (O. . . . . . O) or fluorescein (O ©) to visualize the reactions. The linear regression lines fitted by the method of least squares are: for fluorescein, y = 1.9 log(x)+3.1, the coefficient of determination (r 2) is 0.86; for Evans blue dye, y = 1.7 log(x)+ 2.4, the coefficient of determination (r 2) is 0.85.
10 9
GO 7_ E m / ..¢ / r~ ..J < 3_
0
2_
~•
0
F
~
~
,
•
•O
1 0
~ ~.00164
~ 0.~32
' ' 0.16 0.80 410 ANTIGEN CONCENTRATION ~g/ml
, 20.0
I 100
Fig. 3. Comparisons of wheal sizes obtained with different antigen concentrations using either Evans blue dye (O. . . . . . O) or fluorescein ( O O) to visualize the reactions. The linear regression lines fitted by method of least squares are: for fluorescein, y = 1.7 log(x)+4.5, the coefficient of determination (r 2) is 0.79; Evans blue dye, y = 1.2 log(x)+3.5, the coefficient of determination (r 2) is 0.71.
226 10
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5.
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~,
* 4
i 5
;
-~
,
;
8
,
10
,
11
,
12
EVANS BLUE WHEAL DIAMETER (ram)
Fig. 4. C o r r e l a t i o n of fluorescein visualized a n d E v a n s blue dye wheal sizes o b t a i n e d with given h i s t a m i n e c o n c e n t r a t i o n s on the same a n i m a l s (y = 0.95 ( x ) + 1.12; r = 0.85).
With the antigen dose versus reaction size data (Fig. 3), coefficients of determination were 0.71 (Evans blue method) and 0.79 (fluorescein method). These data showed that the slope of the curve obtained by the fluorescein method was significantly greater than that obtained by the Evans blue method (P < 0.05, paired t-test). Similarly, the intercept of the curve obtained with the fluorescein method was 10.
v
8.
~6-
,~.
•
o
• ~/o,/o
~:4_ z
g2. u. 1
2
4
5
7
8
9
1
11
12
EVANS BLUE WHEAL DIAMETER( r am)
Fig. 5. C o r r e l a t i o n of visualized fluorescein a n d E v a n s blue dye wheal sizes o b t a i n e d with given antigen c o n c e n t r a t i o n s o n the s a m e a n i m a l s (y = 1.2 (x)+0.0004; r = 0.92),
227 30.
-- 2 0
== P O
_= u. 10 ==
,
I
I
0 0.25 1 4 24 T i m e A f t e r Fluorescein Injection ( h )
Fig. 6. Clearance of fluorescence from plasma with time.
significantly less than that obtained with Evans blue ( P < 0.05, paired t-test). Skin test measurements made from tests employing the same doses of histamine in the same animal, comparing fluorescein vs. Evans blue dye are shown in Fig. 4. Similar data for ovalbumin are displayed in Fig. 5. Note that by this method of comparison, also, the fluorescein method appears to be slightly more sensitive (slope 1.2) with antigen but not with histamine (slope 0.95). Correlation coefficients of these comparisons indicate a high strength of association. Biopsies taken from positive skin test reactions to ovalbumin show edema and a sparse non-specific cellular reaction 6 h after antigen injection. After 24 h, a predominantly eosinophil-marked cellular reaction is found. The time course of plasma fluorescence clearance after intravenous fluorescein injection appears in Fig. 6. Fluorescein had disappeared from plasma after 24 h. Skin fluorescence clears within 4 h. Discussion These findings seem to indicate that fluorescein is a reasonable alternative to Evans blue dye for visualization of skin reactions in guinea pigs. The fluorescein assay was slightly more sensitive with antigen but not with histamine. However, results obtained with fluorescein and Evans blue dyes were essentially very similar, and animals could be retested soon after a previous test with fluorescein. The difference in mechanisms by which Evans blue dye and fluorescein appear to operate is of interest. Evans blue dye, initially bound to serum albumin, stains skin in areas of increased local vascular permeability (i.e., positive test sites). Fluorescein, injected immediately after application of the skin test, is apparently excluded from positive skin test sites. The dose of fluorescein routinely employed by us has been 0.5-1.0 ml of a 5% solution with no toxicity noted. As much as 10 ml has been injected without altering
228 test results. O n the other hand, such high doses of fluorescein serve n o useful p u r p o s e a n d can cause toxic reactions in guinea pigs due to renal t u b u l a r necrosis. While the use of somewhat larger t h a n needed a m o u n t s of fluorescein does n o t cause e x p e r i m e n t a l problems, excessive Evans blue dye doses render skin tests difficult to measure. I n our protocols we have repeated fluorescein injections at weekly intervals for up to 6 weeks i n over 100 a n i m a l s with n o a p p a r e n t harm. Fluorescein appears potentially a d a p t a b l e to a variety of skin test procedures. G o o d PCA results in guinea pigs have b e e n obtained, although we have n o t rigorously c o m p a r e d Evans blue dye a n d fluorescein P C A assays.
Acknowledgement The authors gratefully acknowledge the s u p p o r t of Dr. G,L. W i n n . This work was also supported i n part b y the Veterans A d m i n i s t r a t i o n a n d by O k l a h o m a Allergy Clinic, Inc.
References Cain, W.A., C.P. Cox, B.E. Pennock and J.H. Wells, 1980, Int. Arch. Allergy Appl. Immunol. 63, 361. Chasen, S., 1979, in: BMDP-79: BiomedicalComputer Programs P Series, eds. W.S. Dixon, M.B. Brown, L. Engelman, J.W. Franc and R.I. Jennrick (University of California Press, Berkeley, CA) pp. 230-244. Cox, C.P., W.A. Cain, B.E. Pennock and J.H. Wells, 1979, Fed. Proc. 38, 931. Erlich, P., 1881, Dtsch. Med. Wochenschr. 2, 21. Glantz, S.A., 1981, in: Primer of Biostatistics (McGraw-Hill, New York) pp. 180-228. Maurice, D.M., 1967, Invest. Ophthalmol. Visual Sci. 6, 464. Neller, J.L. and E.R. Schmidt, 1945, Ann. Surg. 121,328. Ovary, Z., 1958, Progr. Allergy 5, 459. Pennock, B.E., W.A. Cain, R.M. Rogers and J.H. Wells, 1980, Am. Rev. Resp. Dis. 121, 88. Pennock, B.E., C.P. Cox, R.M. Rogers, W.A. Cain and J.H. Wells, 1979, J. Appl. Physiol. 46, 399. Stein, M.R. and C.W. Parker, 1971, Am. J. Ophthalmol. 72, 861. Wollheim, E. and K. Lang, 1931, Verh. Dtsch. Ges. Inn. Med. 43, 134.