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Toad test for pregnancy
Toad test for pregnancy KONSTANTIN Monterey,
E.
ARENSBURGER,
PH.D.
California
T H E great accuracy of the very convenient male Rana pipielzs frog test for detection of pregnancy, described by Hodgson,‘, ’ is considerably lowered during the summer months because of the animal’s refractoriness during that period of the year. According to Mayer-Samson3 the refractoriness of Rnna pipiens during the winter and fall months is between 0.2 and 1.0 l~r cent, while during the summer season it rises to 11.2 per cent. A toad test, as described below, has significantly lowered false-negative results. This test is based on the above-mentioned Rana pipiens frog test. However, with certain modifications, more efficient results can be obtained, especially during the summer season.
saline, containing 300 U.S.P. units per milliliter, was added to 2 ml. of clear serum and stored overnight in the refrigerator at 3-O to So C. The serum was placed in a water bath of 37’ C. for 10 minutes prior to the injection. The treated toad was kept at room temperature out of reach of direct sunlight. The testing was performed during the summer season from June 15 to September 30. Rana pipiens and the toads were exposed to the same t-reatment under identical conditions. The toads B,ufo boreas halophilus and Bufo moodhousii were used and both responded equally well. l?esults Tests conducted by Hodgson’s method’, ’ on 190 clinically proved pregnant patients produced 20 false-negative results (10.5 per cent). At the same time, the toad test gave 7 false-negative results (3.7 per cent). Many positive results in both series were obtained within less than 30 minutes. No false-positive response was produced. When serum was injected immediately
Method Healthy toads which had been kept in a rt+ri,gerator at ,3,’ to 8’ C. for a period not cxxcerding 10 clays were used. The toads were removed from the refrigerator and exposed to room temperature for 30 minutes prior to the injection. Toads were weighed and examined for the absence of spermatozoa. The previously prepared serum was injccted into the dorsal lymph sac of each toad at the approximate rate of 1 ml. pe1 10 grams of its weight. Subsequently, the animals were examined every 30 minutes for the presence of spermatozoa. The abbthnce of spermatozoa after 3 hours was ron
Table I. Incidence of error injected immediately and refrigeration
,~est
Frog test Toad test a26
/ N&;‘~
90 90
“,Iy&y
9 3
when after
serum overnight
1. .Ls!Ty!“!!g!
10.0
3
3.3
1
3.3 1.1
Volume Number
83 6
Toad
Table II. Incidence varying
quantities
of error with of hyaluronidase False-negative
150 U.S.P. units of No Test
tests
Frog test Toad test
60 60
of
hyaluronidase No.
5 2
the use of
results
300 U.S.P. -
unitsof hyaluronidase
%
8.3 3.3
No.
1
%-
1
1.7
0
0
after clotting of the blood, 9 of 90 cases in the Rana pipiens series and 3 of 90 cases in the toad series produced false-negative results. Yet, in 6 of 9 cases in the Rana pipiens series and in 2 of 3 cases in the toad series, the same serum produced only positive results when kept refrigerated over night (Table I). In view of Seifter’s4 studies showing nontoxicity of hyaluronidase in animals receiving not more than 200,000 times the maximum proposed therapeutic dose, concentration of hyaluronidase was increased as suggested by Hodgs0n.l In 5 of the 60 cases in the Rana pipiens series, and in 2 of the 60 cases in the toad series, the addition of hyaluronidase containing 150 U.S.P. units per milliliter produced false-negative results. Addition of hyaluronidase in a 300 U.S.P. unit concentration to the same serum gave one falsenegative result in the Rana pipiens series (Table II). Application of this method to urine rather than serum produces equally reliable results. In 35 tests conducted by the conventional Rana pipiens method with the application of the Scott technique of urine concentration as recommended by Cutler,5 3 false-negative results were obtained. In the toad test, the application of the same concentrate gave positive results. All test animals were in very good condition after the injection. Summary
I n general, the excellent frog test described by Allison6 and Hodgsonl? 2 loses its accuracy during the summer season.
test
for
pregnancy
827
The toad test described above has the marked advantage of being accurate throughout the year including during the summer season when its percentage of error is reduced markedly. The technique of the toad test is not more complicated than that of the frog test. Should an inadequate supply of toads not warrant the use of that test throughout the year, it can be recommended for at least the summer season. The use of serum rather than urine is preferred during the summer season as well as during the remainder of the year.
% u/I IO9A?cSt32I -
Fig. 1. Seasonal
comparison of per cent of error in Rana pi&ens and toad tests. Solid line, per cent of error in frog test (Rana pipiens), according to Mayer-Samson.3 Broken line, per cent of error in toad test.
828
Arensburger
REFERENCES
1. 2. 3. 4.
Hodgson, J. E.: Am. J. Clin. Path. 25: 1096, 1955. Hodgson, J. E.: J. A. M. A. 153: 271, 1953. Mayer-Samson: Science 3: 231, 1950. Seifter, J.: Ann. New York Acad. SC. 52: 1141, 1950.
5. 6.
Cutler, J. N.: J. Lab. & Clin. 1949. Allison, R. M.: Surg. Gynec. 4.46, 1954. 685 Belden St. Monterey. California
Med. &
34:
554,
Obst.
98:
E.
ARENSBURGER,
PH.D.
California
T H E great accuracy of the very convenient male Rana pipielzs frog test for detection of pregnancy, described by Hodgson,‘, ’ is considerably lowered during the summer months because of the animal’s refractoriness during that period of the year. According to Mayer-Samson3 the refractoriness of Rnna pipiens during the winter and fall months is between 0.2 and 1.0 l~r cent, while during the summer season it rises to 11.2 per cent. A toad test, as described below, has significantly lowered false-negative results. This test is based on the above-mentioned Rana pipiens frog test. However, with certain modifications, more efficient results can be obtained, especially during the summer season.
saline, containing 300 U.S.P. units per milliliter, was added to 2 ml. of clear serum and stored overnight in the refrigerator at 3-O to So C. The serum was placed in a water bath of 37’ C. for 10 minutes prior to the injection. The treated toad was kept at room temperature out of reach of direct sunlight. The testing was performed during the summer season from June 15 to September 30. Rana pipiens and the toads were exposed to the same t-reatment under identical conditions. The toads B,ufo boreas halophilus and Bufo moodhousii were used and both responded equally well. l?esults Tests conducted by Hodgson’s method’, ’ on 190 clinically proved pregnant patients produced 20 false-negative results (10.5 per cent). At the same time, the toad test gave 7 false-negative results (3.7 per cent). Many positive results in both series were obtained within less than 30 minutes. No false-positive response was produced. When serum was injected immediately
Method Healthy toads which had been kept in a rt+ri,gerator at ,3,’ to 8’ C. for a period not cxxcerding 10 clays were used. The toads were removed from the refrigerator and exposed to room temperature for 30 minutes prior to the injection. Toads were weighed and examined for the absence of spermatozoa. The previously prepared serum was injccted into the dorsal lymph sac of each toad at the approximate rate of 1 ml. pe1 10 grams of its weight. Subsequently, the animals were examined every 30 minutes for the presence of spermatozoa. The abbthnce of spermatozoa after 3 hours was ron
Table I. Incidence of error injected immediately and refrigeration
,~est
Frog test Toad test a26
/ N&;‘~
90 90
“,Iy&y
9 3
when after
serum overnight
1. .Ls!Ty!“!!g!
10.0
3
3.3
1
3.3 1.1
Volume Number
83 6
Toad
Table II. Incidence varying
quantities
of error with of hyaluronidase False-negative
150 U.S.P. units of No Test
tests
Frog test Toad test
60 60
of
hyaluronidase No.
5 2
the use of
results
300 U.S.P. -
unitsof hyaluronidase
%
8.3 3.3
No.
1
%-
1
1.7
0
0
after clotting of the blood, 9 of 90 cases in the Rana pipiens series and 3 of 90 cases in the toad series produced false-negative results. Yet, in 6 of 9 cases in the Rana pipiens series and in 2 of 3 cases in the toad series, the same serum produced only positive results when kept refrigerated over night (Table I). In view of Seifter’s4 studies showing nontoxicity of hyaluronidase in animals receiving not more than 200,000 times the maximum proposed therapeutic dose, concentration of hyaluronidase was increased as suggested by Hodgs0n.l In 5 of the 60 cases in the Rana pipiens series, and in 2 of the 60 cases in the toad series, the addition of hyaluronidase containing 150 U.S.P. units per milliliter produced false-negative results. Addition of hyaluronidase in a 300 U.S.P. unit concentration to the same serum gave one falsenegative result in the Rana pipiens series (Table II). Application of this method to urine rather than serum produces equally reliable results. In 35 tests conducted by the conventional Rana pipiens method with the application of the Scott technique of urine concentration as recommended by Cutler,5 3 false-negative results were obtained. In the toad test, the application of the same concentrate gave positive results. All test animals were in very good condition after the injection. Summary
I n general, the excellent frog test described by Allison6 and Hodgsonl? 2 loses its accuracy during the summer season.
test
for
pregnancy
827
The toad test described above has the marked advantage of being accurate throughout the year including during the summer season when its percentage of error is reduced markedly. The technique of the toad test is not more complicated than that of the frog test. Should an inadequate supply of toads not warrant the use of that test throughout the year, it can be recommended for at least the summer season. The use of serum rather than urine is preferred during the summer season as well as during the remainder of the year.
% u/I IO9A?cSt32I -
Fig. 1. Seasonal
comparison of per cent of error in Rana pi&ens and toad tests. Solid line, per cent of error in frog test (Rana pipiens), according to Mayer-Samson.3 Broken line, per cent of error in toad test.
828
Arensburger
REFERENCES
1. 2. 3. 4.
Hodgson, J. E.: Am. J. Clin. Path. 25: 1096, 1955. Hodgson, J. E.: J. A. M. A. 153: 271, 1953. Mayer-Samson: Science 3: 231, 1950. Seifter, J.: Ann. New York Acad. SC. 52: 1141, 1950.
5. 6.
Cutler, J. N.: J. Lab. & Clin. 1949. Allison, R. M.: Surg. Gynec. 4.46, 1954. 685 Belden St. Monterey. California
Med. &
34:
554,
Obst.
98: