Notes on the 8 and 24 hour Frank-Berman pregnancy test

NOTES ON THE 8 AND 24 EOUR FRANK-BliWUN TEST ROSE Ii

BERMAN,

PREQEYANCY

B.A., NEW YORK, N. Y.

T

HE Frank-Berman pregnancy test, first described in 1941, is the first successful pregnancy test using rats with results in 8 and 24 hours. At this time a review of the test based on experience with many thousands of tests, of the precautions to be followed, and a general discussion of the role of a pregnancy test are indicated. The great number of inquiries received from physicians for help in interpretation of tests plus the great number of incorrect results that physicians have received from laboratories using varying tests and techniques have prompted this review. Technique of Test.-

1. The test animals required are two immature female rats, weight 50 grams (weight 45 to 60 grams). 2. Two subcutaneous injections of test urine are made, a 5 ml. dose at each injection. 3. The animals are sacrificed next morning with illuminating gas, the ovaries dissected out and read grossly with the naked eye. range,

The urine to be injected is first centrifuged to bring down extraneous matter-erystals, urates. white blood cells, epithelial cells, etc.-which are not necessary for the test and the clear supernatant urine which contains the hormone is used. The animals are given two subcutaneous injections, 5 ml. at each injection, one injection being given in the morning, the second injection in the afternoon, with a minimum time interval of 4 hours between injections. The injection needle is inserted under the loose skin of the rat, the needle being pointed toward the tail of the animal. After the injection, massage the skin downward at the injection site to help spread the injected urine over a larger surface. The following morning, the animals are killed with illuminating gas. Keep the animals in the lethal chamber with gas entering the chamber until they are dead, not just gasping. The ovaries are then dissected out via the abdominal route. The ovaries are read grossly with the naked eye, It will facilitate matters to place the dissected ovaries on a piece of slightly moistened gauze and read immediately. A negative ovary is small, pale, and creamy in color or slightly pinkish, the surface of the ovary showing minute colorless follicles. A positive ovary is usually, but not always, enlarged, and is entirely reddened or may have many distinct red spots on it. In the 24 hour period, luteinization in the ovary does not occur. It takes 48 hours for the ovary to become luteinized. It is absolutely necessary to use illuminating gas to sacrifice the animals since the anoxia following exposure to carbon monoxide affects the capillaries of the ovaries, giving the characteristic reddened effect to positive ovaries. Microscopic sections of the 24 hour test ovary show that the characteristic hyperemic zone is due to hyperplasia of the theta interna cells with marked multiplication and dilatation of the ingrowing capillaries. It is apparently the chorionic gonadotropic hormone which affects the vascular system of the ovary and with the use of illuminating gas the typical carbon monoxide effect on the ovary is therefore produced. Since 3.5 per cent of the rats are refractory, the test must be done on two rats. Occasionally, in a rat one ovary will be negative and the other ovary positive. This is a positive pregnancy test. The test, if performed as described and all preeautions followed, is 100 per cent accurate. 440

FRANK-BERMAN Precazlt~ions There he valueless.

PREGNANCY

TEST

441

to Be Followed.-

are

certain precautions which The instructions to be given

must be observed by the patient patients for the test are:

1. Take no medication-hormones, Prostigmine, antihistamines, amides quinine, ergot-for 96 hours prior to test. (These substances kill the test animals. Toxic urines cannot be detoxified.) toxic

2. Drink no alcoholic to the test animal.)

beverages

for

24 hours

prior

t,o test.

or the test

laxatives, are toxic (Excreted

may

sulfonand will alcohol

is

3. Save first urine on arising, put into refrigerator immediately. Keep in refrigeratoi until brought to laboratory. (D o not restrict fluids for the purpose of concentrating tho urine. The concentrated urine will either kill the animals or make it difficult to read the ovaries. There is sufficient concentration in the overnight normal concentration. It is unwise to use any random specimen of urine. The patient may have had a diuretic such as coffee, t.ea, cola, etc., which will induce urination with a subsequent diiute specimen, or the patient may be a low hormone excreter and the hormone excreted in a random xpecimen may therefore be too little to be picked up by the animals. It is necessary to keep urine refrigerated since the gonadotropic hormone deteriorates at room temperature.) 4. Bring

a 2 ounce

specimen

The urine specimens urine it should be allowed shock the animals. Urine terioratiou of the hormone. Llydat,id

Xole

and

to the laboratory

in the morning.

should be kept refrigerated at all times. Before injecting the to stand at room temperature for about 20 minutes so as not to can be kept in the refrigerator for several weeks without do-

Chorionepithelioma.-

In cases of suspected chorionepithelioma or hydatid mole, a urine nancy test is necessary as a diagnostic procedure. A normal pregnancy tive reaction with as little as 0.1 ml. urine. Therefore, positive results are indicative of a hydatid mole or chorionepithelioma since in both these is a great excess of chorionic gonadotropic hormone excreted. In suspected pregnancy test with a 10 ml. urine dose is done, If this is positive, following dose ranges of urine: 5 ml., 1 ml., 0.1 ml., 0.05 ml. In making prepare them so that the total amount injected into each rat is 10 ml.: Dilution Dilution Dilution Dilution

A, B, C, D,

titration pregmay give a posibelow this level conditions there cases, a routine proceed with the up t,he dilutions.

5 ml. dose-15 ml. urine + 15 ml. distilled water 1 ml. dose4 ml. urine + 36 ml. distilled water 0.1 ml. dose4 ml. from Dilution B + 36 ml. distilled water 0.05 ml. dose-15 ml. from Dilution C + 15 ml. distilled water

If sary. must ferent

a positive reaction is obtained with the 0.05 ml. dose, further dilutions are neres Proceed with 0.025 ml. dose, 0.0125 ml. dose, 0.00625 ml. dose, etc. Extreme care be taken to wash the syringe and needle very carefully between injections of dif, dose ranges to prevent the carry-over from one dose range to another.

Repeated pregnancy tests must be done in all cases of hydatid mole until the nancy test is negative. If after the. removal of the hydatid mole, repeated pregnancy are negative followed by a positive pregnancy teat or if the pregnancy tests remain tive, the possibility of the transformation of the mole to chorionepithelioma must he ously considered. Ectopic tive. tive centa

PI-egna?&cy

and

0 ther

Conditions.-

In cases of suspected eetopic pregnancy, the pregnancy test may The test will be positive only if the chorionic villi are attached. pregnancy test does not signify the absence of an eetopic pregnancy. In cases of are retained

abortion, or if

positive fragments

preg. tests posiseri

pregnancy of chorionic

tests villi

will be obtained are still retained

be negative Therefore, if

or poni a nega-

fragments of plaand attached.

Use of Blood

Seiwm.-

The

Frank-Herman pregnancy test may simpler procedure for the patient to bring in blood withdrawal. However, in the following serum: 1. 2. physician 3. 4. associated serum. in the

also be done with blood serum. Jt is a a urine specimen than to be subjected to cases it is necessary to do the test with

If the patient has cystitis, the urine will kill the animals. If the patient is under medication that will prove toxic to the animals and the needs an immediate pregnancy test. If the patient is bleeding, urine may prove toxic. If the patient is diabetic, the urine specimen may be very dilute because of the polyuria. In diabetes, it is therefore essential to do the pregnancy test with

The serum afternoon, General

is administered making a total

by subcutaneous of 2 ml. serum

for

injection, 1 ml. each rat.

in the

morning

and

1 ml.

Comments.-

As reported in our first publication, results can also be obtained in 8 hours. Howe,ver, since a negative result in 8 hours cannot be considered a true negative unless confirmed Another factor to be considered by the 24 hour test, the value of the 8 hour test is limited. is the time element involved for laboratory personnel. Since urine specimens will arrive at the laboratory at varying time intervals, it is impractical to start tests at varying times and then report back to the laboratory at varying time intervals to kill and autopsy the animals. The 24 hour test has time latitudes that will fit into the ordinary working day of a laboratory. The animals ideally can be given the first injection at 10 to 11 A.M. and the seeond injection at 4 to 4:30 P.M. If urine arrives later, injections can be given at 1:00 P.M. and 5:OO P.M. If a urine specimen arrives later in the afternoon and laboratory personnel are available to report back to the laboratory in the evening, animals can be injected at 6:00 P.M. and again at 1O:OO P.M. All animals are sacrificed the next morning at 8:00 to 8:30 A.M. This therefore makes the 24 hour pregnancy test a 16 to 24 hour test. Time ranges below 16 hours are not reliable and dose ranges below 10 ml. are also unreliable. Since the levels of hormone excretion differ from patient to patient, 10 ml. was adopted as the correct dose range to cover all types of excreters. It is usual for a woman to suspect pregnancy when menstruation is delayed. It is therefore very difficult to determine the earliest time at which pregnancy can be detected. The earliest pregnancy we were able to pick up was a ten-day pregnancy, counting cycle day This case was picked up in the course of research on ten as the first day of pregnancy. control cases-normal menstruating women, cases of primary and secondary amenorrhea, Our diagnosis of pregnancy, which amazed the patient who had menopause cases, etc. not yet had a chance to “miss” menses to suspect pregnancy, was confirmed. It should be pointed out also that there are more cases of early abortion than are We had many cases that we could follow very closely suspected in so-called “sterility” cases. where positive pregnancy tests were obtained followed by “menses. ” Immediate examination of these women was made and whatever was found on the examining glove finger was sectioned. In all cases, chorionic tissue was found on section. Before attempting to perform the pregnancy test, it is absolutely imperative that many tests with known negative and positive urines be performed to learn to read the ovaries carrectly. Role

of

the ‘Pregnancy

Test.-

The pregnancy test is used to diagnose pregnancy, the presence of retained fragments in the male as of placenta or chorionie tissue, hydatidiform mole, and chorionepithelioma The pregnancy test will not predict abortion, will not indicate the prog well as the female. ress of the pregnancy, will not indicate whether the fetus is dead or alive, will not definitely

Volume

64

Number

2

establish nite role patient.

FRANK-BERMAN an ectopic pregnancy, in the interpretation

PREGNANCY

nor will it tell of the pregnancy

Summary

TEST

the ovulation time. test in conjunction

443 The with

physician has a detlthe symptoms of the

and Conclusions

1. The Frank-Berman 24 hour pregnancy test, which consists of injecting two immature with 10 ml. urine, is described. 2. The Frank-Berman pregnancy test is used to diagnose pregnancy, hydatidiform mole. . ehorionepithelioma, and the presence of retained products of conception in aborting patients. 3. If all conditions described for the test are followed, the test is 100 per cent accurate.

rats

Reference Frank,

R. T., and

Berman,

BERMAN CLINICAL 131 WEST 58~~

R. L.:

LABORATORY STREET

Aix.

J. OBST.

& GYSEC.

42: 492, 1941.