Reprinted From

The American Journal of Clinical Pathology, Vol. 13, No. 9 , September, 1943

THE PHENOLPHTHALIN TEST FOR THE DETECTION OF "OCCULT" BLOOD

ALEXANDER O. GETTLER & SIDNEY KAYE

From the Toxicology Laboratories of the Chief Medical Examiner's Office of
New York City


One of three tests is used frequently by clinical laboratories for the detection of "occult" blood
in feces, urine, spinal fluid and gastric contents, namely the guaiac, benzidine and orthotolidin test.
These tests are not specific for hemoglobin (blood) since many other substances, as indicated in the
table, give a similar color reaction. Oxidizing agents in general, or substances which catalyze oxidation
reaction will produce a similar green or blue color. The phenolphthalin test, first described in 1903, if
properly carried out is much more reliable and specific for hemoglobin than the three tests in general
use. In the chemical laboratories of Bellevue Hospital we have made a comparative study of all four
tests for several years and are convinced that the phenolphthalin test is the most specific for
hemoglobin. Since the test may well be labeled "the forgotten test" we take this opportunity to bring it
to the attention of clinical laboratories and also forensic chemists and serologists.

The phenolphthalin test was first used by Kastle and Shedd
1
in 1902, for the detection of plant
oxydases. In 1903, Meyer
2
used it for the determination of the oxydase content of leucocytes from
leucemic blood and from pus. Meyer also mentions its use for the detection of blood in urine. DeLearde
and Benoit
3
applied it to the detection of blood in urine, feces, gastric juice, and for the forensic proof of
blood stains . In 1911, Boas
4
published his studies in which he compared the sensitivity and the
specificity of the phenolphthalin, the guaiac and the benzidine test, as reagents for the detection of occult
blood in feces.

PREPARATION OF THE PHENOLPHTHALIN REAGENT


200 ml. of distilled water, 20 Gm. of sodium hydroxide, I Gm. of phenolphthalein are placed into
a 500 ml. Erlenmeyer flask. When completely dissolved, 20 Gm. of granulated zinc (20 to 30 mesh) are
added. Using a reflux condenser, to prevent evaporation, the mixture is now slowly boiled until the red
color of the alkaline phenolphthalein disappears leaving a colorless solution. This may take two to three
hours of boiling. The phenolphthalein is thereby reduced to phenolphthalin which is colorless in alkaline
solution. When cool the solution is placed into a brown bottle, and stoppered with a rubber stopper. Some
of the zinc is added; this will aid in keeping it in the reduced form. The reagent if kept in a cool dark place
will keep for years.

-2-

THE PHENOLPHTHALIN TEST IN DETAIL

An aqueous extract of clothing, weapon or any material suspected of containing blood is made
with a minimal volume or one or two ml. portions of urine, spinal, fluid, thin fecal suspension, or gastric
contents are placed into a test tube and boiled for about 30 seconds in order to destroy any oxidases
that may be present. The specimen is then cooled. About five drops of the phenolphthalin reagent are,
added, followed by about three drops of 3 per cent hydrogen peroxide. A pink to red color indicates the
presence of blood . The intensity of the color its a rough indication of the quantity of blood present. This
color will fade within 1/2 hour. If the pink-red color persists indefinitely it is due to copper interference.

It is perhaps needless to state that fine test for blood in feces and in gastric contents should
only be applied if the patient was one a meat and fish free diet for from 3 to 4 days. Bleeding as seen
in hemorrhoids, as well, as the admixture of menstrual blood is to be considered in the interpretation
of the result.

INTERFERENCES WITH EACH OF THE FOUR TESTS FOR BLOOD

In order to determine which of the four color tests for blood (hemoglobin) is the most specific,
they were applied to a number of substances as indicated in the table.


MATERIAL OTHER THAN BLOOD GIVING POSITIVE
REACTION WITH*

' Phenolphthalin ' Benzidine ' Guaico Test ' o-Tolidin
' Test ' Test ' ' Test

Milk ' ' blue ' green ' green
Saliva ' ' ' light blue ' light green
Pus. (Hb. free) ' ' green-blue ' green ' green-blue
Copper sulfate / ' Red(persists) ' blue ' blue ' green
Ferric chloride ' ' ' blue ' blue
Permanganate / ' ' violet ' green-blue ' blue-green
Iodides ' ' blue 'blue-green ' green
Bromides ' ' ' '
Ferrocyanide ' ' ' '
Ferricyanide / ' ' blue ' light green ' green
Formaldehyde ' ' light blue ' '
Salicylate ' ' ' '
Sulfonamides ' ' ' '
Bismuth subnitrate ' ' ' '


* Alcoholic solutions of the benzidine, guaiac and o-tolidin were used.
/ Dilute solutions of the chemical reagents were used:
/ Upon adding phenolphthalin reagent to dilute permanganate or dilute ferricyanide solution
a red color results, but this becomes colorless upon adding the hydrogen peroxide.
The tabulated results in the table clearly indicate that several substances give the same green
to blue color reaction with the guaiac, benzidine and orthotolidin tests, that is obtained with blood. In
the case of the phenolphthalin test, only cupric ions may interfere. However, if the pink-red color is due
to blood, the color fades fairly rapidly, whereas if it is due to a copper salt, the red, persists indefinitely.
Also, fortunately copper is of very rare occurrence and can easily be detected by simple tests. It is there-
fore evident, that the phenolphthalin test is much more reliable and specific for the detection of blood
than the benzidine, guaiac and orthotolidin tests. When phenolphthalin reagent is added to
permanganate or ferricyanide solutions a red color is produced, but this becomes colorless upon the
addition of the hydrogen peroxide.
SENSITIVITY OF TESTS FOR OCULT BLOOD
A comparison of the sensitivity of the four color tests used .for the detection of hemoglobin
was found to be: guaiac test, one part in ten thousand, benzidine test, one part in one million,
orthotolidin test, one part in ten million, phenolphthalin test, one part in ten million (if sample is
fresh), and one part in one million (if sample is old and decomposed).
SUMMARY
1. The phenolphthalin test for "occult "blood, is described, in order to bring it to the
attention of clinical laboratories again.

2 . The phenolphthalin, benzidine, guaiac; and orthotolidin tests have been studied as to
their reliability and specificity for the detection of blood in urine, feces, gastric, contents and spinal
fluid; also clothing, weapons, and other suspected materials of police interest.
REFERENCES
(1) KA.STLE, J. H., and.SHEDD, O. M.:
Phenolphthalin as a Reagent for Oxidizing
Ferments. Am. Chem. Jour., 26: 526, 1901.
(3) ICE LEARDE and BENOIT: De la recherche
chimique du sang dans lee secretions
organiques,. Compt. rend. soc. de biol.,
64:1048, 1908.
(4) BOAS, I.: Erie phenolphthalinprobe als
Reagens auf okkulte Blutungen des
Magendarmkanals. Deutsche. med.
Wchnschr. 37:62, 1911.
(2) MEYER, E.: Beitrage zur Leukocytenfrage.
Munchen. mod. wchnschr., 35: 1489, 19 03 .