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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.

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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

 

' o-Tolidin

'

Test

'

Test

' Guaico Test '

'

Test

Milk Saliva Pus. (Hb. free) Copper sulfate / Ferric chloride Permanganate / Iodides Bromides Ferrocyanide Ferricyanide / Formaldehyde Salicylate Sulfonamides Bismuth subnitrate

'

' green ' light blue ' green ' blue ' blue ' green-blue 'blue-green ' ' ' light green ' ' ' '

 

'

' blue ' ' green-blue ' blue ' ' violet ' blue ' ' ' blue ' light blue ' ' '

' green ' light green ' green-blue ' green ' blue ' blue-green ' green ' ' ' green ' ' ' '

'

' Red(persists)

'

'

'

'

'

'

'

'

'

'

* 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.:

(3)

ICE LEARDE and BENOIT: De la recherche

Phenolphthalin as a Reagent for Oxidizing Ferments. Am. Chem. Jour., 26: 526, 1901.

chimique du sang dans lee secretions organiques,. Compt. rend. soc. de biol., 64:1048, 1908.

(2)

MEYER, E.: Beitrage zur Leukocytenfrage. Munchen. mod. wchnschr., 35: 1489, 19 03 .

(4)

BOAS, I.: Erie phenolphthalinprobe als Reagens auf okkulte Blutungen des Magendarmkanals. Deutsche. med. Wchnschr. 37:62, 1911.