Our experience with Brachet test on twenty-four leukemic patients has shown a high degree of reliability of the test for differentiating acute leukemias. A standard method has been described which is simple enough to be carried out routinely. The test, however, is not without pitfalls and need be interpreted with some caution.
From the fact that urine hydrolysis can be closely simulated by the enzymatic action of pure DNase solution, it is suggested that the urine factor responsible for the nuclear lysis is DNase excreted in the human urine. The possible mechanism and implication of the test have been discussed in relation to the results obtained.</p

Miyoshi, Isao

Sanada, Hiroshi

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 16, Issue 4 1962 Article 5AUGUST 1962Brachet Test (Urine Hydrolysis Test) as an Aidto Differentiation of Leukemic CellsIsao Miyoshi∗ Hiroshi Sanada†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Brachet Test (Urine Hydrolysis Test) as an Aidto Differentiation of Leukemic Cells∗Isao Miyoshi and Hiroshi SanadaAbstractOur experience with Brachet test on twenty-four leukemic patients has shown a high degreeof reliability of the test for differentiating acute leukemias. A standard method has been describedwhich is simple enough to be carried out routinely. The test, however, is not without pitfalls andneed be interpreted with some caution. From the fact that urine hydrolysis can be closely simulatedby the enzymatic action of pure DNase solution, it is suggested that the urine factor responsible forthe nuclear lysis is DNase excreted in the human urine. The possible mechanism and implicationof the test have been discussed in relation to the results obtained.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLActa Med. Okayama 16. 225-231 (1962)BRACHET-TEST (URINE HYDROLYSIS TEST) AS ANAID TO DIFFERENTIATION OF LEUKEMIC CELLSIsao MIYOSHI and Hiroshi SANADADepartment of Internal Medicine, Okayama University Medical SchoolOkayama (Director: Prof. K. Hiraki)Received for publication, July 5, 1962Better selection of antileukemic agents, which is only possible on the exactknowledge of the type of leukemia, is of considerable importance for a prognosticevaluation of a given leukemic patient. Nevertheless, it is not necessarily easyto determine the type of leukemia, particularly of the acute type.It appears, therefore, any technic which will aid in differentiating im-mature leukemic cells deserves attention. Being stimulated by the reports ofTHOMA1 and GARDNER et al.,2,3 we have investigated the validity of Brachet-test in differentiating leukemic cells. The test is based on the fact that exposureof blood films to human urine results in the differential extraction of the nuclearchromatin.It is the purpose of this paper to report our experience with the Brachet-teston twenty-four leukemic patients who were admitted to our Medical Service.Factors influencing the test were also studied.MATERIALS AND METHODSA slight modification of the methods described by THOMA1 and GARDNERet a12• was emplyed. Five peripheral blood or bone-marrow smears were pre-pared on a leukemic patient. They were fixed in methanol for three minutesand air-dried. Approximately 100 ml. of freshly voided urine from a normal orleukemic individual was collected in a beaker, and its pH was adjusted to 6.4with either O.lN NaOH or O.lN HCl. And the urine was heated to 60°C in awater bath. Four of the 5 blood films were immersed in the urine for 2, 4, 6and 8 minutes, respectively, before they were taken out and rinsed thoroughlyin running tap water. After being air-dried, the blood films were stained withMay-Giemsa or methyl green-pyronin stain. The one remaining slide wasstained as control without urine hydrolysis.Apart from urine, pure enzyme solutions were made up with crystallizedDNase and RNase ( x 1) obtained from Worthington Biochemicals Corp., U. S. A.They were used in a concentration of 200 fl.g. per ml. of double-distilled water.The RNase solution was handled as with urine, but because of the limited2251Miyoshi and Sanada: Brachet Test (Urine Hydrolysis Test) as an Aid to DifferentiationProduced by The Berkeley Electronic Press, 1962226 1. MIYOSHI, and H. SANADAamount of the expensive DNase, the method was modified as follows. TheDNase solution stored in the refrigerator was pre-heated to 37 QC prior to eachuse. A few drops of the solution were put on the methanol-fixed blood films inthe humidified incubator at 37 QC. The duration of the enzyme action wasempirically set to from one to two hours. Following this treatment, the slideswere stained with May-Giemsa stain as before.RESULTSStained blood films were examined microscopically to see the degree ofurine hydrolysis. Regardless of the source of the specimens, normal or leukemic,it was shown that the nuclear chromation of neutrophilic cells was lysed, whilethe nuclei of lymphocytes, monocytes or eosinophils were unaffected. Amongthe four slides exposed to urine for different lengths of time, one slide was se-lected for microscopic observation, in which distinction between lysed neutro-phils and unaffected lymphocytes or eosinophils was most apparent. Maturesegmented neutrophils and their precursors as well showed lysis of the cellnuclei. This characteristic behavior of the neutrophilic series provided the basisfor differentiating myelogenous leukemia from other types of leukemia (Figs. 1,2 and 3).Brachet test was performed on twenty-four leukemic patients who wereconsecutively admitted to our Medical Service since June 1961. Correlationbetween the clinical diagnosis and degree of urine hydrolysis is shown in Table1. They comprised 6 cases of acute lymphocytic leukemia, 8 cases of acuteTable 1. Urine Hydrolysis of Leukemic Cells in Various Types of LeukemiaI Blasts I More mature forms I No. cases~--~_ D.egree of hydrolysis I + +-- -I+ +-- - IChn.dlag. -----__Acute lymph. leuk. 6 6 6Acute myelo. leuk. 8 7 1* 8Chronic myelo. leuk. 6 6 6Monocytic leuk. 1** 3 1** 3 4Total 24* Brachet negative neutrophilic cells possibly showing treatment effect.** From the standpoint of Brachet-test, this case was regarded as acute myelogenous leukemia.myelogenous leukemia, 6 cases of chronic myelogenous leukemia, and 4 casesof monocytic leukemia. The clinical diagnosis was established by a simultaneoususe of various technics including May-Giemsa staining, phase contrast and flu-orescence microscopy, vital staining, and bone marrow tissue culture6• Except2Acta Medica Okayama, Vol. 16 [1962], Iss. 4, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol16/iss4/5Brachet Test 227one case of monocytic leukemia, the diagnoses by the Brachet-test and by thesemethods were in full agreement on the other twenty-three leukemic patients.In this instance of monocytic leukemia, urine hydrolysis revealed lysis of thenuclei of some of the blast cells and all of the more mature leukemic cells. Fromthe standpoint of the Brachet-test, therefore, the case was considered to be acutemyelogenous leukemia rather than monocytic leukemia.Attempts to analyze some of the factors influencing urine hydrolysis led tqthe following observations.1. Influence of pH.The pH of urine in the range of 6.0 to 6.8 gave an equally satisfactorydifferential extraction of the cell nuclei. However, at pH 7.0, rupture of thenuclear membrane and diffusion of the chromatin material into the surroundingarea were seen.2. Inactivation of urine.Heating urine at 75 QC for 15 minutes completely deprived the urine of thelytic action on the nuclear chromatin. Standing urine at a room temperaturefor 24 hours, likewise, resulted in the almost complete loss of an active agent.3. Use of sodium chloride solution.A 0.2 M sodium chloride solution was used in place of urine in the otherwisesame experimental condition, but no nuclear change was observed. The slidewhich was kept immersed in the saline solution for one hour, however, showeddiffusion of the nuclear material into the adjacent area.4. Effect of DNase.The differential extraction of the leukocyte chromatin by means of urinecould be closely simulated by pure DNase solution in distilled water, whereaspure RNase sotution in distilled water caused no comparable alteration of thecell nuclei in any leukocyte series.DISCUSSIONThe original investigators1.6-9 ascribed the differential nuclear lysis of leu-kocytes to the action of RNase and DNase. They considered that the nuclei ofneutrophilic cells are rich in RNA and are susceptible to the action of RNase,while the nuclei of lymphocytes, monocytes or eosinophils contain more DNAthan RNA, being susceptible to DNase. Our experiment as well as GARDNER'S2failed to produce a typical nuclear lysis with pure RNase solution. The reasonfor this discrepancy is not clear, but it is possible that, the crude RNase whichLAVES and THoMA7 extracted from the cow's pancreas might have contained asmall amount of DNase. Moreover, with our present-day knowledge of cyto-chemistry, it is hard to believe that RNA is a major nucleoprotein of neutrophilic3Miyoshi and Sanada: Brachet Test (Urine Hydrolysis Test) as an Aid to DifferentiationProduced by The Berkeley Electronic Press, 1962228 1. MIYOSHI, and H. SANADAcells. Methyl green-pyronin staining after urine hydrolysis demonstrated failureof the nuclei of neutrophilic cells to take up a green color of methyl green, in-dicating loss of DNA from the cell nuclei.From the finding that sodium chloride solutions in various concentrationsalso caused removal of the chromatin material, SPRAGUE et al. 10,11 regardedurine as a mere salt solution. As stated earlier, we could not induce a clear-cutlysis of the cell nuclei with a 0.2 M sodium chloride solution, and we feel, asGARDNER et afl. thought, that the effect of sodium chloride solutions is a non-specific dissolution of the nuclear material.It should be kept in mind that not all of the myeloblasts appearing in acuteor chronic myelogenous leukemia show a complete lysis of the nuclear chro-matin. Some are quite susceptible to urine hydrolysis but some are entirelyretistant to it. In some cases, the degree of nuclear lysis varied considerablyamong myeloblasts, and the percentage of the susceptible myeloblasts differedfrom case to case. From the fact that promyelocytes are uniformly susceptibleto the action of urine, those myeloblasts similarly susceptible are presumablycloser to promyelocytes in their maturation level, having the same DNA com-position as promyelocytes. It may be said, therefore, that the degree of urinehydrolysis of myeloblasts in a certain case depends on the level of maturationwhere the myeloblasts are proliferating. If myeloblasts are closer to stem cells,they are hardly susceptible to urine hydrolysis whereas if they are closer to pro-myelocytes, they are as susceptible as promyelocytes.Blood cells may be separated into two groups depending on the susceptibilityof the nuclear chromatin to urine hydrolysis (Table II). However, this distinc-Table H. Susceptibility of Blood Cells to Urine HydrolysisSusceptible cellsMyeloblastsPromyelocytesMyelocytesMetamyelocytesBand formsSegmented neutrophilsBasophilic seriesMature erythroblastsTheir mitotic formsResistant cellsLymphatic seriesMonocytic seriesEosinophilic seriesPlasma cellsReticulum cellsMegakaryocytesPlateletsImmature erythroblastsTheir mitotic formstion is not an absolute one, and with longer urine hydrolysis, even the nucleiof lymphocytes, monocytes or eosinophils show lysis of the chromatin material.It is important, therefore, to be sure that the slide under examination is repre-4Acta Medica Okayama, Vol. 16 [1962], Iss. 4, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol16/iss4/5Brachet Test 229sentative of the maximum contrast between lysed neutrophils and resistantlymphocytes.The mechanism involved in the urine hydrolysis of the nuclear chromationis not entirely clear. It has been shown that the action is influenced by the pHand temperature of the urine. The urine factor, whatever it may be, thermo-labile and can be inactivated by heating at 75 QC for 15 minutes. Treatment ofblood films with pure DNase solution has given a similar result. From theseobservations, it appears reasonable to assume that an unidentified substanceresponsible for urine hydrolysis would be DNase excreted in the human urine.What is the difference, then, in DNA composition between neutrophils andother leukocytes, resulting in the differential nuclear extraction? At the presenttime, we do not seem to have enough evidence to answer this question.In the peripheral blood of one patient with acute myelogenous leukemiawho was heavily treated with steroid hormones prior to the time Brachet testwas done, there were some Brachet negative mature and immature neutrophiliccells. It could not be determined whether the emergence of the Brachet negativeneutrophils in this patient was due to the use of steroid hormones or an indica-tion of biochemical alterations of the nucleoprotein by leukemic transformationper se. It may be of note that these Brachet negative neutrophils were per-oxidase negative. THOMA1 and LAVES6 reported X-ray irradiation therapy in leu-kemia reduced the susceptibility of neutrophilic cells to urine hydrolysis. POLLIet a1'2. presented evidence for the presence of cross-linking between DNA mo-lecules induced by Myleran in chronic myelogenous leukemia. NOWELL13 ob-served inhibition of human leukocyte mitosis by prednisolone in vitro. In viewof these reports, it is possible that such antileukemic agents as steroid hormonesand Myleran which are most widely used today, act on the DNA level, althoughtheir precise mode of action is unknown.SUMMARYOur experience with Brachet test on twenty-four leukemic patients hasshown a high degree of reliability of the test for differentiating acute leukemias.A standard method has been described which is simple enough to be carried outroutinely. The test, however, is 'not without pitfalls and need be interpretedwith some caution.From the fact that urine hydrolysis can be closely simulated by the enzy-matic action of pure DNase solution, it is suggested that the urine factor res-ponsible for the nuclear lysis is DNase excreted in the human urine.The possible mechanism and implication of the test have been discussed inrelation to the results obtained.5Miyoshi and Sanada: Brachet Test (Urine Hydrolysis Test) as an Aid to DifferentiationProduced by The Berkeley Electronic Press, 1962230 1. MIYOSHI, and H. SANADAACKNOWLEDGEMENTThe authors wish to express their thanks to Prof. K. Hiraki for his helpful advice.REFERENCES1. THOMA, K.: tlber die Verwendung von Ribonuclease (Brachet-Test) in der h~matologischenDiagnostik. Acta haemat. 4, 81, 19502. GARDNER, E. Jr., WRIGHT, C. and WILLlAMS, B. Z.: An evaluation of the urine hy·drolysis test for primitive white blood cell differentiation, ]. Lab. & CUn. Med. 55, 883,19603. IDEM.: The use of desoxyribonuclease as an aid to differentiate primitive white blood cells.Blood 18, 102, 19614. BRACHET, J.: La localization des acides pentosenucleiques dans les tissus animaux et lesoeufs d'Amphibiens en voie de developpement. Arch. Bioi. 53, 207, 19425. HIRAKI, K., OFU]I, T. and SUNAMI, H.: The method of tissue culture (mainly the bonemarrow) and a simple method of observing living tissue. Acta Med. Okayama 10, 99,19566. LAVES, W.: Uber ein neues System der Leukozyten. Munch. med. Wschr. 93, 210,19517. LAVES, W. und THoMA, K.: Zur Cytochemie der eosinophilen Leukocyten und der Lym-phocyten. KUn. Wschr. 95, 377, 19518. LAVES, W., THoMA, K. und OBERDORFER, A.: tlber ein einfaches Verfahren zur zyto-enzymatischen Vntersuchung von Blut- und Knochenmarkausstrichen und seine BedeutungfUr das Stadium leuk~mischer Zellen. Wien. klin. Wschr. 64, 4, 19529. LAVES, W.: tlber die Anwendung von Enzymen in der H~matologie und die physio-logische Bedeutung der Nucleasen fUr periphere Leukozytensteuerung. Acta haemat. 8, 1,195210. SPRAGUE, C. C., GREEN, R. T. and CARRERA, A. E.: Differential extraction of nucleo-protein from human leukocytes. ]. Lab. & CUn. Med. 50, 955, 195711. BURDETTE, W. J,: Etiology and Treatment of Leukemia, p.79, Mosby, St. Louis, 195812. POLLI, E. E., RosoFF, M., di MAYORCA, G. and CAVALIERI, L. F.: Physicochemicalcharacterization of deoxyribonucleic acids from human leukemic leukocytes. Cancer Re·search 19, 159, 195913. NowELL, P. C.: Inhibition of human leukocyte mitosis by prednisolone in vitro. CancerResearch 21, 1518, 19616Acta Medica Okayama, Vol. 16 [1962], Iss. 4, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol16/iss4/5Brachet Test 231Fig. 1. Bone marrow smear of acute lymphocyticleukemia showing lysis of the nucleus of a bandform and unaffected lymphoblasts.Fig. 2. Bone marrow smear of acute myelogenousleukemia showing complete lysis of the nuclei ofthe neutrophilic series, a basophil and a erythro-blast in contrast to unaffected lymphocyte.Fig. 3. Bone marrow smear of monocytic leukemiashowing the intact nuclei of monocytes, monoblasts,a lymphocyte and a eosinophil, while the nucleusof a segmented neutrophil is completely lysed.7Miyoshi and Sanada: Brachet Test (Urine Hydrolysis Test) as an Aid to DifferentiationProduced by The Berkeley Electronic Press, 1962

Brachet Test (Urine Hydrolysis Test) as an Aid to Differentiation of Leukemic Cells

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