Thymol Turbidity test, Takata-Ara's test, and Gros' test in Icteric Conditions

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  • Acta Medica Scandinavia. Vol. CXXXIV, fasc. I, 1949.

    Department 11, the Kommunehospital, Copenhagen. (Physician-in-Chief: H. Heckscher, M.D.)

    Thymol Turbidity test, Takata-Aras test, and Gros test in lcteric Conditions

    15Y

    I NGER- LOU ISE MAR N ER .* (Submitted for publication Julie 10, 1948.)

    Introduction.

    Since, in 1906, Bauer introduced the galactose tolerance test as a liver function test, no small number of such tests for estimation of the condition of the liver have been proposed, in consequence of an ever increasing interest in the diagnosis of diseases in liver and bile ducts. These various proposals show tha t it has been difficult to attain to a satisfactory test. In papers published within recent years the conditions of the serum proteins play a great part in the diagnosis of liver diseases.

    In 1930 Jezler pointed out the great importance of the Takata-Am test for the diagnosis of liver diseases. This test has since been extensively used, particu- larly so in the Scandinavian countries. I ts value has become further established through numerous investigations, especially through Hafstroms comprehensive work.

    All investigators agrec tha t the Takata-Ara trst is of importance particularly by being generally positive in cirrhosis of tlic liver, i.c. positive i n 90 to 100 pcr cent of the cases (Jezler, Hafstrom, Buck, Godtfredsen, Ivcrsen, and Grindsted). In the diagnosis of acute hepatitis, on the other hand, we cannot rely on this test, i t being often negative in this disease (see Table). The various statements concerning the frequency of positive reaction in hepatitis differ considerably. The reaction is always negative in eases of obstructive jaundice, where the liver is damaged but little.

    * Nsrrebrogade 142, Copenhagen (Dellmdrk).

  • THYMOL TURBIDITY TEST, TAKATA-ARAS TEST, AND GROS TEST IN ETC. 41

    Gros, through his works in 1939 and 1940 with the so-called Gros test, showed that marked flocculation was elicited by addition of 1 to 10 drops of Hayems solution to serum from patients with a positive Takata-Ara test. The intensity of this flocculation was in the main parallel to the strength of the Takata-Ara test. Gros found, however, that the reaction was positive more often than the Takata-Ara test in the case of hepatitis. Hence he thought that i t would also be of importance for the diagnosis of this disease. In 1940 Stolte modified the test, showing that Na2S0, in Hayems solution plays no part for the reaction.

    The thymol turbidity test is of a more recent date, having been introduced by Maclagan in 1944. Like the Takata-Ara test and the Gros test, it is a globulin precipitation test. The test has in the course of these few years got to play an increasing part among the serological liver function tests, because, unlike the other tests, i t has proved very sensitive to the changes taking place in blood serum during acute hepatitis.

    There is reason to mention that the thymol turbidity test, which, besides, is very specific, has given a positive reaction in ab. 75 per cent of cases with infectious mononucleosis (I. L. Marner, Iversen & Raaschou). Iversen and Raaschou have, moreover, found a positive reaction in measles in 71 per cent of the examined cases.

    Own Investigations. In the present investigation Takata-Aras test, Gros test, and the thymol

    turbidity test were done simultaneously on 536 patients with jaundice. Each of these tests was performed 2754 times, the majority of the patients having been examined once weekly during the period of illness. All the patients had been admitted to one of the three medical departments or one of the two sur- gical departments of the Kommunehospital (Municipal Hospital), Copenhagen, or to the Blegdamshospital, Copenhagen.

    Principles of Procedure. The Takata-Ara test was performed in Jezlers modification, only without

    addition of fuchsin, which seems of no significance (van Ginkel and Hafstrom). 1 ml of 0.9 7 NaCl is pipetted off in 8 dwarf test tubes. 1 ml of serum is added

    in tube No. 1; the tube is shaken and the mixture transferred to the next tube. On this principle 1 ml is still transferred from tube t o tube after careful mixing. Next 0.25 ml of a 10 yo Na,CO, solution (prepared from anhydrous Na2C0,) is added in each tube, followed by addition of 0.3 ml of a 0.25 Yo HgCl, solution, after which the tubes are shaken. Reading after 24 hours standing a t room tem- perature.

    After Godtfredsen maximum precipitation in 3 tubes or more is indicated by +++. Marked precipitation in 3 tubes or more by ++. Beginning precipita- tion in 3 tubes or more by +.

  • 42 IN GER-LOU1 SE MARNER

    light > 1 month . . . . 127 41 (32%) moderate 1-2 months 51 (31%)

    Thymol Number Takata-Ara's Gros' Test Turbidity

    jerrminedl p2:t:ve 1 Positive 1 Positive Test 24 (19%) 121 (95%) 36 (22%) 137 (83%)

    Subchronic hepatitis . . . . . . . . . . . . . .I 31 I 26 (84%) I 24 (78%) I 30 (97%) Total I 31 I 26 (84%) 1 24 (78%) I 30 (97%)

    Chronic hepatitis . . . . . . . . . . . . . . . . . . (

    Cirrhosis of the liver { rz:F ' ' ' ' . ' 1 26 I 26 (100%) I 23 (88%) I 26 (100%)

    Total I 26 [ 26(100%) I 23 (88%) 1 26 (100%) 6 3 3 1 . . . . . .

    complication 5 1 1 2 L L U l l C -metastases 1 1; 1 ," (42%) I + metastases (8%) 1 0 2 1 1 Obstruction

    Total I 44 1 11 (25%) 1 2 (5%) I 5 (11%) Metastatic cancer of the liver . . . . , . I 21 I 10 (48%) 1 9 (43%) I 10 (48%) -___

    Total I 21 1 10 (48%) 1 9 (43%) 1 10 (48%) Stasis of the liver (Mb cordis) . . . . . .) 10 I 2 (20%) I 1(10%) I 3 (30%)

    Total 1 10 I 2 (20%) 1 1(10%) I 3 (30%) , \Veils disease . . . .. . . . . . . . . . . . . . . . . . 1 2 1 0 I 0 I Total 1 2 I 0 . I 1 I 0

    I 1

    Gros' test in Stolte's modification: 1 ml of 0.9 % NaCl is added to '/z ml of serum. Titration by addition by drops of 0.1 yo HgCI, till beginning turbidity. Then, after standing for 30 seconds, i t is tried whether small writing can be read through the turbidity. If this is difficult or impossible the titration is concluded. Thc titration value is indicated direct as the number of millilitres of HgCI, applicd. Values below 1.5 ml are positive.

    The thymol turbidity test: 0.05 ml of serum is added to 3 ml of a buffer (pH 7.8). prepared from barbitone, sodium barbitone, distilled water, and thymol. After thorough mixing, standing a t room temperature for half an hour. Reading is then undertaken in Pulfrich's photometer. Filter s. 72, 1 cm cuvetfes. Distilled water is used as fluid for comparison. The thymol turbidity value is indicated

  • THYMOL TURBIDITY TEST, TAKATA-ARAS TEST, AND GROS TEST IN ETC. 43

    direct as the extinction value, or in Maclagan units (the photometer can be standardized by means of simple protein solutions). The reaction is regarded as positive a t values above 0.15 (Maclagan units above 4).

    The examined 536 icteric patients can be classed within the following groups (see Table).

    Diagnosis.

    A diagnosis of hepatitis was here made on the basis of the usual criteria: Period of indisposition, successively increasing and decreasing jaundice with acholic faeces and choluria, a more or less typical clinical course, as well as absence of all signs and symptoms suggestive of a different origin of the jaundice.

    Tke cases were regarded as belonging to the group of acute hepatitis (even if the period of illness extended over 3 months), when clinical recovery was obtained while a t the same time the laboratory analyses showed no signs of a permanent liver damage.

    A special group comprises the cases in which, because of a certain influence of medicinal ireaiment or because of pregnancy, the possibility could not be denied of impaired resistance to liver infection or of a direct toxic damage. It is, however, as likely as not that, despite the complicated course, the cases were otherwise ordinary cases of hepatitis.

    Of the 10 patients constituting the group of patients under the influenceof medicinal treatment 9 had been treated with arsphenamine for syphilis less than 1 month prior to the occurrence of jaundice. In the 10th case large doses of cincho- phen had been given on account of uratic arthritis. The number and strengths of the positive reactions found in these cases corresponded approximately to the results of the tests for acute hepatitis in the other patients.

    The thymol turbidity test was positive in 5 out of 7 cases of hepatitis during pregnancy, while the Takata-Ara test and the Gros test were negative in all 7 cases. The positive thymol reaction cannot be explained as due to the pregnancy itself, for thymol turbidity tests done on 66 gravidae (after the 6th month of pregnancy) gave positive reaction in 1 case only, a patient who had shortly before been suffering from acute hepatitis. Hence the most likely explanation is one of light hepatitis in these 7 gravidae.

    The group of subchronic hepatitis is not sharply demarcated from those of acute and chronic hepatitis. In such cases only continued observation for a considerable length of time can show whether the disease is progressive. The disease is, how- wer , likely to cause chronic liver damage in the majority of these cases.

    The group of chronic hepatitis comprises, besides the cases verified by autopsy (14), also such cases where the clinical course showed a steadily deterioiating general state of health, while a t the same time the laboratory analyses revealed signs of increasing liver damage. In 2 of the 12 cases not submitted to autopsy the diagnosis was based on liver biopsy.

  • 44 INGER-LOUISE XIARNER

    The diagnosis of cirrhosis of the liver was in the ))certain)) cases verified by autopsy. In the dikelys cases the diagnosis was based on the clinical picture, liver biopsy, and demonstration of oesophageal varices.

    All diagnoses of obstructive jaundice were verified by operation or autopsy. By ))complication)) is here understood cholangitis, and possibly liver abscess formation.

    Kesults.

    It appears from the Table that of the three tests the thymol turbidity test was the one which most often gave positive reaction in the case of acute hepatitis. The difference was smaller when the cases of hepatitis assumed a more protiacted character, so as to be classed in the groups of subchronic or chronic hepatitis. Here also the Takata-Ara test and the Gros test gave positive reaction in the majority of the cases. In the cases of chronic hepatitis the Takata-Ara test was positive in 100 per cent, the Gros test positive in 88 per cent, and the thymol turbidity test positive in 100 per cent. Thus, the three tests gave almost parallcl results.

    In the only case examined here of acute yellow atrophy the patient died 10 to 12 days after the jaundice had manifested itself. All 3 tests gave highly positive reactions. The diagnosis was verified by autopsy.

    Previous investigators found, as mentioned before, positive Takata-Ara test in 90 to 100 per cent of the cases of cirrhosis of the liver. Grindsted found a positive Gros test in 12 out of 12 cases, and Maclagan a positive thymol turbidity test in 13 out of 13 cases. Although the present series did not have such a high per- centage of positive reactions (75 per cent), the Takata-ha test was also here positive more often than the two other tests, The values found in the Takata- Ara positive cases (55 per cent) were generally not so high as the corresponding values in chronic hepatitis.

    In 7 of the 20 cases of cirrhosis of the liver there was, simultaneously with a highly positive Takata-Ara test, found a completely negative thymol turbidity test. In 3 of these cases there was a past history of chronic alcoholism. (In 2 of the latter a Takata-Ara test and a thymol turbidity test were performed also on the ascitic fluid. Here, too, the Takata-Ara test proved to give a strongly positive reaction, whereas the thymol turbidity test was completely negative). In 1 of these cases the post-mortem examination revealed cirrhosis of the livcr attended by excessive hepatic steatosis.

    There is, perhaps, reason to remark that no more than 2 of the cirrhosis cases had a past history of hepatitis, and that the Takata-Ara test, as well as the Gros test, and the thymol turbidity test gave positive reactions in both these cases.

    The group of patients with cirrhosis of the liver thus contained cases differing serologically from each other. However, the significance of this fact from a patho- genetic, and possibly an aetiological point of view still remains an open question.

  • THYMOL TURBIDITY TEST, TAKATA-ARAS TEST, AND GROS TEST I N ETC. 45

    Both the Takata-Ara test and the Gros test gave reactions in all the 18 cases of uncomplicated obstructive jaundice due to gallstones. But in 1 case, where the jaundice had persisted for 9 months, and where the liver had hecome greatly enlarged because of stasis, the thymol turbidity test was found to give a slightly positive reaction in 3 of the 13 tests done here.

    In the cases of metastatic cancer of the liver the three tests gave almost the same results: more frequent and stronger reactions the greater the parenchyma damage.

    In stasis cirrhosis due to cardiac disease the 3 different tests were weakly positive only.

    As for the 2 cases of Weils disease, both the thymol turbidity test and the Takata-Ara test were negative, while the Gros test was positive in one of the cases, however, only on a single examination.

    Conclusions.

    The question of the value of these three tests for the differential diagnosis between the different diseases associated with jaundice -this question constituting the nucleus of the whole problem - can, perhaps, best be answered by a review of the results of the three tests in these different diseases. Previous investigations together with those presented here show as follows (see Table):

    In acute hepatitis the thymol turbidity test is most often positive (86 per cent positive), while the Takata-Ara test most often is negative (33 per cent positive), and the Gros test likewise most often negative (22 per cent positive).

    In acute (subacute) yelZow atrophy all tests give positive reactions. In protracted (ssubchronic))) hepatitis all three tests are positive in the majority

    of the cases (Takata-Ara test 84 per cent, Gros test 78 per cent, thymol turbidity test 97 per cent).

    In chronic hepatitis the Takata-Ara test has been found positive in 100 per cent, the Gros test in 88 per cent, and the thymol turbidity test in 100 per cent.

    In cirrhosis of the liver the facts are in a way the reverse of those in acute hepatitis, the Takata-Ara test being here more often positive (75 per cent) than the Gros test (35 per cent) and the thymol turbidity test (55 per cent).

    In obstructive jaundice due to gallstones all three tests give negative reactions in the great majority of cases, as long as there is found no cholangitis with conse- quent liver damage.

    In obstructive jaundice due to cancer (in pancreas or stomach) all three tests likewise seem to give negative results, a t least for long periods, not to become positive till the occurrence of secondary liver damages,

    In metastatic cancer of the liver all three tests will fairly often give positive reaction. The latter seems to bear a direct proportion to the parenchyma damage.

  • 46 INGER-LOUISE MARNER

    Thymol Ara,s Test Gros Test Turbidity

    Takata- 1 Test - - +(+) + + ++ ++ ++ +++

    f+ ++ - - +++ -

    In stasis of the liver due to cardiac disease all three tests may in a small proportion

    With regard to Weils disease the results seem uncertain. The conclusions that may perhaps be drawn from the facts stated here appear

    from the following small Table, which, however, should be accepted with some reserve, the reactions being by no means law-directed.

    of cases give a weakly positive reaction.

    suggestive of acute hepatitis ) x 1) --f subchr. hepatiti5 ) 1) chronic hepatitis ) 1) cirrhosis of the liver , I) obstructive iaundice

    Summary.

    Takata-Aras test, Gros test and the thymol turbidity test have been done on an average once weekly on 536 patients with jaundice, which means that each test has been performed 2754 times. The thymol turbidity test is the one which is most often positive in acufe hepatitis. By examination of 381 cases of acute hepatitis a positive Takata-Ara test was found in 33 per cent, a positive Gros test in 22 per cent, and a positive thymol turbidity test in 86 per cent.

    Among 31 cases of subchronic hepatitis a positive Takata-Ara test was found in 100 per cent, a positive Gros test in 88 pev cent, and a positive thymol turbidity test in 100 per cent.

    Among 20 cases of cirrhosis of the liver a positive Takata-Ara test was found in 75 per cent, a positive Gros test in 35 per cent, and a positive thymol turbidity test in 55 per cent. In 7 of the cases there was found a highly positive Takata-Ara test and a t the same time a negative thymol turbidity test, in 2 of these, both in serum and in the ascitic fluid.

    This fact suggests that cases of cirrhosis of the liver differ serologically. Among 23 cases of obsfructive jaundice due to gallstones a positive Takata-Ara

    test was found in 4 per cent, a positive Gros test in 4 per cent, and a positive thymol turbidity test in 8 per cent.

    Among 21 cases of obstructiue jaundice due to cancer the Takata-Ara test was positive in 48 per cent, the Gros test in 5 per cent, and the thymol turbidity test in 10 per cent.

    Among 21 cases of mefasfatic cancer of fhe liver the Takata-Ara test was positive in 48 per cent, the Gros test in 43 per cent, and the thymol turbidity test in 4.8 per cent.

  • THYMOI. TURBIDITY TEST, TAKATA-ARAS TEST, AND GROS TEST IN ETC. 47

    References:

    Brante, Gunnar: Svenska Lakartidningen 43: 2661, 1946. - Buch, Holger: Disputa- tion 1942. - Cohen, Ph. P. & F. L. Thompson: Journ. of Lab. and Clin. Med. 32: Nr. 5, 475, 1947. - Godtfredsen, Erik: Nord. Med. 5: 131, 1940. - Grindsted, Tage: Nord. Med. 31: 1879, 1945. - Gros, Walther: Klin. Wochenschr. 18: 781, 1939. - Gros, Walther: Klin. Wochenschr. 19: 130, 1940. - Hafstrsm, Torsten: Acta med. scand. Suppl. 62, 1935. - Iversen, K. & F. Raaschou: Nord. Med. 38: 824, 1948. - Jszler, Adolf: Zeitschr. f . klin. Med. 111: 48, 1929. - Jezler, Adolf: Verhandl. der Deutsch. Gesell. f . innere Med. 51. Kongress 1939. - Jezler, Adolf: Zeitschr. f . klin. Med. 114: 739, 1930. - King, h. J. & G. A. D. Haslewooh: The Lancet Nov. 14: 1153,1936. - Kunkel, H. G. & C. L. Hoagland: Proceed. of the SOC. for Exper. Biol. and Med. 62: Nr 2, 1946. - Ley, A. B., J. Lewis & C. Davidson: The Journ. of Lab. and Clin. Med 3: 910, 1946. - Maclagan, N. F.: Xature 154: 670, 1944. - Maclagan, N. F.: British. Journ. of Exp. Path. 25: 234, 1944. - Maclagan, N. F.: The Biochem. Journ. 39: XI, 1945. - Mac- lagan, N. F.: The Biochem. Journ. 39: XXII, 1945. - Martin: British. Journ. of Exp. Path. 27: 363, 1946. - Recant, L., E. Chargaff & F. M. Hanger: Proceed. of the SOC. for Exp. Biol. and Med. 60:245, 1945. - Shank, R. E. & C. L. Hoagland: The Journ. of Biol. and Clin. Med. 162: 133, 1946. - Watson, C. J. & E. L. Rappaport: The Journ. of Lab. and Clin. Med. 30: 983, 1945.