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The Weltmann reaction and the paper electrophoretic analysis of the serum globulins
VOL.
3 (1958)
531
CLINICA CHIMICA ACTA
THE WELTMANN
REACTION AND THE PAPER ELECTROPHORETIC
ANALYSIS
T. D. ULLMANN,
OF THE SERUM GLOBULINS
J. KLEEBERG
Laboratory of CLinica,l Research,
AND
E. HEIMANN-HOLLAENDER
Rothschild-Hadassah
University-Hadassah
University Hospital and The Hebrew
Medical School, Jerusalem
(Israel)
The introduction into the clinical laboratory of the quantitative analysis of the serum proteins by paper electrophoresis has caused many clinicians to abandon the older empirical, nonspecific methods of judging the properties of the serum proteins from their coagulative behaviour during heating or after addition of various salts. However, one of these empirical tests, the Weltmann reaction’, has for practical and theoretical reasons deservedly retained its place in many laboratories. The clinical value of this test lies in the fact that it is a reliable diagnostic aid in a great variety of conditions, characterized either by inflammation, exudation, or necrotic processes, which cause a shortening of the Weltmann coagulation band (W.C.B.), a “shift to the left”, or by proliferative and fibrotic processes, which are accompanied by a broadening of the W.C.B., a “shift to the right”2-6. From the theoretical point of view, the advantage of this test as compared with other protein tests, is that its results are not influenced by the total protein concentration, but only by increases in the proportions of the globulin fractions, either the y-globulins, or the a- and the ,&globulins. Thus it has been shown by various investigators4-7 that the shortening of the W.C.B. is caused by an increase in the a- and ,&globulin fractions, whilst the broadening of the W.C.B. is due to an increase in the y-globulin fraction. Likewise, it has been pointed out 5 that a simultaneous increase in all these fractions causes a “veiling” of the result of the Weltmann Reaction, i.e. an apparently normal W.C.B.; a finding which, by its contrast with the clinical condition and with the positive results of other tests, may be also of high diagnostic value89 B. The value of the Weltmann reaction as a diagnostic tool could be increased even more if the conditions leading to the shift of the W.C.B. into one direction or the other could be defined still better. Thus, it would be of interest to examine the influence on the Weltmann reaction of each serum globulin fraction separately, not only when it is present in increased quantity, but also when its quantity is diminished. One approach to this problem would be the statistical comparison of a sufficiently large number of results of the Weltmann reaction with those of simultaneously carried out electrophoretic serum analyses, based on the proportions existing between all the three main globulin fractions rather than on the absolute concentration of one fraction or the other. Another approach would be to examine sera which are deficient in one of the globulin fractions, but the only well known disorder with a complete lack of one of the globulin fractions is the rare condition called Agammaglobulinemia. In 1947, one of the present authors (J.K.) started to compare the results of the References p. 534
VOL. 3 (1958)
T. D. ULLMANN et al.
532
Weltmann reaction with those of an electrophoretic analysis of the serum proteins, using the method of TISELIUS. This study had to be abandoned, however, for technical reasons, in spite of promising results lo. The following study is based on the paper electrophoretic analysis of normal and pathological sera, in comparison with the results of the Weltmann reaction carried out on the same sera. In addition, the serum of a patient with Agammaglobulinemia* was examined, before and after the proportion between the originally present a- and j3-globulins and an extraneous y-globulin (Immune Serum Globulin, Lederle) had been changed in vitro. MATERIAL AND METHODS 1. Eighty-two non-selected serum samples were examined by the Weltmann reaction and the same samples were subjected to paper electrophoretic analysis. The Weltmann reaction was performed as described in WELTMANN’S original papers i and mentioned also in various books I-6. The paper electrophoresis was carried out in duplicate on Whatman No. I paper strips in 0.01 M barbiturate buffer with a pH of 8.6, at 0.3 mA for 14 h. Amido Black IO-B was used for staining. The quantity of the fractions was evaluated by mechanical reading on a recording densitometer (“Densigraph”, A. Thomas) and the areas under the density curves were measured with a Keuffel-Esser planimeter. The whole material was divided into ro groups, according to the results of the Weltmann reaction, from “W.C.B. 0” to “W.C.B. 9” (there was no serum resulting in “W.C.B. IO” in this group), and for each of these groups the average values of albumin, o-globulin, B-globulin and y-globulin, as well as the average ratios for o/p, a/y, p/y and (a + /?)/y were calculated. II. In the part of the study dealing with the serum of the patient with Agammaglobulinemia, the following procedure was adopted: The serum was mixed in vitro with various proportions of Immune Serum Globulin, resultinginmixtures having a y-Globulin content of o, 5, IO and 15%. The Weltmann reaction was performed on these mixtures. RESULTS
I. lables I and II contain the average values for the separate protein fractions, and for the proportions existing between the various globulin fractions, for each of the IO groups obtained by the Weltmann reaction. TABLE VALUES
AVERAGE
W.C.B.
FOR ALBUMIN
EACH
OF THE
“WELTMANN
0
I
No.
Number of samples Albumin y0 a-globulin O/a /l-globulin o/0 y-globulin y0
AND
GROUPS
MAIN
CALCULATED
2
3
4
No.
a/B o/Y B/r (a + B)/r
FRACTIONS
BAND”
TABULATED
FOR THE
0-9
__ 6
5
8
7
9
2
I
6
II
19
r5
8
2
32 40 14 ‘4
36 21 2G
7 28 26 22
II
22 45 20 13
33 24 18
37 21 17
38 20
40 15
32 17
15
38 17 ‘7
18
*I
23
25
28
29
16 29
'5 37
FROM
THE
GROUPS
W.C.B.
GLOBULIN
COAGULATION
TABLE RATIOS
I
o
I
2.3 3.8 1.6 5.1
2.9 2.9 I.0 3.9
AVERAGE
VALUES
“WELTMANN
II
FOR EACH
COAGULATION
OF THE BAND”
GLOBULIN
FRACTIONS
FOR
THE
O-9
._
3
4
5
6
7
8
9
0.8
I.2
1.4 I.3 2.6
1.3 I.1 0.8 1.8
1.2 0.9 0.7 1.6
1.3 0.7 0.6 I.3
1.0
1.3 2.1 3.4
0.7 0.7 I.2
0.9 0.5 0.6
I.I 0.5 0.4
I.0
0.8
2
* The clinical history of this patient has been published”. The serum of the patient contained no y-globulin at all, as shown by paper electrophoresis and as confirmed by moving boundary electrophoresis (TISELIUS) carried out in the Weizmann Institute of Science, Rehovoth (Israel). References p. 534
VOL. 3 (1958)
PAPER ELECTROPHORESIS
OF SERUM GLOBULINS
533
From these tables it is readily apparent that the average percentage values for the albumin fractions do not show any constant variation with the increasing number of the W.C.B. The same holds true, of course, for the percentage values of the sum of the globulin fractions, and therefore also for the albumin/globulin ratio. The percentage values of the a-fraction and also of the B-fraction show a certain tendency to fall as the W.C.B. number rises, but on the other hand, the figures for the y-fraction increase markedly, from 13-37%, ;ts the W.C.B. goes up from o-g. The figures calculated for the ratio between the a- and the p-fraction appear to be in no way related to the results of the Weltmann reaction. The ratios a/y and /3/yfall steadily, but in a not very pronounced manner, with the rising number of the W.C.B. 1 his tendency, however, becomes much more pronounced and consistent for the ratio calculated between the sum of the a- and ,8globulins, and the y-globulins, (a + /I)/r. In the following table (Table III) this ratio is shown compared with the results of the Weltmann reaction, the latter being divided for the sake of greater simplicity, only into 4 groups instead of the usual II groups (O-IO). This table conta.ins also the results of a statistical evaluation of the differences found. TABLE
III
(see text) Groups Comprising the W.C.B. Number of cases Ratio of (a + B)/y
I Nos.
o-2
9 3.92
SD.
II
III
IV
3-5 29 I.94
6-7 34
S-9
I.27
I.OI
79\,
1.61,
(p = 0.001)
IO
/o.44\,I,6/oJ9
2.3 (p = 0.02)
(p = 0.1)
i- values 5.3 (p < 0.001)
The ratio (a + ,9)/y shows a steady decline from j.gz-1.01 from group I to IV and statistical evaluation of the results shows that there is a significant difference between the groups I and II, II and III, and I and IV. It appears from this analysis that the ratio between the (a + /I)-globulins and the y-globulin is the deciding factor for the behaviour of the W.C.B., rather than the ratio between either the a- or the ,!?-globulin alone and the y-globulin. II. The Weltmann reaction carried out on the serum of the patient with Agammaglobulinemia gave the following results (Table IV). TABLE Mixture
of serum containing
100
95
5
IV Result of W.C.B.
3 5-6
IO :;
I5
87
534
T. D. ULmAhw et al.
VOL. 3 (1958)
This experiment shows clearly that in the presence of normal amounts of a- and /?-globulins, the lack in y-globulin causes a “shift to the left” in the Weltmann reaction and that this may be reversed by the addition of y-globulin. Thus, this experiment is an additional proof of the results based on the statistical analysis presented in Part I, that the results of the Weltmann reaction are closely related to the ratio existing between the a- and @-globulin fractions and the y-globulin fraction. COMMENT
Comparison of the results of the paper electrophoretic analysis of 82 sera with those of the Weltmann reaction carried out on the same samples, as presented in Part I of this study, furnishes statistical proof of the previously made observation2’ 4-7 that the results of the Weltmann reaction reflect quantitative changes in the various globulin fractions of the serum. From this study it appears, that it is the relative proportion existing between the globulin fractions, rather than their absolute values, which influence the outcome of the Weltmann reaction. The deciding ratio seems to be that existing between the combined a- and /?-globulins and the y-globulin. Thi: is also illustrated by the results of the experiment reported in Part II, in which the W.C.B. was shown to shift back to normal when y-globulin was added in vitro to a serum originally free from y-globulin and showing a “shift to the left”. On the basis of these studies it may be stated that the Weltmann reaction can furnish us with semi-quantitative information concerning the various serum globulin fractions. This property enhances the value of the reaction for the clinician, apart from its main value, which consists in the diagnostic help to be derived from it in many clinical conditions.
The results of a paper electrophoretic analysis of 82 sera were compared with the results of the Weltmann reaction carried out on the same samples. This comparison showed that a “shift to the left” is connected with an absolute and relative increase in the a-and /3-globulin fractions, whilst a “shift to the right” is connected with an absolute and relative increase in the y-globulin fraction. This relation was further confirmed by an in vitro experiment, in which the serum from a patient with Agammaglobulinemia was mixed with increasing amounts of y-globulin, resulting in a progressive increase in the number of the Weltmann Coagulation Band. The value of the Weltmann reaction as a semi-quantitative estimation of the serum globulin fractions is pointed out. REFERENCES 0. WELTMANN. Med. Klinik lMunichi 26 ~IO?O\ 240: 0. WELTMANN AND C. V. MEDVEI. ’ ’ _- ’ ’ 2. klin. Med., ;18 (1931) 670. ’ AND F. BODART. Blutk6r~erchensenkunr. _ Koaaulationsband und Blutbild, 4. Aufl., 2 R. KLIMA _ Urban und Schwarzenberg, .Wien, ‘947. 3 S. A. LEVINSON AND R. P. MACFATE, ClinicalLaboratory Diagnosis, 5th ed., Lea and Febiger. Philadelohia. 1056. 4 G. RIVA: Da; S~&meiweissbild, Huber Verlag, Bern, 1957. AND C. WUNDERLY. Die Bluteiweisskdrfierdes Menschen. 3. Aufl., Benno Schwabe 5 F. WUHRMANN und Co., Basel/Stuttgart, 1957. 6 H. ROSEGGER, Ergeb. inn. Med. u. Kinderheilk., 57 (1939) 183. S. SCHERLIS AND 6. S. LEVY, Bull. Johns Hopki&Hosp.. 71 (1942) 24. ?3 B.-S. DJORDJEVIC AND A. ROTOVIC, Arch. maladies coeur et vaisseaux, 42 (1949) gog. 9 J. KLEEBERG AND A. EINHORN, rgth Sci., Congr. Palestine Jewish Med. Assoc., 1946. J. KLEEBERG,A.KATCHALSKYAND N.S~~~~~~~~,unpublishedobservations. IO II A. FREUNDLICH AND Z. SHAMIR, Harefuah, 48 (1955) 264. I
Received
March z4th,
1958
3 (1958)
531
CLINICA CHIMICA ACTA
THE WELTMANN
REACTION AND THE PAPER ELECTROPHORETIC
ANALYSIS
T. D. ULLMANN,
OF THE SERUM GLOBULINS
J. KLEEBERG
Laboratory of CLinica,l Research,
AND
E. HEIMANN-HOLLAENDER
Rothschild-Hadassah
University-Hadassah
University Hospital and The Hebrew
Medical School, Jerusalem
(Israel)
The introduction into the clinical laboratory of the quantitative analysis of the serum proteins by paper electrophoresis has caused many clinicians to abandon the older empirical, nonspecific methods of judging the properties of the serum proteins from their coagulative behaviour during heating or after addition of various salts. However, one of these empirical tests, the Weltmann reaction’, has for practical and theoretical reasons deservedly retained its place in many laboratories. The clinical value of this test lies in the fact that it is a reliable diagnostic aid in a great variety of conditions, characterized either by inflammation, exudation, or necrotic processes, which cause a shortening of the Weltmann coagulation band (W.C.B.), a “shift to the left”, or by proliferative and fibrotic processes, which are accompanied by a broadening of the W.C.B., a “shift to the right”2-6. From the theoretical point of view, the advantage of this test as compared with other protein tests, is that its results are not influenced by the total protein concentration, but only by increases in the proportions of the globulin fractions, either the y-globulins, or the a- and the ,&globulins. Thus it has been shown by various investigators4-7 that the shortening of the W.C.B. is caused by an increase in the a- and ,&globulin fractions, whilst the broadening of the W.C.B. is due to an increase in the y-globulin fraction. Likewise, it has been pointed out 5 that a simultaneous increase in all these fractions causes a “veiling” of the result of the Weltmann Reaction, i.e. an apparently normal W.C.B.; a finding which, by its contrast with the clinical condition and with the positive results of other tests, may be also of high diagnostic value89 B. The value of the Weltmann reaction as a diagnostic tool could be increased even more if the conditions leading to the shift of the W.C.B. into one direction or the other could be defined still better. Thus, it would be of interest to examine the influence on the Weltmann reaction of each serum globulin fraction separately, not only when it is present in increased quantity, but also when its quantity is diminished. One approach to this problem would be the statistical comparison of a sufficiently large number of results of the Weltmann reaction with those of simultaneously carried out electrophoretic serum analyses, based on the proportions existing between all the three main globulin fractions rather than on the absolute concentration of one fraction or the other. Another approach would be to examine sera which are deficient in one of the globulin fractions, but the only well known disorder with a complete lack of one of the globulin fractions is the rare condition called Agammaglobulinemia. In 1947, one of the present authors (J.K.) started to compare the results of the References p. 534
VOL. 3 (1958)
T. D. ULLMANN et al.
532
Weltmann reaction with those of an electrophoretic analysis of the serum proteins, using the method of TISELIUS. This study had to be abandoned, however, for technical reasons, in spite of promising results lo. The following study is based on the paper electrophoretic analysis of normal and pathological sera, in comparison with the results of the Weltmann reaction carried out on the same sera. In addition, the serum of a patient with Agammaglobulinemia* was examined, before and after the proportion between the originally present a- and j3-globulins and an extraneous y-globulin (Immune Serum Globulin, Lederle) had been changed in vitro. MATERIAL AND METHODS 1. Eighty-two non-selected serum samples were examined by the Weltmann reaction and the same samples were subjected to paper electrophoretic analysis. The Weltmann reaction was performed as described in WELTMANN’S original papers i and mentioned also in various books I-6. The paper electrophoresis was carried out in duplicate on Whatman No. I paper strips in 0.01 M barbiturate buffer with a pH of 8.6, at 0.3 mA for 14 h. Amido Black IO-B was used for staining. The quantity of the fractions was evaluated by mechanical reading on a recording densitometer (“Densigraph”, A. Thomas) and the areas under the density curves were measured with a Keuffel-Esser planimeter. The whole material was divided into ro groups, according to the results of the Weltmann reaction, from “W.C.B. 0” to “W.C.B. 9” (there was no serum resulting in “W.C.B. IO” in this group), and for each of these groups the average values of albumin, o-globulin, B-globulin and y-globulin, as well as the average ratios for o/p, a/y, p/y and (a + /?)/y were calculated. II. In the part of the study dealing with the serum of the patient with Agammaglobulinemia, the following procedure was adopted: The serum was mixed in vitro with various proportions of Immune Serum Globulin, resultinginmixtures having a y-Globulin content of o, 5, IO and 15%. The Weltmann reaction was performed on these mixtures. RESULTS
I. lables I and II contain the average values for the separate protein fractions, and for the proportions existing between the various globulin fractions, for each of the IO groups obtained by the Weltmann reaction. TABLE VALUES
AVERAGE
W.C.B.
FOR ALBUMIN
EACH
OF THE
“WELTMANN
0
I
No.
Number of samples Albumin y0 a-globulin O/a /l-globulin o/0 y-globulin y0
AND
GROUPS
MAIN
CALCULATED
2
3
4
No.
a/B o/Y B/r (a + B)/r
FRACTIONS
BAND”
TABULATED
FOR THE
0-9
__ 6
5
8
7
9
2
I
6
II
19
r5
8
2
32 40 14 ‘4
36 21 2G
7 28 26 22
II
22 45 20 13
33 24 18
37 21 17
38 20
40 15
32 17
15
38 17 ‘7
18
*I
23
25
28
29
16 29
'5 37
FROM
THE
GROUPS
W.C.B.
GLOBULIN
COAGULATION
TABLE RATIOS
I
o
I
2.3 3.8 1.6 5.1
2.9 2.9 I.0 3.9
AVERAGE
VALUES
“WELTMANN
II
FOR EACH
COAGULATION
OF THE BAND”
GLOBULIN
FRACTIONS
FOR
THE
O-9
._
3
4
5
6
7
8
9
0.8
I.2
1.4 I.3 2.6
1.3 I.1 0.8 1.8
1.2 0.9 0.7 1.6
1.3 0.7 0.6 I.3
1.0
1.3 2.1 3.4
0.7 0.7 I.2
0.9 0.5 0.6
I.I 0.5 0.4
I.0
0.8
2
* The clinical history of this patient has been published”. The serum of the patient contained no y-globulin at all, as shown by paper electrophoresis and as confirmed by moving boundary electrophoresis (TISELIUS) carried out in the Weizmann Institute of Science, Rehovoth (Israel). References p. 534
VOL. 3 (1958)
PAPER ELECTROPHORESIS
OF SERUM GLOBULINS
533
From these tables it is readily apparent that the average percentage values for the albumin fractions do not show any constant variation with the increasing number of the W.C.B. The same holds true, of course, for the percentage values of the sum of the globulin fractions, and therefore also for the albumin/globulin ratio. The percentage values of the a-fraction and also of the B-fraction show a certain tendency to fall as the W.C.B. number rises, but on the other hand, the figures for the y-fraction increase markedly, from 13-37%, ;ts the W.C.B. goes up from o-g. The figures calculated for the ratio between the a- and the p-fraction appear to be in no way related to the results of the Weltmann reaction. The ratios a/y and /3/yfall steadily, but in a not very pronounced manner, with the rising number of the W.C.B. 1 his tendency, however, becomes much more pronounced and consistent for the ratio calculated between the sum of the a- and ,8globulins, and the y-globulins, (a + /I)/r. In the following table (Table III) this ratio is shown compared with the results of the Weltmann reaction, the latter being divided for the sake of greater simplicity, only into 4 groups instead of the usual II groups (O-IO). This table conta.ins also the results of a statistical evaluation of the differences found. TABLE
III
(see text) Groups Comprising the W.C.B. Number of cases Ratio of (a + B)/y
I Nos.
o-2
9 3.92
SD.
II
III
IV
3-5 29 I.94
6-7 34
S-9
I.27
I.OI
79\,
1.61,
(p = 0.001)
IO
/o.44\,I,6/oJ9
2.3 (p = 0.02)
(p = 0.1)
i- values 5.3 (p < 0.001)
The ratio (a + ,9)/y shows a steady decline from j.gz-1.01 from group I to IV and statistical evaluation of the results shows that there is a significant difference between the groups I and II, II and III, and I and IV. It appears from this analysis that the ratio between the (a + /I)-globulins and the y-globulin is the deciding factor for the behaviour of the W.C.B., rather than the ratio between either the a- or the ,!?-globulin alone and the y-globulin. II. The Weltmann reaction carried out on the serum of the patient with Agammaglobulinemia gave the following results (Table IV). TABLE Mixture
of serum containing
100
95
5
IV Result of W.C.B.
3 5-6
IO :;
I5
87
534
T. D. ULmAhw et al.
VOL. 3 (1958)
This experiment shows clearly that in the presence of normal amounts of a- and /?-globulins, the lack in y-globulin causes a “shift to the left” in the Weltmann reaction and that this may be reversed by the addition of y-globulin. Thus, this experiment is an additional proof of the results based on the statistical analysis presented in Part I, that the results of the Weltmann reaction are closely related to the ratio existing between the a- and @-globulin fractions and the y-globulin fraction. COMMENT
Comparison of the results of the paper electrophoretic analysis of 82 sera with those of the Weltmann reaction carried out on the same samples, as presented in Part I of this study, furnishes statistical proof of the previously made observation2’ 4-7 that the results of the Weltmann reaction reflect quantitative changes in the various globulin fractions of the serum. From this study it appears, that it is the relative proportion existing between the globulin fractions, rather than their absolute values, which influence the outcome of the Weltmann reaction. The deciding ratio seems to be that existing between the combined a- and /?-globulins and the y-globulin. Thi: is also illustrated by the results of the experiment reported in Part II, in which the W.C.B. was shown to shift back to normal when y-globulin was added in vitro to a serum originally free from y-globulin and showing a “shift to the left”. On the basis of these studies it may be stated that the Weltmann reaction can furnish us with semi-quantitative information concerning the various serum globulin fractions. This property enhances the value of the reaction for the clinician, apart from its main value, which consists in the diagnostic help to be derived from it in many clinical conditions.
The results of a paper electrophoretic analysis of 82 sera were compared with the results of the Weltmann reaction carried out on the same samples. This comparison showed that a “shift to the left” is connected with an absolute and relative increase in the a-and /3-globulin fractions, whilst a “shift to the right” is connected with an absolute and relative increase in the y-globulin fraction. This relation was further confirmed by an in vitro experiment, in which the serum from a patient with Agammaglobulinemia was mixed with increasing amounts of y-globulin, resulting in a progressive increase in the number of the Weltmann Coagulation Band. The value of the Weltmann reaction as a semi-quantitative estimation of the serum globulin fractions is pointed out. REFERENCES 0. WELTMANN. Med. Klinik lMunichi 26 ~IO?O\ 240: 0. WELTMANN AND C. V. MEDVEI. ’ ’ _- ’ ’ 2. klin. Med., ;18 (1931) 670. ’ AND F. BODART. Blutk6r~erchensenkunr. _ Koaaulationsband und Blutbild, 4. Aufl., 2 R. KLIMA _ Urban und Schwarzenberg, .Wien, ‘947. 3 S. A. LEVINSON AND R. P. MACFATE, ClinicalLaboratory Diagnosis, 5th ed., Lea and Febiger. Philadelohia. 1056. 4 G. RIVA: Da; S~&meiweissbild, Huber Verlag, Bern, 1957. AND C. WUNDERLY. Die Bluteiweisskdrfierdes Menschen. 3. Aufl., Benno Schwabe 5 F. WUHRMANN und Co., Basel/Stuttgart, 1957. 6 H. ROSEGGER, Ergeb. inn. Med. u. Kinderheilk., 57 (1939) 183. S. SCHERLIS AND 6. S. LEVY, Bull. Johns Hopki&Hosp.. 71 (1942) 24. ?3 B.-S. DJORDJEVIC AND A. ROTOVIC, Arch. maladies coeur et vaisseaux, 42 (1949) gog. 9 J. KLEEBERG AND A. EINHORN, rgth Sci., Congr. Palestine Jewish Med. Assoc., 1946. J. KLEEBERG,A.KATCHALSKYAND N.S~~~~~~~~,unpublishedobservations. IO II A. FREUNDLICH AND Z. SHAMIR, Harefuah, 48 (1955) 264. I
Received
March z4th,
1958