CA1129319A - In vitro testing method for the diagnosis of malignant tumours - Google Patents

Abstract

Abstract An in vitro testing method for the diagnosis of malignant tumours, preferably human tumours, comprising determining the mobility variation of cellular blood components contacted with a migration inhibition factor (MIF), wherein the MIF is secreted from lymphocytes being sensisized against tumour tissue, whereby subjecting, as said cellular blood components, the lymphocytes as a part of a leucocyte suspension to be examined prior to a migration inhibition test. The lymphocytes are contacted with an antigen capable of stimulating them to produce MIF. The leucocyte migration is inhibited and fixed after a predetermined period of time. The leucocytes migrate in a capillary intermediate layer having a thickness of from 1 to 20 microns and being at least partially filled with liquid. Said intermediate layer is formed between a glass surface and an agar layer. The invention further refers to the construction and preparation of a testing plate or dish.

Description

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The present invention relates to an in vitro testing method for the diagnosis of malignant tumours, prefe-rably human tumour~ comprising determining the mo~ility varlation of cellular blood components contacted with a migra~ion inhibition factor (MIF), wherein the MIF
is secreted from lymphocytes being sensitized against tumour tissue. Such MIF is not secreted when the lymphocytes come from patients who do not suffer from malignant tumo~,,r(malignoma) since these lymphocytes are not sensitized and, therefore, fail to release MIF when contacted with tumourantigen.

It is kno~m from literature (~ouwes, F. R. et al.;
Verh. Deutsche Ges. inn. Med., Vol. 82, J. F. Bergmann-Verlag, Munich) that the early diagnosis of malignant diseases can be made by means of the so-called electro-phoresis mobility test (EMT). The principle of the EMT is based upon the fact that the velocity of migration (mobility) of ~pecific bodies li. e. macrophagesJ
granulozytes and other blood corpuscles) visible in the electric ield of a special microscope (zytophero-meter) is only varied when these bodies come lnto ., .. , . . .......... ~ . , .
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- 2 -contact with lymphokines of sensitized lymphocytes.
It is hereby assumed that lymphocytes which are sen-sitized against a specific antigen (so-called T-lymphocytes) release a series of soluble factors, i.
e. the so-called lymphokines. An 1mportantone among these factors is the migration inhibition factor (MIF3.
This factor affects the capability of migration of blood corpuscles, especially of macrophages and granulocytes, and provides for their arresting and accumulation at the place where the antigen inducing the sensibilization is present. In vivo, this represents a possibility to destroy the tumour.

Lymphocytes of patients suffering from malignoma react in specific manner by releasing the so-called soluble factors, when contacted with a basic protein obtained from the brain substance on the one hand, or from carcinoma tissue on the other hand. The basic proteins causing the specific reactions of the lymphocytes, are termed encephalitogenous factor (EF) and basic protein from carcinoma tissue (CaBP). They are produced in accordance with known per se methods (Caspary, E. A. et al., Brit. Med. 1971, pages 61~

- 617, and other publications; compare Douwes F. R., loc. ~it.).

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The results of blood test with the EMT as described in literature reveal that the test allows an almost 100 % differentiation of the samples to be made so as to determine whether the samples have been taken from an organism affected by malignant (tumour) diseases or from a healthy organism or an organism affected by non-malignant diseases, respectively. All of the patients tested and suffering from (malignant) tumour provided a positive lymphocyte response, i. e. the lymphocytes yielded a factor resulting in a measurable and characteristic variation of the mobility of indicator cells (compare Douwes, F. R., loc. cit.). Control diagnoses with patients affected by non-malignant diseases did not provide such lymphocyte response.
Incorrect results could be traced back to specific causes. Accordingly, the EMT can be considered to be the presently most sensitive test for cancer.

It is considered to be disadvantageous in the perform-ance of the test that qualified control of an instrument being relatively complicated to operate, i. e. the zytopherometer, is required. The personnel okserving and measuring the speed of migration must be highly trained and supervised very carefully. Owing to di-minishing personnel attention, the operators can be 31~29;~i9 engaged with the work for a limited period of time only.
Accordingly, the use of the EMT is restricted to highly specialized institutes. The analysis of the test is made while the blood corpuscles are in motion, i.e. dynamically.
"Stopping" of the experiment is not possible; therefore, repeated measurements cannot be taken, either.

It is the object of the prescnt invention to provide a testing method for the diagnosis of malignomae, which method is as general and unspecific as possible a manner with respect to the type of the malignomae and with a high degree of accuracy or reliability signals the presence of malignomae, but owing to its priciple may be used or performed in any medical laboratory, while lending itself particularly for use in mass examination for preca~tional diagnosis of cancer. The result of the test should be readily verifiable. The test should be adapted to the analyzed statistically and recorded without any intermediate protocolling.

Thesc objects are solved in a testing method as defined above by subjecting as said cellular blood components the lymphocytes as a part of a leucocyte suspension to be examined prior to a known per se migration inhibition test wherein the , . ~
. :

lymphocytes are contacted with an antigen capable of stimulating them to produce MIF, and inhibiting and fixing the leul~ocyte migration after a predetermined period of time.

Various migration inhibition tests are known which are principally suitable for the examination in accordance with the basic object. Among other methods, the so-called capillary technique by S0BORG and BENDI~EN is known. For the realization of the inventive idea, it is necessary to detect the mobility of the indicator cells by "freezing" a state of migration reached a~ter a given period of time.

According to the invention, preferably the propagation or variation of mobility of the lQ~ocytes within an extremely fine "open" capillary layer of about 1O
microns ~m) thickness is measured. A test system has been found to be particularly suitable, which comprises a dual-layer element, namely a carrier or substrate made of an inert medtum such as e.g. glass, and an agar-agar being saturated with nutrients for ~lood cells. A particularly suitable agar is one sold under the tradename Agarose as produced by the firm Behring-Werke of Marburg (Germany).

' llZ93~9 Preferably, the intermediate layer between the agar and the carrier layer is partially filled with liquid.
This layer has a thickness of about 1 to 20 microns.
Advantageously, an incubation period in the MIT of between 10 and 30 hours is chosen.

The test is performed with a sample of leucocyte-rich supernatant phase ("buffycoat") obtained from human blood and containing a fixed number of leucocytes of between 104 and 107 per sample. These standard values are approved and tried values which do not require an excessive quantity of blood to be taken from the patients.

The buffycoat sample is mixed with an antigen solution in a ratio of from 1 : 50 to 1 : 10. To this end, antigen solutions may be used which have been obtained by a method disclosed by CASPARY and FIELD or by means of a 3 M KCl extraction according to MELTZER. As the starting material, human malignoma tissue from surgical operations or human brain substance are employed.

However, it is also feasible to yield antigen solution from tumour cells grown in vitro.

The testing method according to the invention i6 not only applicable to an unspecific malignoma examination, llZ~3~

but may be extended also to an organo-specific examination. If antigens are employed which have been recovered from carcinomous tissue of specific ~rgans, lymphocytes of patients affected by the same organic carcinomae react in the indicated manner.
It has been disclosed in literature that antigen solution obtained, for instance, from mammary carcinoma according to CASPARY and FIELD or MELTZER, result in a specific reaction exclusively ~ith respect to lymphocytes coming from persons affected by mamary carcinoma.

In the case that a positive immune response in a general test is present, i.e. in the case of suspected carcinoma, it is also possible to perform a series of tests by us1ng organo specific antigens, and thereby to determine within a relatively short per~od of time, what organs might be affected by carcinoma.

It is of great importance that the so-called immune response, i.e. the production of the slowing effect in the mo~ement of the indicator cells in the course of existing tests, can be suprisingly enhanced by adding to the sample solution a ~olution containing the so-called transfer factor. Such factor is known .

~129319 from immunology (compare e.g.B.M. Rosenthal; "Der Transferfaktor und seine therapeutische Anwendung";
Schweiz. med. Wschr., Vol. 104, 1974; pages 1501 -1506). The transfer factor may be produced from normal leucocytes of the blood. This factor represents a subcellular, dialyzable, non-immunogeneous leuco-cyte factor which is responsible for the T-lymphocyte responsive reaction. Transfer factor preparations are commercially available (SCHURA, Krefeld/Germany) which have been prepared by fractionation in liquid oxygen and subsequent gradual ultrafractionation.
One commercially available unit contains transfer factor of about 5 x 101 leucocytes. The preparation is stabilized by means of 1 % solution of human albumin.

It is found that the addition of transfer factor may yield an increase in the immune response. Thus, the discrlmination of the various sample tests is substantially improved by the add~tion of the transfer factor.

Further, the present invention relates to a test dish or plate for carrying out the method, which plate comprises a planar carrier plate of an inert materlal, e.g. glass, havlng coated thereon a solidifled agar , ;-112931~:1 - g layer of from 2 to 5 mm thickness and having at least two substantially circular punched holes of a diameter of the order of from o,5 to 5 mm ~preferably from 1,1 to 3,0 mm). Each punched hole has a capacity of from 5 to 20 and preferably 8 microliters of test substance.

It is essential that the punched holes are cut with such a configuration that uniformity of the capillary migration layer is provided even in the transition area between the punched hole and the layer.

Below, the invention is explained in Examples, and furthermore, the following Figures are added:

igure 1 shows in plan view a test plate according to the invention; and igure 2 shows a test plate in cross-sectional view.

Exam~le 1 A) Prepaxation of the le~cocyte suspension 30 ml of human blood are heparinized with 300 USP
units of Na heparinate. The heparinized human blood ~Z~3 ~

(from a patient) is substrated within a plastic ferrule with 8 ml of 6 percent by weight dextran solution in physiological normal salt solution (molecular weight of the dextran: 75,000). Thereupon, the erythrocytes are allowed to sediment (settle) in a heating cabinet for about 45 minutes at 37 C. The leucocyte enriched supernatant phase (buffyco2t) is pipetted off and mexed with an equal volume of 0,9 percent by weight NaCl solution and then centrifuged at a centrifugal force of 750 G's. The pellet deposited in the centrif-uge is again washed for two tLmes in 10 ml of Hank's solution (pH 7.2) each and suspended in a Tc 199 medium (producer: Serva, Heidelberg). During washiny and suspending, the cells are aqitated by means of a pipette until lumps of cells are no longer visible to the naked eye. Upon dyeing with Turk's solution, the leucocytes arecounted in the Neubauer compartment and asjusted to 2 x 105 cells/microliter in Tc 199 medium. In the average, the sample contains 25 % of mononuclear cells (lymphocytes and monocytes) as well as 75 % of granulocytes. The erythrocyte contamination i~ le s than 1 to 1.

B) Preparation of the anti~en solution For the preparation of the antigen solution, the llZ93~9 encephalitogenous factor (EF) or the basic protein of carcinoma tissue (CaBP) has to be isolated.

The tumour or the brain, respectively, are processed as rapidey as possible under sterile conditions after the surgical removal. Connective tissue and healthy tissue are removed from the tumour. There-after, the tumour or brain tissue it cut into small pieces with sterile scissors (scalpel) and further disintegrated in a bath of ice in a homogenizer. The bumour tissue must not be heated during such processing.
The homogenate is suspended in four times the volume of distilled water and then centrifuged at a force of 23.000 G's. The thus produces pellet is again suspende~ in a fourfold volume of 0.9 ~ NaCl, homogenized and centrifuged at 23.000 G's for 30 minutes. The pellet again suspended in four times the ~olume of dis~illed water is thereafter freeze-dried. The freeze-dried powder is blended with a mixture (2 : 1) of chloro-form and methanol in a ratio of 1 : 10 and agitated for 30 minutes. The mixture is centrifuged for 10 minutes at 600 G's. The supernatant phase is discar-ded.

This process ~tep i8 repeated twlce. The pellet is ~ ~2~31~

then air dried. The dried pellet is resuspended in the fivefold volume of a 5 % NaCl solution in water, and centrifuged at 23,000 G's for 30 min. By adding N/100 NaCl, the pellet again resuspended in a five-fold volume of distilled water is adjusted to a pH
of 3.5. The suspension is slightly shaked and there- ;
after again centrifuged at 23,000 G's for 30 minutes.
The supernant phase is discarded. Again, the pellet is resuspended in a fivefold volume of distilled water which is adjusted to a pH of 2.6 with N/100 ~Cl, and allowed to stand for from 3 to 18 hours.
The pH value is not allowed to vary during this period.
Then, the suspension is centrifuged at 23,000 G's for 30 minutes. The supernant phase is stored, and the pellet is again washed in N/100 NaCl at a pH of 2.6 and centrifuged. The supernatant phase resulting fr~m this step is stored. The two lastmentioned super-natant phaSeS are combined and then dialyzed~distilled water. Hereupon, the product ls subjected to freeze-drying.

An antigen test solution is obtained by dissolving 0.~ mg/ml of the freeze-drled substance in dlstilled water. Principally the same process of preparation is employed for obtaining a solution of the baslc protein 11~2931~

from tumour tissue (CaBP).

C) Preparation of the test plate (compare also Figures 1 and 2) 0.5 g of a specific culture agar (agarose; producer:
Behring-Werke of Marburg) are weighted into a 50 ml Erlenmeyer bulb or flAsk. 22.5 ml of sterile aqua bidestillata are added to the mass.

22.5 ml of the double concentrated Tc 199 medium and 5 ml of plasma obtained from human blood are charged into a second Erlenmeyer bulb. The agarose is dissol-ved in a boiling water bath by being supported by a bracket, under slight circulatory agitation. Hereby, the Erlenmeyer bulb is sealed with a film or stopper to prevent a part of water from evaporating.

When the agarose is completely dissolved, the substance is cooled to 47 C in a water bath. In the s~me bath, the medium-plasma mixture is preheated and added to the agarose under shaking, such that there are now pre ent 50 ml of 1 % agarose ln plain Tc 193 medium containing 10 ~ of plasma.

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With the ald of a preheated discharge pipette, 5 ml each of the agar are filled into circular tissue culture dishes (Petri dishes) 10 having a diameter of 50 mm, which dishes are shown in plan view in Figure 1. Upon solidification of the agar, holes 11 of 2,5 mm diameter are punched out from the agar by means of a telescoping punching canule ~ehring-Werke, Marburg) with the aid of a template. In the punching work, care has to be taken that the agar layer is not released from the glass layer in the punching area, in order that the capillary layer is not increased such that, migration of the leucocytes would no longer be possible.

The pattern of the holes 11 appears from Figure 1.
Of course other configurations are imaginable.
Figure 2 illustrates an enlarged sectional view of the lower part of dish 10. ~s indicated, the agar layer 1 is separated from the glass layer of the dish 10 by a thin capillary layer 3. The various columns of holes are provided e. g. for the following purposes: I
~A) Control (standard) (B) Test ~uspension contain$ng general antigen ;.
(C) Test suspension conatining organo-specific antigen 112931~
- 15 ~

(D) Test suspension containing organo-specific anti-gen and transfer factor.

D) Reaction, incubation and fixation , The following preparations are provided for a test:
1) A preparation containing each 100 microliters of leucocyte suspension according to (A) and 8 micro-liters of antigen suspension according to (B);
2) a preparation containing each 100 microliters of leucocyte suspension and 8 microliters of N/100 NaCl solution (for reference value).

The mixed preparations 1 and 2 are subjected to incu-bation for 60 minutes in a heating cabinet at 37 C
and at 100 ~ humidity. Then, 8 microliters of leuco- :
cyte- and antigen-containiny substance or standard substance, respectively, of the incubated solution are filled into each punched hole. Depending on the configuration of the test plate (number of punched holes~, from 2 to 5 measurement results and from 2 to 5 reference values are available for the analysis of the leucocyte migration and the effect of the factors produced by the lymphocytes and delaying the migration.
~he cell~ contained in the suspension and introduced 112931~

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lnto the punched holes at the start of the test, migrate into the intermediate layer 3 between the agar and the survace of the dish. By incubating the dishes at a temperature of 37 C in the heating cabinet for a period of 18 houxs, such migration is maintained across a definite distance. The leucocytes do not migrate into the agar, but rather take their nutrients therefrom. Incubation is effected in a humidified cabinet at a relative humidity of 100 %.

Following the incubation, the plates are coated with methanol for 15 minutes, and subsequently with for-malin solution (37 percent by weight of methanal) for 10 minutes. Thereafter, the agar is carefully stripped from the bottom of the dish as a membrane. The cells migrated in the intermediate layer are now fixed to the surface of the dish. Upon drying, the cells are dyed ~n accordance with the PappenheLm method, where-by dark-violet halos 4 (m~gratlon halos) are formed on the plate surface (compare Figure 1).

E~ Measuring and determinin~ the results ~slng a graduated mag~ifying glass (Bausch & Lomb), the dlameter of the migration halo is measured. In llZ93~9 such measurement, the polymorpho-nuclear cells discre-tely positioned in the periphery should be detected, too. The arithmetic average of a plurality of mea-suring steps each is formed.

The quotient of the migration area F1 with antigen and of the migration area F2 without antigen yields a "migration index" MI:

MI F1/F2.

A migration index smaller than 0.85 means inhibition of the leucocytes to significant degree and, thus, a positive cell response. The following Table illustra-tes one example of measurement:

Column 0 (mm) F (mm2~ MI
__ _ . _ B 6,75 F1 = 35,8 0,80 A 7,55 F2 = 44~75 (1) 1~93~

Initial experiments with a total of 97 female patienb were conducted to demonstrate the effectiveness of the migration inhibition test in the case of mammary carcionmae. 27 among 46 female patients tested and affected by mammary carcinoma showed an inhibition higher than 15 ~ (= 58.7 %), i. e. the migration index MI is less than 0.85. Among 23 female patients tested and suffering from mastopathia, only four patients showed such inhibition ~= 17 %), whereas in the case of female patients affected by fibroadenoma (fibroma), one female patient out of five (=20 %) showed an inhi-bition. The following Table provides a general summary:

Diagnosis Number of patients Migration index (n)(inhibited) (normal) .
Controls 20 0 (O %) 20 (100 %) Mastopathia cystica 23 4 (17 %) 19 (83 %) Fibroadenoma 5 1 (20 ~) 4 (80 %) mammary cysts 4 0 ( 0% ) 4 (100 ~) Other malignant diseases 9 3 (33 %) 6 ~67 %~
Mammary carcinoma 46 27 (58.7%) 19 (41.3 %) Example 2 Employment of the transfer actor for increasing the lmmune respon~e.

1~29319 It has already been noted that an increase of the im-mune response may be cbtained by the addition of a solu-tion containing transfer factor (producer: SCHURA, Xrefeld3. It is found that with such addition the factor MI is more accentuated as compared to Test -Example 1.

The following test compositions were prepared:
1) 100 microliters of leucocyte suspension + 8 micro-liters of antigen solution 2) 100 microliters of leucocyte suspension + 8 micro-liters of antigen solution + 20 microliters of transfer factor solution (prepared by SCHURA;
1 microliter corresponding approximately to the transfer factor of 5 x 10 leucocytes) 33 100 microliters of leucocyte suspension 4) 100 microliters of leucocyte suspension + 20 micro-liters of transfer factor solution as in composition 2).

Incubation and preparation of the dish are made in the same manner as in Example 1. A dish having four t$mes f$ve punched holes (diameter 2.5 mm) is pre-pared. One column of 5 holes each is charged with 10 microllters o~ test compositlons 1 to 4.

~l~g3~9 Results (Average of five values each):

Composition Column Diameter (mm) F (mm2) MI
_ _ __ _ _ 1 B 6,90 37,4 0,79 2 C 6,05 28,7 0,61 (incl.
transfer-factor)

3 A 7,75 47,2 1,0 standard!

4 D 7,70 46,6 0,99 The low MI of columns of B/C indicates a malignoma case.

Results of the migration inhibition test by using the transfer factor in the determination of gastrointestinal tumors:

Measurements were made in accordance with the m~asuring instruction according to Example 2. No appreciable inhibition was found in a (health) group of patients; the migration index was of the order of 1.02 * O.l. The trans~er fractor did not affect these results. In the group of patients by colonic carcinomae, the migration index amounted to 0.65 ~ 0.2.

` ~ .

l~Z9319 Actually, 11 out of 15 cases tested showed a migration index smaller than 0.85. Upon addition of the transfer factor, the migration index was of the order of 0,61 ~ 0.09. Furthermore, 14 out of 15 patients exhibited an MI of less than 0.85 after the addition of the transfer factor, such that a significant improvement of the test result was obtained in this instance, too. No substantial inhibition could be observed in other groups tested.

Example 3 An antigen solution may be extracted also from tumour cells which have been grown in v1tro in a fermentation culture, with the aid of a buffer and with repeated disintegration of the tissues.

Cells of cerebral tumour are grown _ vitro as follows:
Special care has to be exercised to malntain sterile operating conditions. Preferably, the cells are washed for about 30 seconds in a Han~'s balancing solution.
Agitation should be avoided. All walls and surfaces of the bulb, preferabley a 250 ml bulb of glass, are washed carefully. The solution is made free of the cells ';~

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by centrifuging under cold conditions for a period of 10 minutes. The medium is poured into a beaker glass.
A small quantity of buffered proteinaise encyme solution is added and purged rapidly in order to avoided decom-position of the cells. In the preferred method, up to 2 ml of trypsin solution (EDTA) are added in a bulb.
The trypsin solution is poured away after a purging period of 10 seconds.

Subsequently, the same volume of fresh typsin solution is added. Incu~ation is conducted until it can be seen by observation under the microscope that the cells .
separate from the walls of the chamber. This takes nor-mally from 5 to 10 minutes. A suitable growing medium is added, e. g. 50 ml of a solution of 7 to 10 %
solution of the serum of calf foetuses in 100 ml of F-10 nutrient.

25 ml of the fresh medium including the cells are transferred into a new growing chamber (bulb) for re-productio~. Both chambers are placed into an incubator at 35 C for about seven days. In accordance with the process of this Example, a culture of the artificial tumor celLs ~s divided up to this point into a pair of fresh culture~ approximately every seven days.

1~293~

The mode of process may be repeated in intervals of seven days. Accordingly, the number of cells grown in vitro doubles every seven days.

The medium containing the cells is transferred into a centrifuge tube and centrifuged at 3,000 rpm (~about 2200 G's) in the cold for 10 minutes. The medium is discarded. The cells remaining in the growing chamber are scraped from the chamber walls and washed or purged into the centrifuge tubes with a neutral buffer so-lution. The cells are washed twice with a neutral buffer solution and then again centrifuged under cold conditions at 3,000 rpm (- about 2200 G's). The medium is disposed of. The washed cells are suspended in 10 ml of neutral phosphate buffer until they are prepared for extraction. Then, the washed cells are treated under cold conditions with a sonic dtvice for 20 seconds in order to b~ak up the cells. However, denaturation of the proteins must be avoided as far as posslble. Follo-wing the acoustic irraditaion, the remains of the cells are centrifuged at 30,000 rpm (- about 220 000 G's) for 30 minutes. The supernatant product is decanted. 10 ml of buffer solution are employed for washing the remaining cell residues. Another acoustic irradiation and centrl-fugation are performed~ and the supernatant products ~29319 axe combined. This method is repeated once. The combined supernatant product is prevaporized in order to reduce the volume from about 30 ml to about 6 to 7 ml. A
corresponding portion is removed for the total protein analysis.

By means of a liquid column chromatograph, and with filtering through Millipore Filters, the preparation is than made free of contaminants. Details of the respec-tive process may be gathered from Example 2 of the laid-open German patent application 2,606,257. A polypeptide having a molecular weight of about 10.000 may be ob-tained. An antigen solution is prepared having a con-centration of about 100 mg (milligrams) of the antigen per liter.

The experiments according to Examples 1 and 2 are carried out with the artificially produced antigen. It is found that the artificially produced antigen provides the same effect as the one obtained from body tissue.

le_4 Use of an organo-specific antigen.

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A) The production of the leucocyte suspension is effec-ted in the same manner as in Example 1 - ~.
B) Preparation of an organo-specific antigen solution.

.

Preferably fresh tumour tissue of a mammary carcinoma is cleaned of blood, fatty tissue and binding tissue, and cot into small cubical pieces or strips. All of the subse~uent method steps are performed at a tem-perature of 4 C. The ~issue pieces are disintegrated to pieces of about the size of peas in a threefold volume of a 3 molar potassium chloride solution. The p~
of this coarse suspension is adjusted to 7.4.
Thereafter, the tissue is disintegrated in an Ultra-turrax (Trademark) homogenizer (20,000 rpm). The homo-genisate is allowed to stand for 24 hours. Then, the homogenisate is centrifuged, The supernatant phase ob-tained after 60 minute~ of centrifugation at 4000 G's is dialyzed for 2 hours against a tenfold volume of aqua destillata, and thereafter dialyzed for further 24 hours against a S0-fold volume of 0.9 NaCl solution.
The proteins are precipitated from the dialysate by adding 2 molar ammonium sulfate during a period of 1 hour~, and then separated by centrifugation for 20 minutes at 4,000 G's. Upon suspending the prote~ns llZ93~9 :`

in a 0.9 ~ N~aC1 ~olution, a dialysis against the five-fold volume of aqua destillata for one hour, and a further dialysis against the 50-fold volume of 0.9 %
NaCl are carried out. Following a sterile filtering step through a 0.45 Millipore Micropore filter, the antigen extract is filled into small ~mpules and stored at -20 C. Prior to use, the protein content of every charge is determined by the Biuret method after a preceding TCA precipitation.

C) The same test Plate as in Example 1 is e~ployed.

Three compositions are prepared for the t-st, as follows:
1) Each containing 100 microliters of leucocyte sus-pension from patient blood according to Example 1 - A, + 8 microliters of N/100 NaCl solution . (for column A, standard value).

2) Each containing 100 microllters of leucocyte suspension + 8 microliters of antigen suspension - according to Example 4 - B. I<

3) Each containing 100 microliters of leucocyte su~pension + 8 micxoliters of antigen suspension 11~9319 according to Example 4 - B + 15 microliters of transfer factor solutlon.

The test plate having a hole diamter of 2,5 mm is loaded with 8 mlcxoliters each of the test substance 1 to 3 incubated in accordance with Example 1. The results, each representing the average of five measurements, are as follows:

Composition Column Diameter F (mm2) MI

1 A 7,70 46,5 1,0 2 C 7,00 38,1 0,82 3 D 6,10 29,3 0,63 The result indicates a mammary carcinoma since the MI is clearly below 0.85 and the result becomes still more prominent when the transfer factor is employed.

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l~Z9319 Further examinations show that the results in the case of female patients, as explained in Example 1 t vary still more significantly. Fema~ patien s affected by mammary carcinoma, when the method is applied with the aid of the transfer factor, for more than 9~ %
show a migration index smaller than 0.80. It is only in 10 %of the patients affected by mammary carcinoma that the migration index is above 0.85.

Example 5 The mobility change of lymphocytes, foreign macro-phages and other indicator cells is determined in a capillary test according to S0BORG and BENDIXEN. To this end, an opPn capillary of 10 microns thickness is used. The results correspond to those according to Examples 1 and 2, wherein principally the same prepa-rations or compositions are used.

Concluding remarks The testlng method cannot be applled to cert~in aspects of case. Incorrect by negative results were obtained ln each case fo ~o-called leucoses. At present, 93i9 this phenomenon cannot be explained because it is just leucemic cells that contain plenty of basic proteins. Also, the cellular immunity of such patients is not affected so remarkably that this re~ult could be explained with an affection of the immune system.
Thus, it is also striking that the chronic lymphatic leucemia, same as the acute lymphatic leucemia, fail to yield a reaction whereas lymphosarcomae and lympho-granulomatoses show the same reaction as other mali-gnomae. Prior to the chemotherapy, however, the patients showed a distinct inhibiti~n. This inhibition is eliminated, however, already after the first chemo-therapy cycle, and converted into a incorrectly po-sitive value in the second cycle. Further, wrong results are obtained from patients in tumour cachexy.
Patientæ with rapidly progrediently growing tumours, or patients in the tumour cachexy exhibit wrong re-sults in partO as the cellular immunity is affected tn such instances. Experiments to clear up this question are still being carried out.

Incorrectly positive results were obtained ~n first line in the case of inflammatory and degenerative neurologic disea~es, for instance multiple sclero31s.
The reason was seen by Caspary and Field in a cross ~ J ~;~ i""~

reaction between the EF and the sensibilization of the lymphocates by brain tissue.

Bowever, wrong results may be easily eliminated by pre-examination such that the abovementioned dise-ases do not represent any impediment to using the test.

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Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An in vitro testing method for the diagnosis of malignant tumours, preferably human tumours, comprising determining the mobility variation of cellular blood components contacted with a migration inhibition factor (MIF), wherein the MIF is secreted from lymphocytes being sensitized against tumour tissue, characterized by (a) subjecting, as said cellular blood components, the lymphocytes as a part of a leucocyte suspension to be examined prior to a known per sec migration inhibition test wherein the lymphocytes are contacted with an antigen capable of stimulating them to produce MIF;
(b) measuring the rate of migration of the leucocytes in a capillary intermediate layer having a thickness of from 1 to 20 microns and being at least partially filled (saturated with liquid;
(c) adding to said sample an antigen solution according to CASPARY and FIELD or a 3 M KCl extract of tumour tissue according to MELTZER in a ratio (volume sample to antigen solution) of from 1 : 50 to 1 : 10;
(d) adding to said antigen solution, a transfer factor solution; and, (e) inhibiting and fixing the leucocyte migration after a predetermined period of time.

2. The testing method as claimed in claim 1 including the step that the migration inhibition test is for a duration of between 10 and 30 hours, at a temperature of from 35°C to 37.5°C and a relative humidity of from 90% to 100%.

3. A test plate for carrying out the method according to claim 1, characterized by a planar carrier plate or dish of an inert material having applied thereon a solidified ager layer of a thickness of from 2 to 5 mm, a capillary migration layer being between surface of the plate or dish and agar layer, said migration layer having a thickness of between 1 - 20 µm, and said ager layer being provided with at least two substantially circular punched holes having a diameter of from 0.5 to 5 mm, preferably 2.5mm.

4. The test plate as claimed in claim 3 wherein the inert material is glass.

5. The test plate as claimed in claim 3 or 4, characterized in that said punched holes are cut such that uniformity of the capillary migration layer exists even in the region of the transition from the respective punched hole to said capillary layer.

6. A test plate for carrying out the method according to claim 2, characterized by a planar carrier plate or dish of an inert material having applied thereon a solidified ager layer of a thickness of from 2 to 5 mm, a capillary migration layer being between surface of the plate or dish and agar layer, said migration layer having a thickness of between 1 - 20 µm, and said ager layer being provided with at least two substantially circular punched holes having a diameter of from 0.5 to 5 mm, preferably 2.5mm.

7. The test plate as claimed in claim 6 wherein the inert material is glass.

8. The test plate as claimed in claim 6 or 7, characterized in that said punched holes are cut such that uniformity of the capillary migration layer exists even in the region of the transition from the respective punched hole to said capillary layer.