Propagation of thrombocytes?
To memory of Ludmila, our dear wife and mother, her sufferings were the reasons of this research
There are some theories which describe the thrombocytes formation mechanism. They are based on some experimental facts. Some researches suppose that thrombocytes are the final product of the transformations by this or that way in the megakaryocytes of the marrow. The fact of the presence of the disintegrating small megakaryocytes and leukocytes let us assume that thrombocytes may be formed in the lungs and blood vessels .
The other researchers suppose that thrombocytes may be formed during the changes of the erythrocytes and are the final result of the erythrocytes existence .
Both trends have to vote there is a biological phenomenon: the transformation of cytoplasm of cell (nuclear or not) lias very high grade of organization, perfects copy performing and unique properties. The result cell is extremely different from the initial one. The best position has the flow theory" .
The transformation of the erythrocytes into thrombocytes is the super phenomenon. The most researchers try do not see it because in this case they have to recognize the possibility of the development downward (from bole cell to erythrocyte) and then upward (from erythrocyte to thrombocyte).
What is the mechanism of the thrombocytes formation? What it is realized by? How is it realized?
No one of the theories answer these questions.
The thrombocytes was supposed (Osier, 1874) are the bacteria able to grow and propagate themselves.
In this case the phenomenon disappear: the thrombocytes penetrate into the megakaryocytes or leukocytes and grow and propagate themselves there, all that happen with the erythrocytes is cellular parasitism.
How to prove that thrombocytes grow and propagate themselves? How identify the thrombocytes?
The thrombocytes are extremely polymorphous. So if you get the thrombocytes from the blood, sow them in nutrient medium and get their growth it does not mean this culture is the culture of thrombocytes. The cultivation conditions determine the cell morphology so you are to prove that the final culture may has under the definite conditions the morphology of thrombocytes in all its multiformity. It is not possible to use the conventional identification technique because you have nothing to compare: you are to prove the presence of the thrombocytes culture, and you can not compare this culture with the thrombocytes from blood because you do not know exactly what relate to the thrombocytes, to the plasma of blood, to the cytoplasm of megakaryocytes or leucocytes.
The way of the biochemical identification also is not correct because the cultivation conditions determine the biochemical properties of the culture and the different kinds may produce albumens the same or similar by characteristics. Serological investigations are difficult because of you do not know which receptors on the thrombocytes membranes are primary and which are secondary. The last must be absent in the culture from the synthetic nutrient solution. In addition there is no exact determination of the thrombocytes. The thrombocytes are determined in connection with the blood: it is the rest without erythrocytes, leucocytes and the cells which are obviously bacteria or protozoa. So all the certain characteristics of the thrombocytes are average. Strictly speaking one should suppose all we consider as thrombocytes may be the heterogeneous formations.
Let us consider under the reproduction of formations their ability to propagate themselves, to increase the quantity in the volume: under the growth-the increment of their mass as result of the synthesis of the initial material they consist of simultaneously with the exhaustion of the surroundings. To prove the thrombocytes are able to grow and propagate themselves let us monitor all significant period of time under the conditions close to the natural ones. More over we pay attention to the transition from one morphological variety to another if it exist.
If it will turn out that most varieties by their morphology and place of location (plasma, leucocytes, erythrocytes) grow and propagate themselves, that morphological forms are mutual transitional, properties of the cells arc equal and the same like thrombocytes (if we can say it exactly) we can say it proved that thrombocytes are able to grow and propagate themselves. We should consider the plasma
of blood and cytoplasma of cells as natural surrounding.
There was prepared the dilution of the whole blood with the 0, 95% sodium chloride aqueous solution. The relationship was 1: 200. The dilution was prepared very quickly to prevent blood coagulation. Just after the diluting the drop of blood was placed in a chamber. The chamber was right angled cylinder of 12-mm diameter and 0, 08-mm height. A circular groove was made in the center of polished parallel planed glass plate. The circle was etched by hydrofluoric acid; the duration of the process was determined by necessary chamber depth. The chamber was covered by glass. The cover glass was selected by interference rings. The above described construction of the chamber provides its sterility and gas exchange with the environment simultaneously because the clearance is comparable with the length of the light wave; it provides the ability of steam sterilization; it prevents the distribution irregularity of the particles on the botton of the chamber under the gravity forces and give the possibility to use the immersion system.
For comparison the observations were performed with the squashed drop blood and the smears on the glass or acrylic plastic.
Calculation of the thrombocytes and their aggregates was made 10 minutes later after the filling of the chamber. There were calculated the thrombocytes of all sizes including powdered. The chambers with net were used for calculation. There was the approximate quantitative evaluation of the therombocytes of different shapes and sizes: cocci-, bacillus-shaped, chains and irregular. The thrombocytes attached to the erythrocytes and leucocytes with the mobile graininess were noted. This calculation was made every two hours.
The average figures for 3 years show that within first 46 hours the number of thrombocytes increased sharply; there were cases of 15 times increasing of the thrombocytes amount mostly due to the mobile bacillus, chains and cocci. Next 2472 hours there was no significant increasing of amount. The prognosis of this period of time is difficult and we could not determine exactly what is it based on. It was noted that it is different for two consecutive portions of blood, fop various people, it depends on the health condition and on the part of body where the blood was taking away; it depends on the seasons: the period is shorter in and autumn.
Owing to what the sharp increasing of the free thrombocytes in chamber is occur within the first hours of experiment?
Perhaps there is the reserve of the thrombocytes connected with something where they become free later. The observations show us three sources: erythrocytes, leucocytes and thrombocytes themselves.
There were erythrocytes with naturally deformed membrane in the fresh dilution of blood and sodium chloride hypertonic solution. They were different to the horn-shaped erythrocytes which were similar to the ball-crumpled sheet of paper. Those erythrocytes quiver episodicaly and start to move intensive and abruptly. This time their shape was changed and sometimes it became visible the chainor bacillis-shaped thrombocytes attaclied to them (see Photo 78). During some hours of continuous observation one could see the bacillus- or chain-shaped thrombocytes detachment together with the next jerk. A speed of detachment was up to 60 micrometer/sec. Just after the detachment the erythrocytes stoped move and his shape became similar to the normaly deformed one. From one erythrocyte it could be separated up to three thrombocytes.
The attachment of thrombocytes to the erythrocytes is visible on every blood smear. Some researchers consider it the smear preparation technic deffect.
Observing the squashed drop of blood from the tip of rat tail in those part of preparation where the single erythrocytes in plasma are visible we can see the chains of thrombocytes attached to the erythrocetes winch moves like on the Photo 2-4.
The chamber with fresh dilution of blood and 3 % sodium chloride solution was placed on the microscopic stage on the electric heating coil. The temperature was measured by thermocouple. The chamber was heated up to 55 grad. C and stand up to 5 minutes, then the temperature was decreased to 30 grad. C. Here we can see exactly that horn-shaped erythrocytes get the shape of disc and the bacillus and chain-shaped thrombocytes straighten from them. The thrombocytes were fastend to the erythrocytes by polar side and stand together within 2472 hours movig, growing and propagating. Afterwards they start free existence in the solution. Other thrombocytes (chainshaped with various links) was attached to the erythrocytes by thin appendixes almost by every link. Usually the erythrocyte had the form of sickle.
The real picture of the thrombocytes defeat on the erythrocytes appears during the treatment of the blood smears by the vapour of dichlorethane in order to remove fat from membrane before painting (see photo 14).
We noted the cells among leucocytes which had the optical dense graininess in cytoplasma moving continuously relatively to the nucleus. Watching them we noted that some of them were destroyed: the membrane was breaked through by thrombocytes (cocus shaped and short chains). The same picture was observed for the blood smear preparation and their horizontal fixing by mixture of alcohol and ether. We could not find the leucocytes disitegration with the going out of thrombocytes in the smears fixed by heating.
The allowance of the blood smears was made before their fixing to check that thrombocytes go out from leucocytes during smear preparation. Fresh blood smear was placed on the glass sticks in the flat-bottomed cup without drying. The bottom of the cup was covered by water. The covered cups were placed in thermostat (constant temperature cabinet) at a temperature of 37 grad. C. The allowance was made 4 and 8 hours, after that the smears were dried on air, fixed by heating and paited. Checking the smears it turned out the amount of disintegrated leucocytes was up to 5 times more than in the control preparation. The smears were made from the consecutive drops of blood by random selection. Prolongation of the allowance time till 8 hours did not result in the significant increasing of the amount of the disintegrated leucocytes.
In the sodium chloride blood dilution we noted the oval thrombocytes from 6 to 15 micrometer long; the shape could change to round till 4 micrometer thick. In some of them there were bacilli and coccie moving sinuously within the limits of thrombocyte. The thrombocytes had up to 4 inclusions of bacilli, coccie or chains. To find out the analogy in the smears the last were dried, treated by vapour of dichlorethane within 5 minutes and painted.
We drew a conclusion that thrombocytes (bacillus-, chain- and cocus-shaped) were into the bigger thrombocyte because:
a/Curving sinuously the bacillus moves against a background of bigger thrombocyte and do not overstep the limits of it. Approaching the visible border of the cell the main movement of the bacillus get a covering which change it. This change repeats the form of the borber similar to the form of the bacillus with time lag. The movement of the bacillus has no significant obstacle though it take place not in the whole space of the bigger thrombocyte.
b/ Supposition that bacillns is placed on the surface of the bigger thrombocyte is getting away because the measurement of the bacillus position by height show that in is placed lower the top surface of the bigger thrombocyte membrane.
c/ Supposition that bacillus is placed under the bigger thrombocyte is groundless because in this case the bigger thrombocyte have to move the same intensive as bacillus due to interaction of the glass-bacillus-bigger thhrombocyte system. This movement was observed but very seldom in case of bacillus vertical location. The character of this movement show the very poor interaction between bacillus and the bigger thrombocyte.
d/ In a definite moment of time the bacillus jump out from the thrombocyte. It is impossible to foresee this moment, usually it take place during the first 46 hours since the filling of the chamber, when the bacillus go out from bigger thrombocyte it become more thin and it is optical density decrease. While the bacillus go out no rejection of the bigger thrombocyte is observed. Let us consider as object for the following researches those part of blood we call thrombocytes. It does not belong to the erythrocytes, leucocytes, well-known protozoa and bacilli, it can move actively and change its form within a part of second. The thrombocytes presents in a free condition in the solution, into the cells and on their surface.
Motion of the thrombocytes
To separate the active and thermal motion of the thrombocytes we used the theory of the last. The displacement of the particles in time and the radii of the displacement were determined by cine film.
The analysis of the shorted motion showed us that thrombocytes of all forms and sizes including motion are able to move active (see Photo 3, 4, 9, 12).
The observations show that thrombocytes moves mainly owing to the change of the cell shape looking like a volumetrical wave.
To analyse the changes with thrombocytes during their motion the chamber was filled with the dilution of blood and 3% sodium chloride aqueous solution. The chamber was placed on the microscopic stage equlped with the electric heating coil and held 5 minutes at a temperature of 55 C, then the temperature get down till the room one.
During the process of heating and holding the thrombocytes up to 10 micrometer long got the round shape. They were from 1 to 2 micrometer thick, the appendixes were not always visible, the surface was smooth. They moved within the first 1525 minutes then stoped. In a day one can see that in one case from twenty the thrombocytes start to grow; it accompany with their motion and changes of the shape. Three variants were observed:
a/ The shape of the thrombocyte has no significant changes in the horizontal plane. It occur the changing of the width of cell from 0, 5 to 3 micrometer with a period of time from a few minutes to some hours. It looks like if the thrombocytes breathe. Their optical density changes considerably: while the width of cell increase the thrombocytes become visible the same like erythrocytes, while the width decrease the thrombocytes become almost invisible.
b/ The shape of thrombocyte is round in the cross-section, without significant changes. It get an oval projection with a changeable size of up to 1/3 of the cell diameter. Optical density of the projection is lower than the thrombocyte. Changes of the projection size have no constant period: during 1015 minutes the projection pulsate with a frequency up to 1/3 Hz changing in size in 23 times, then it stand still when the projection may disappear for 1/22 hours. After that everything repeats again. Insignificant displacement of the cell was observed.
c/ The shape of the thrombocytes is oval changing from round to badly stretched. Those changes are accompanied with significant notion of the cell. The contrast range of this thrombocyte is very low, like for stroma of erythrocyte. The changes of the cell shape are accompanied with the volumetrical wave: the front part of the cell is expanding, the rear part is compressing and the cell get the shape of pear, then the middle part is expanding and the end parts are compressing, then the rear part is expanding and the front part is compressing and so on. The optical density of the expanded part is higher than of the compressed part. Axis of symmetry of the cell may be cirved.
In 23 day in one case from a hundred the division of this thrombocyte by constriction was observed. This division was not always expressed clear. But there were some cases when the thrombocyte was divided on two or more oval shaped cells of different sizes connected with each other by the thin pipes. Every link of this chain was changing its shape as it is described above. It results in the sinuous motion of the chain. The elements of the chain are not locked in step with each other: we could see that one or some elements do not move together with the others.
Observing the thrombocytes in the chamber with fresh blood dilution after 6 hours since their filling one can see the chains and bacilli which moves sinuously without significant changes of the shape. The translational motion may be superposed by rotary component. The bacillus can move without significant curve and change its movement direction sharply to the opposite. The speed reaches up to 60 micrometer per second. The bacilli, chains and coccii can move with a few hours for relatively large distance: The most mobile are the formations with the length in order of 107 nm; the longer formation the less mobility. Usually the very long formations are unmovable in general and only some parts of them moves.
When the chain or bacillus moves sinuously it get the volumetrical wave close to sinusoid. This wave show us the absence of the strong flexible connection between the parts of the moving cell. The visible character of the movement may be described as result of the oscillations composition. There are harmonic oscillations propagating on the flexible thread and oscillations of some parts (both longitudinal and lateral) generating by approaching impulse with phase lag and with weak flexible connection. Sometimes the motion is observed when only part of the bacillus or chain is changing.
Sometimes we can see the change of the shape of thrombocyte accompanied with the short jerks. This suggest an idea to us that there is something moving inside the cell more dense than the cell itself. That must be accompanied by the change of the centre of mass. This change together with the change of shape determine the mechanism of the cell motion.
So we suggest that thrombocytes move due to displacement of the centre of mass and change of the shape. Also it follows from the analysis of the motion that parts of the chains and perhaps bacilli and threads are the independent or semi-independent cells.
The thrombocytes can suck the enviroment in but do not use the ejection as jet propulsion.
Growth and lateral division
As it mentioned above after 46 hours the density of the thrombocytes in chamber stabilized on the definite level for the period of time up to two weeks or even more. For this period time the number of moving thrombocytes could become 10 times less, the motion became inert, a lot of thrombocytes laid up motionless on the bottom of chamber. This period is difficult foresee and we could not find exactly what it is determined by.
The period of quantitative stability is changing with a period of the increasing of the thrombocytes amount in a sequence close to geometric with a ratio 23 and a period of 4 6 hours. In the chamber one can see: the increasing of the free thrombocytes amount and their growth or the growth and division of the bacilli and chains attached to the erythrocytes. Growing and dividing thrombocytes moves continuously. If they stop to move that only for a short period of time. We did not observe the growth and division of the unmoveable thrombocytes so we consider the active motion of the thrombocytes as a sign of their life.
In the second case the free thrombocytes almost do not grow, they start their development later. The chain-shaped thrombocytes with a length up to 50 micrometer begin to grow from the surface of erythrocytes. Often they are very thin and low-contrast and a little shaking of the chamber may result in tneir disintegration (see Photo 24). They are tightly connected to the erythrocytes, the chain is able to move the erythrocyte on the distance equal to the radius of it in a fraction of a second. In time the erythrocyte become weak; when nothing remains but only stroma the thrombocytes start an intensive motion and lose touch with erythrocyte. Usually the only cocus-shaped thrombocyte left by erythrocyte.
It was noted that if within the first hours of experiment the number of the free thrombocytes in the dilution increase low (up to 3 times) later the thrombocytes develop generally in connection with erythrocyte. The presence of the antibiotics in the solution or increasing of the sodum chloride concentration promote this.
The period of the intensive growth and division changes with a period of quantitative stability and then decreasing of the free thrombocytes amount occur simultaneously with the growing or reducing of their dimensions.
Let observe the changes with bacillus shaped thrombocyte attached to erythrocyte. At the beginning of the observation the bacillus is optically homogeneous and almost transparent. It's growing longwise. In some time (about 4 hours) an optical condensation appears in the bacillus. This condensation get the whole central part of the bacillus along the main axis of symmetry. Within about 30 minutes the condensation divides for some parts. The number and sizes of the parts are not constant. Usually the short and thick bacilli get two parts.When the dividing of the optical condensation was over it began the dividing of the cell itself. Generally the lateral constriction corresponds to the division of the middle part. The process was over in 46 hours and the bacillus turned into the chain of coccie or short bacilli connected with each other by the thin threads of up to 2 micrometer long and I micrometer thick; The optical condensation in the middle of the cell disappear. All this time the periods of rest changes with the periods of motion. After the division the chain start to move actively. This motion usually result in the breaking of the chain. We could not find the breaking without previous motion. About one case from hundred one can see the branching of the chain. The growing up of the brach was a result of its growth and division. Only the thrombocytes unmoveable completely had no growth and division. So we suggest that the thrombocytes are able to grow and propagate themselves by lateral gemmation. The thrombocytes which had no growth and division are not an independent morphological variety but the cells which belong to the different varieties and mostly are able to grow and propagate themselves.
Propagation of thrombocytes by gemination
Observing the culture of thrombocytes in the chamber it was noted the formatting of the daughter cells inside the thrombocytes. Under the first consideration this process is similar to the formation of the bacterium spores.
a/ An optical condensation appears inside the cell, the place of its location is arbitrary. It moves inside the cell and go out from the polar end (see Photo 10).
b/ After the going out the daughter cells the maternal cell not always die but continue to move, grow and divide.
c/ The formation of the daughter cells was observed not only in the separate throbocytes but in the colonies of every type.
d/ The formation of the daughter cells occur simultaneously with the growing and lateral division of the maternal ones.
e) The formation of the daughter cells was not determined by poor conditions of cultivations. Very often it occur in the fresh resowing and very seldom in the old cultures.
f) The season dependence was noted: the formation of the daughter cells was more distinct in spring and autumn.
We conclude from the above-said that the thrombocytes have the second type of propagation themselves by gemmation: formation of the daughter cells inside the maternal one. It is the natural way of propagation for the colonies of thrombocytes. Formation of the daughter cells and their further behaviour are similar to the formation of the migratory cells of the colonial suctorial infusoria so we adopted the term gem-ma for the daughter cells of the thrombocytes. We suggest the supposition that the thronbocytes propagate themselves by lateral division and intracellular gemmation.
Painting of the thrombocytes
Prepare the smear of blood on the normal glass. Dry it on the air. Fixate the definite part on film. Fixate it in the fresh alcohol-ether dilution of the possible less volume. Dry and photograph the same place again. Paint and look through the same place with and without immersion system. The comparison show us that the number of the thrombocytes in the smear decrease after every operation. If we put the mixture of alcohol and ether under microscope we can see the moving cocus and bacillus-shaped thrombocytes. There are suspended thrombocytes in a drop of cedar oil. The same picture was observed if the culture of thrombocytes was painted in the smear. We treated the thrombocytes by the vapours of dichlorethane for better penetration of the paint. The smear was dried by air and treated by the vapours of dichlorethane within 35 minutes and then was painted conventionly. The hemolysis of the erythrocytes on the stroma. The graininess is distinct. The thrombocytes are better attached to the glass.
Nutrition of the thrombocytes
The cells formated as result of the growth and division of the thrombocytes are able to suck the surroundings into through the suckers. The cells may attachs to erythrocytes, for example. The chain may be attached to the erythrocyte not only by its first link but by every separate part simultaneously. The place of connection between thrombocytes and erythrocytes is able to take a significant load; the jerk of chain may result in the displacement of the erythrocyte equal to its radius in a fraction of a second. The attachment of the cells to the glass were observed. The cells had rotary motion describing a cone at a frequency about 4 Hz. We can see these projections on the photo 10.
The erythrocytes attached to the thrombocytes become weak but the thrombocytes grow and propagate themselves. When only stroma of the erythrocyte remains the threads breaks down and attachs to the unaffected erythrocyte. And so on.
The membrane of the cell is dense, the paint penetrate poor through it. The best penetration may be obtained by removal of the fat from the membrane. That means the membrane has low penetration for the nutrient substances.
So we suggest that the thrombocytes have holozoic nutrition and their canal system is the digestion system .
Cultivation of the thrombocytes
We made the sowing of the thrombocytes in those mediums:
a) the whole blood dilution with water or 1-5% sodium chloride aqueous solition;
b) meat-peptonic broth;
c) salt basis with the albumens and carbohydrates;
d) salt basis with the hemoglobin and carbohydrates;
e) salt basis with a set of the amino acids and carbohydrates;
f) the above-mentioned mediums with agar.
It were used for the sowing:
a) the whole blood;
b) the thrombocytes selected from the hemolysed blood;
c) the thrombocytes selected from blood dilution with 1-5% sodium chloride aqueous solution;
d) the thrombocytes selected by above-mentioned ways and washed a few times with water or 1% sodium chloride solution.
The selection was made by differential centrifugation and/ or filtration.
In all cases the same picture was observed as mentioned above. The sowing of the thrombocytes selected from the blood on the nutrient mediums always gave us a period of time when there was no significant increasing of the cell amount and no phase of division as well as no growth were determined. This period for the big vessels is longer than for the chamber and it reaches up to 4 weeks and more for conventional method of flask cultivation. And in this case the growth was determined not by the turbidity of medium but by observation of the squashed drops or the painted or not smears with a microscope.
The thrombocytes mainly settle out on the botton of the vessel and because they need oxygen hardly the cultivation should be performed in a thin layer or using the aeration with mixing in order to help the thrombocytes with division.
Let us dilute the blood with distilled water and connect all the hemogroups with carbon monoxide. In this case it appears that thrombocytes are able to stay under the vacuum up to 18mm HC. It appears when the hemogroups are connected the development of the thrombocytes decrease sharply, they grow thicker, the propagation get slower as well as motion activity.
Amino acids formula of the synthetic nutrient medium was determined by the following method.
Native blood was diluted with 2% sodium chloride solution, the dilution was centrifugated. The filtrate was 3 times washed properly in 2% sodium chloride solution using the centrifugation. The final filtrate containing erythrocytes, leucocytes and thrombocytes was placed in a distilled water. The concentration of the cells were constant. The final solution stood during 6 months at a temperature of 37°C, adenosine triphosphoric acid was added to solution periodicaly. After 6 months the solution was drained through the 0, 3 micrometer millipore filter. Hemogroup was not determined by the spectral analysis. Application of the partition chromatography method on paper show us there are the definite set of amino acids and light peptides in the solution. There were no albumens. After peptides disintegration by acidic hydrolysis the exact analysis of the amino acids was made both quantitative and qualitative.
The amino acids formula of the hemoglobin is known so subtraction of the amino acids remained in the solution give us the amino acids utilized by thrombocytes.
We should point out that composition of the utilized amino acids corresponds to the surplus oft those acids in the blood of the patients sick with rheumatism in acute phase .
The salts composition was chosen under the generally accepted view on the metabolism in the organic cells close to the blood formula. The vitamins were chosen by the same way. The carbohydrates were chosen by the trial method. The solution of salts, amino acids and vitamins was prepared and divided on two parts. Then the definite carbohydrate was added in both parts. Those carbohydrates were checked: saccharose, rhamnose, dulcite, inositol, maltose mannitol, sorbitol, xylose, lactose, arabinose, glucose. The best assimilation was noted for mannitol, worse for inositol and saccharose, worst for dulcite, the rest carbohydrates were not assimilated by thrombocytes.
The amino acids composition was not checked for their necessity.
Adding of agar resilted in the obtaining of the solid nutrient medium. In this case the thrombocytes dilute agar and may use it as the source of carbohydrates.
Some colonies of the thrombocytes have up to 5 mm diameter. Sometimes they are very small and are visible by microscope only. They are semi transparent and have the round shape. The surfase is bright and smooth. The texture is grainy. The colour is light-bronze, it is visible for the young the trombocytes only under the light; they look like a drop of water under the wettability condition. The profile is convex, the boundary is even, at a dense sowing of the colony the boundaries merge together. The consistence is jelly-like. They poor emulsify in the water. Theu do not develop into agar. Bringing the solid medium into the chamber result in the active motion of the thromdocytes in the colony one can see by microscope
The cultivation was performed in a test tube with a tap closed by a rubber plug with a hole where a glass pipe reaching the botton of the test tube was inserted. The lower end of the pipe has a hole with 0, 01 mm diameter. A ceramic bacterial filter for air sterilization was inserted into the plug from the outside. The tap was closed by dense cotton-wool plug and a clutched rubber pipe was puled on it. This devise was sterilized in an autoclave within half an hour at a pressure of 1, 5 atm. The air was drawn off through the outlet pipe. The cultivation was also performed in the flat-bottom flask closed by cotton wool-and-gause plug.
To obtain the thrombocytes for sowing 0, 05 ml blood was taking from finger. The skin was treated by petrol, formalin, alcohol and iodine. The blood in taking was made in a table box under ultra-violet radiation where only donor hand was puted in through the glove. The prick was made by the syringe containing the solvent. Then this solution was mixed and poured into the centrifuge test tube. The blood solution was centrifugated during 5 minutes at acceleration of 4 m/sec2. Over-sediment liquid was poured into clear centrifuge test tube and re-centrifugated during 10 minutes at accleration of 24 m/sec2. The over-sediment liquid was sucked out, 0, 1 ml of the liquid was remained in test tube.
The contents of the test tube was mixed and transfered into a nutrient medium. The test tube with the thrombocytes in the nutrient medium was placed in a constant-temperature cabinet in the inclined position at a temperature of 37°C. The insufflation of the nutrient medium by air was made during 2 minutes every two hours. For this purpose the previously pressed rubber ball was connected by the rubber pipe to the outlet pipe. When the clutch was released small air bubbles came through the nutrient medium from the central tube. In this case the test tube was placed verticaly. The rubber ball was not allowed to release completly. The rubber pipe was clutched and the test tube was placed in the constant-temperature cabinet inclined again.
The growth was checked in a squashed drop by 600x microscope. If the growth developed the sowing in the agar medium was performed to check the purity and to obtain the pure culture of the thrombocytes.
The medium was taken from the test tube by the loop, spread over the agar surface. The sowing was made by the thorough spreading of the medium over the surface of another cup by the same loop. Both cups were placed in the constant-temperature cabinetat a temperature of 37°C. After 48 hours the colonies were observed in microscope at 105x magnification. Some colonies were transfered onto the test tube with nutrient medium and were cultivated as it is described above. For this period of time the thrombocytes develops to a density of 1, 2x 109 per one ml of medium at the sowing of one 4 mm diameter colony in 10 ml of medium.
The sowing of the thrombocytes from the blood or the blood itself in the agar nutrient medium result in the growth of the colonies very seldom. The better results 50% - were obtained fir the thin sowing with a thorough spreading. The colonies up to 0, 4 mm were stretched along the touch of palette-knife. Those colonies are visible in microscope due to the dilution of agar. Re-sowing of the well-developed thrombocytes on the nutrient medium either liquid or solid gave the fast growing of the culture. The visible growth of the colonies and the dimness of the medium occured within a day.
Perhaps the long period of the development for the thrombocytes getting from the blood is used for the release of those thrombocytes from the obstacles for development presenting in the blood itself and on the surface of the thrombocytes. It should be checked whether those obstacles are eliminated by the thrombocytes or it occur naturally. An analysis of the medium where the thrombocytes were developed tells us that thrombocytes are able to split any peptide links.
The presence of the obstacles was confirmed by:
a) This period decrease if the thrombocytes are washed a few times with 1% sodium chloride solution before their sowing from blood to the liquid nutrient mediums.
b) This period is absent for sowing of the thrombocytes which have grown in the chamber or in the test tube to the liquid or solid nutrient mediums.
c) This period was reduced up to one day if the medium where the thrombocytes were cultivated was added to the nutrient medium before the sowing of thrombocytes from the blood. The added medium was sterilized by filtration.
d) The sowing density increasing of thrombocytes but. not the blood resulted in the reduction of this period.
It may be thrombocytes secrete some combinations which reduce or eliminate the obstacles for thrombocytes development.
During the cultivation of the thrombocytes in liquid nutrient mediums into the chambers and flasks growing of the threads up to 200 micrometer long and up to 3 micrometer thick was observed. The threads had separated graininess, the cases of constriction were very seldom, some of them were poor expressed. They were sinuous-shaped and periodically start to move inertly, sometimes it was possible to observe the motion of the ends only.
The threads were not always spreading over the bottom of the chamber, sometimes they were branching out (see Photo 11). The balls of the interiaced threads were noted. The same balls were found in the blood smears of the patient sick with rheumatism in acute phase.
We consider those threads as a complex of the cells colonies, which have general plasma with the signs of uncompleted division. Some cells are determined not always clear by the separated graininess because of the different phases of the mitotic development. Every part of the thread is able to grow and divide independently, the plane of division for all cells is determined by the general axis of orientation. It happened not always which result in the branching out the colony. In this case the growing and division of all parts of the thread occur what is clear visible by direct obcervation. For example, the process of growing and division of the chains and bacilli was described above. If the medium was intermixed no growing of the threads was observed so we may consider the intermixing as additional factor for completing of the division with disintegration.
For cultivation on the liquid nutrient mediums in the chambers and flasks the growing of the thicker bacilli with a length up to 30 micrometer and variable thickness from 4 to 10 micrometer were observed. These bacilli had projections of the different length and orientation. The growing of these bacilli occured in the unpredicted direction mainly along the mayor axis. The branching out was observed. There was the general plasma for the light microscope, the granules were distributed unevenly. They were not observed in the intermixed mediums even if by aeration. They began formation when the intermixing stoped. A careful observation showed us the small thrombocytes up to 0, 5 micrometer long moving in the plasma, the going out into surrounding was noted.
We consider these bacilli as well as threads as colonies of the thrombocytes which have the general orientation of division partly. In the culture of thrombocytes from the liquid nutrient mediums there were colonies of the thrombocytes where orientation of growth and division was lost completly. These colonies have indefinite, mostly flat shape with projections. In the light microscope their plasma looks almost homogeneous, their graininess is distributed unevenly. There are small thrombocytes in plasma moving inside the colony, sometimes the going out from the colony occur. During the preparing of the smears mainly the coagulation of the colony occur. The mixed colonies were observed.
We could not find what determine the development of this or that kind of colony.
There are some photos of the thrombocytes made by electron microscope at 5000 x magnification (Photo 10, 13). The thrombocytes were cultivated during 4 weeks on the hemolysed blood. Vacuum deposition of the contrast preparation was made by platinum (cathode sputtering) at the angle of incidence of 70°. The spraying was made by a thin layer to remain the electron transparence of the object.
There is a chain of thrombocytes on me Photo 10. It is striking the inconstancy of the shape and sizes. The individuals have unequal ends: one of them is oval, the other one is obtuse which point their specialization. The chain is oriented evidently.
In would be noted while analysing the photoes:
1. During drifting of the thrombocytes on the special film for electron microscopy it appeared that the film is covered with a layer of albuminous molecules from the solution where the thrombocytes have developed though they were washed by distilled water with the following centrifugation three times to remove them.
2. The preparation was treated by vacuum three times.
3. The preparation was treated by heat radiation and platinum atom bombardier during the cathode sputtering.
4. The preparation was checked and photographed twice by electron microscope, in this case it was bombarded by fast moving electrons. The obvious sing of this is bend of the special film for electron microscopy.
The thrombocytes have no flagella. The formations on the photographs looking like flagella are the cracks in reality.
The mobility of the thrombocytes was checked before the preparation was put on the film for electron microscopy, so they died during the process of fixation.
Because of there is no photograph of the microcuts and the magnification is low it's not possible to make the conclusion about membrane texture and intracellular organization of the cultivated thrombocytes. But we can make some suppositions:
1. The outlines of the cells are indistinct. In every case the cell is surrounded by electron dense edging, density get low toward the centre of the cell. The edging turn into the light closed curve which does not correspond to the boundary of the cell. The curve determine bean-shaped area filled with substance which has irregular density. Perhaps distribution of this substance depends on the stage of the cell development. The character of the distribution is different for the different cells but mostly the substance is concentrated to the poles of the outline. It looks like the ovular type nucleus.
2. All cells except the gemmae are stretched. Their poles are not equal. One pole is oval and it is very close to the light inner outline. The opposite obtuse pole has uneven border, it is far from the light outline. The dark zone edging the cell become wider at the obtuse side and its density is higher. There are folds placed perpendicular to the axis of symmetry of the cell. The most cells have formations at the obtuse side looking like short flagella with the ball-shaped ends.
3. The quantitative analysis of the presented photographs tells us that thickness of the outer dense layer in the narrow place is about 104 nm. But it is known that thickness of the three-layer membranes is about 10 nm which is by a three order of magnitude less. Even if we shall suppose the presence of the fourth layer there will be no correspondence. Especially if we pay attention to the fact that the thickness of the layer by the poles reach up to 5xl05 nm. The light inner layer does not repeat the outlines of the cell and seems more definite, bean-shaped, with distinct outlines. The light curve has a thickness of 5xl04 nm which does not correspond again to the known thicknesses of membranes. If we shall suppose that the light curve is the result of the shrinkage of the cell there is no correspondence again between the light curve and the border of the cell (see Photo 13).
We may suppose cautiously that the ends of the thrombocytes are specialized by function. The cell has one nucleus at least most probably ovular type surrounded with relatively thin layer of cytoplasma limited with elastic membrane. The birth of gemmae exist for the thrombocytes.
The characteristics of the culture of thrombocytes
The cultivated thrombocytes are painted Gram-positive. There is catalase in the cells and solution, the ammonia is exuded, no hydrogen sulphide presents, no ihdole is exuded. Agar is diluted perhaps in order to obtain dulcite. There is hemosiderin in the cells and colonies. No cellulose was found.
The thrombocytes cultivated on the artificial nutrient medium and the medium itself contains cholesterin. The concentration of the cholesterin in the solution is up to 40 times less than in the cells.
If we shall put the cultivated thrombocytes or the medium for cultivation into the blood stabilized by heparin the coagulation of the blood will occur. If we shall leave this clot of blood together with the thrombocytes the clot will be diluted completly in 34 days. Adding of the ether extract from the beef meat in the artificial nutrient medium result in the primary development of the pear-shaped thrombocytes with the sizes up to 5 times greater than without extract. The same situation is for the sowing of the blood on the meat-peptonic broth. Adding of the fresh blood into the artificial nutrient medium containing the cultivated thrombocytes cause very fast penetration of the thrombocytes into the leucocytes within a few seconds. The leucocytes expand and disintegrate. The culture of thrombocytes remains for a period up to 5 years both in liquid and solid nutrient mediums. If we shall keep the smear of blood on the glass, in an air surrounding, with no fixation, at a humidity of 8090%, the thrombocytes will develop using the substances on the glass for nutrition (3 years).
The thrombocytes formate no spores and cysts.
The cells cultivated on artificial nutrient medium stand for a day in distilled water. Then they were put into the 0, 1 M oxalic acid solution has 14 C. Five minutes later the thrombocytes were separated by centrifugation. The obtained thrombocytes were washed 3 times in 0, 9% sodium chloride solution and 3 times in distilled water. Every time the vessels were changed. Radioactivity was checked by counters. The level of radioactivity was equal to the natural background, it was detected only by photograph method. The preparation was put on the aluminium base placed on the photoplate for radioactive researches. The time of exposure was one day. The washed thrombocytes were placed in an artificial nutrient medium at a temperature of 37° C. Observations show that during first four days the marked thrombocytes grow and propagate themselves more actively than control ones. Then the culture dies. The large pear-shaped formations prevails in the culture.
Signs for identification
They are obtained from the human and animals blood.
Morphology and cytology features.
1. The shapes and sizes are variable. The shape is cocus-like, bacillus-like, with polar specialized ends, disc-like. May exhaust pseudopodium. The sizes are from 0, 4 to 10 micrometer.
2. Combination of cells. The cells may joint and develop in a colony: chain-shaped which can branch out; like cells of any shape with a solid cytoplasma where small cells move relatively free and may go out. The size of colony: the chains may have length up to 200 micrometer, the cells are practically unlimited in sizes.
3. The motion is active in any age due to changing of the size and shape of the cell.
4. There is no flagella. May exhaust pseudopodium in order to connect to the leucocytes, erythrocytes, to each other and to glass.
5. The nutrition is holozoic. After the catching of the food the sizes become greater, after the rejection the volume get lower. The digestion system is chanal one. The inlet foramens are placed on the ends of the pseudopodium.
6. Graininess presents.
7. Formate no spores and cysts.
8. Propagate themselves by lateral division and intracellular gemmation.
9. No capsules observed.
11. Viable in 0, 1 M hydrochloric acid dilution.
12. May penetrate in leucocytes.
13. May develop in leucocytes. The leucocytes become greater, the mobile graininess appears, them disintegration of the leucocytes with going out of thrombocytes occur.
Features of growing on the solid and liquid nutrient mediums
1. Some colonies of the thrombocytes have up to 5 mm diameter. Sometimes they are very small and are visible only by microscope. The shape is round. They are semi-transparent. The surface is bright and smooth. The texture is grainy. The colour is light-bronze; the young thrombocytes look like a drop of water under the wettabillity condition. The profile is convex. The boundary is even. At the dense sowing of the colony the boundaries merge together. The consistence is jelly-like. They poor emulsify in the water. They do not develope into agar.
2. The above-described colonies are visible along the boundaries of the stroke sowing, the colonies merge together in the centre of the stroke.
3. Very weak even dimness occur in the liquid medium. The dimness fall out in time. Shaking the medium one can see the flakes which are easy disintegrated. Very often especially for the long primary development of the blood sowing the growth is visible in the squashed or dried drop only.
Physiological and biochemical characteristics.
1. The growing on the following carbohydrates and alcohols were checked: saccharose, rhamnose, dulcite, inositol, maltose, mannitol, sorbitol, xylose, lactose, arabinose, glucose.
2. Use: mannitol, inositol, saccharose and dulcite.
3. Acidity decrease.
4. Dilute agar.
5. Ammonia is exuded.
6. No indole is exuded.
7. Catalase presents.
8. No hydrogen sulphide is exuded.
9. Grow on the artificial nutrient medium containing: hexacyanoferrat II potassium, leucine, serine, tripton, cysteine, histidine, arginine, glutamic acid, methionine, pro-line, asparagine, lysin, glutamine, amino-acetic acid.
10. Contents hemosiderin.
11. Contents serotonin.
12. Have anti-heparin activity.
13. Cause blood coagulation.
14. Cause retraction of the blood clot.
15. Cause lysis of the thromb.
16. Contents cholesterin.
17. Need oxygen.
18. Grow at a temperature 0 to 42°C.
19. Sensitive to radioactivity.
20. Grow in the concentrated sodium bicarbonate solution.
21. Grow in the concentrated sodium chloride solution.
22. Presence of the animal fats promote the growing.
23. May use the animal albumens.
24. They are resistant to drying.
25. There are substances in the blood braking the development..
26. There are substances in the blood causing the reaction of agglutination.
27. The period of development of the culture from the blood is up to 4 weeks under the condition of weak dilution. The period depends on the health condition, it decreases considerably during rheumoat-tack.
28. The activity depends on the season. It is hingher in spring and autumn. During these periods the propagation mostly occur due to migratory cells.
29. They are sensitive to chemical inhomogenity, to changing of the electric potential, to changing of illumination, to pressure. The sharp changing of these characteristics result in reduction of the cells.
30. Activity is changeable within a day. Probably belong to the type of protozoa, class of infusoria, order of colonial suctorial infusoria.
Using conventional method of cultivation it is possible to obtain the culture of cells from the blood of every human which are able to grow and propagate themselves by lateral division or intracellular gemmation. The blood itself, its plasma or cytoplasma of the blood elements are used like a nutrient medium.
This culture is very polymorphous. The observations of the growing on the different mediums including artificial ones show the complete correspondence with the thrombocytes in the whole blood. All the forms observed are mutualy transitional.
The biochemical characteristics of the culture of cells correspond completly with the thrombocytes which have no plasma origin. There are antibodies to this culture in the blood.
All morphological varieties of the thrombocytes are able to grow and propagate themselves except individuals which are not able to active motion and changing of the shape. It is clear from the continious observation.
So we suggest that the thrombocytes of human and animals are able to grow and propagate themselves. They may conduct both bycellular and intra-cellular parasitism forming the colonies or exist in the plasma of blood or in any solution containing the sufficient set of nutrient substanses.
These properties of the thrombocytes determine some positions which need the further verification. The main them are:
1. The possibility of the thrombocytous inflammations in any part of macroorganism.
2. The thrombocytes as potential parasites of the blood system.
3. The present classification of the leucocytes is determined mostly by the development of the thrombocytes into them.
4. The albumens produced by thrombocytes, the thrombocytes themselves or their membranes may be used as medicine.
5. Only insignificant part of the thrombocytes presents in the plasma of blood.
6. The thrombocytes and hemostasis.
7. The ways and means of fight against thrombocytes. 8: The thrombocytes and rteumatism.
9. The thrombocytes and marrow.
10. The thrombocytes and bones.
11. The thrombocytes and tooth pulp inflammation.
12. Symbiosis of thrombocytes and macroorganism.
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