Erythrocytes

ERYTHROCYTES (from Greek erythros-red and kytos-cell), red blood cells, specially modified cells that constitute the main mass of the formed elements of blood and give it its usual color. The number of E. is usually determined per 1 mm³ of blood, with the generally accepted norm being 5 million per 1 mm³ in men and 4.5 million in women. Both of these figures were apparently established in the 1850s by Vierordt and Welker through very imperfect (although extremely complex) manipulations and on single individuals. Subsequent research, thanks to significant improvement in the technique of counting E. and an increase in the number of subjects studied, has substantially clarified our understanding of the content of erythrocytes in the blood of healthy people and, above all, about the upper and lower limits of this norm. At the same time, attention is drawn to the similarity of data obtained in different countries of Europe and America, which differ from each other both in racial and climatic aspects. Table 1. Blood composition in men. Author, year of study, subjects studied, place Number of studies Number of erythrocytes in thousands minimum maximum average Friedrichson (no Rieux) Osgood (in students), USA, 1928...... Horneffer (in students), Germany, 1928.... Horneffer (in soldiers), Germany, 1928 .... Foster and Johnson (in students), USA, 1931 Chernyak (in medical workers), Yalta, 1926 . . Simagina (in Red Army men),Astrakhan, 1931 20 137 20 20 115 19 29 4 100 4,480 4,370 4,400 6,400 5,530 5,580 5,930 5,200 5,072 5,390 5,000 4,920 5,263 5,198 4,870 Table 2. Blood composition in women. Author Number of studies Number of erythrocytes in thousands minimum maximum average Friedrichson...... Wintrobe........ Osgood and Haskins . . Bie and Muller..... Chernyak......... 28 50 100 10 12 4,450 4,300 4,120 5,540 5,300 4,903 4,930 4,800 4,740 4,836 The most probable reasons for discrepancies between the data of various authors are, obviously, on the one hand, socio-domestic factors (profession, nutrition of the subjects studied), and on the other hand, the insufficiency and inequality of the number of studies. Summarizing all available data for each sex group and thus establishing both extreme and average figures for the content of E. in normal blood, we obtain (approximately): 360 men: minimum-4,400 thousand, maximum-6,400 thousand, average-5,200 thousand; 200 women: minimum-4,100 thousand, maximum-5,500 thousand, average-4,800 thousand. Since the content of E. is usually determined in blood from the pulp of the finger, less often from the earlobe or from the cubital vein, the question naturally arose: to what extent do the figures obtained reflect the content of E. in other parts of the vascular system, in particular in arterial blood. At present, it can be considered established that with normal blood circulation, the content of E. in the skin capillaries and peripheral veins coincides with their content in arterial blood (Table 3). Table 3. Content of E. in blood (according to Kurt Hofmeier). Disease Stomach ulcer Healthy . . . . Neurasthenia Stomach ulcer Artery Vein 4,020 5,010 4,270 4,180 3,960 4,980 4,310 4,410 Finger pulp 3,960 5,040 4,350 4,470 With circulatory disorders, accompanied by stagnation in the veins and capillaries, the number of E. in them can significantly exceed their content in arterial blood (decompensated heart defects, vasomotor neuroses). In addition, numerous studies on the question of blood 'depots', shock, etc. have shown that both under normal and pathological conditions, in a number of areas (spleen, subpapillary plexus of the skin, liver, mesenteric veins), various changes occur in different vessels, as a result of which they either retain large masses of E. or release them into the general bloodstream. Among the numerous factors causing these changes, the most ordinary ones should be especially noted, which include: the position of the subject's body, movements, physical work, food intake, drinking, cooling, conditioned reflexes associated with the time of day. This leads to the obligation to study the blood composition under certain standard conditions (in the morning, in bed, on an empty stomach), non-compliance with which is a source of errors for a number of authors. Counting E. in a chamber shows only their concentration in the blood. In order to determine their absolute content in the body, it is necessary to take into account the mass of blood (see Blood) and perform the corresponding calculation. There are no special studies devoted to this question, but the necessary figures can be extracted from works on the mass of blood. It turns out that in healthy men, the absolute number of E. in the circulating blood can vary between 20 and 40 trillion (or from 360 to 550 billion per 1 kg of weight) and averages 27 trillion (or 450 billion per 1 kg of weight). In women, the corresponding figures are 20 trillion (from 16 to 28 trillion) and 350 billion per 1 kg of weight (from 280 to 420 billion). While the content of E. in the blood of different healthy people can be very different, their content in the blood of each individual differs by significant constancy, fluctuating within only 1-5% (of course, provided that standard research rules are observed) over long periods of time. However, this constant value is not an expression of any fixed, once and for all laid down mass of E. in the individual, but represents the result of continuously occurring processes of breakdown and regeneration of E. in the body. In the literature, this circumstance is often recorded in the form of a scheme: amount of E = regeneration-breakdown, but this scheme excessively simplifies all connections and cannot explain the constancy of blood composition. In reality, we observe not an arithmetic relationship, but the most complex process in which regeneration and breakdown are in continuous interaction with each other and with the amount of E., and are also subject to the most diverse endogenous and exogenous influences. We are still very far from knowing all the details of this complex interaction, however, some aspects of it can already be considered established. In particular, it was already known at the end of the 19th century that the hemolytic processes occurring in the body have a stimulating effect on erythropoiesis; in recent years, thanks to experiments by a number of authors (Verzar, Dorle, Seyderhelm, Tammann, M. Ono), it has been revealed that the stimuli for erythropoiesis are the breakdown products of E.: protein substances of the stroma, lipoids (cholesterol, lecithin), ergosterol, hemin, bilirubin. All these substances exert their effect both when they are formed in the body during the normal or enhanced work of hemolytic organs (see Hemolysis), and when the breakdown of E. occurs in the bloodstream under the influence of one or another poisons or parasites, or outside the vessels-in hemorrhages, and finally when they are introduced into the body from outside-parenterally or even enterally. Regarding the dependence between the amount of E. and their regeneration, it has long been known that significant blood loss leads to an increase in the regenerative activity of the bone marrow, and experiments with artificial plethora and with systematic blood transfusion have established their inhibitory effect on the bone marrow. On the other hand, Chernyak's data indicate a decrease in hemolysis in anemias due to blood loss, and Adler's data indicate an increase in hemolysis in various polyglobulinemic conditions. Numerous studies on the influence of the internal secretion glands on blood formation, despite the presence of disagreements, still allow us to conclude that the thyroid gland stimulates the activity of the bone marrow (Unverricht, Bose, Zondek, Hoskins, Jellinek) and that the sex glands of females stimulate erythropoiesis in them (but not in males) (Josam, Deneke, Nigst); regarding the other glands, no definite data is available. This should also include the hormonal inhibition of the bone marrow by the spleen, established by Hirszfeld at one time, as well as the recent data on the connection between blood formation and substances obtained from the liver and stomach. Among exogenous factors, the influence of oxygen concentration in the surrounding air was studied earlier than others: a decrease in O₂ leads to enhanced activity of the bone marrow, an increase in O₂-inhibition of blood formation, up to complete cessation (Kuhn, Aldenhoven). On this basis, Koranyi, Bens, and partly Morawitz explained the enhanced regeneration of E. after blood loss as a consequence of reduced O₂ tension in the blood. Despite the significant number of works, the significance of climate features, ultraviolet, solar radiation and other meteorological factors remains completely unclear. In contrast to this, some progress has been made in studying the influence of nutritional factors on blood formation thanks to the works of Whipple and his colleagues, Benedict, Pearce, Kessler, Verzar, Parsons and many others (Benedict, Pearce, Parsons).

As a result, at present, the stimulating effect on blood formation is known from beef and chicken liver, beef, kidneys, various types of greens, fruits (apricots, apples, grapes, plums).-Finally, in relation to certain substances, it has been established that they have a stimulating (or inhibiting) effect simultaneously on both blood formation and blood breakdown; thus, Adler and Morawitz and Kühl established that proteins and meat enhance the breakdown of E. Since this parallelism of action is in complete accordance with the close relationship between the hematopoietic and hemolytic systems, it is highly probable that further research in this direction will reveal similar factors for a number of other substances and factors.-This brief list of interactions is of course far from complete, but it nevertheless gives an idea of the complex mechanism that ensures the constancy of the composition of the blood of a healthy person. The flexibility of this mechanism is very significant and is able to balance very long and intense exogenous and endogenous influences; however, it is not limitless: in some cases, the organism is forced to give up its positions and the number of E. changes either in the direction of decrease (anemia) or in the direction of increase (poliglobulia, polycythemia). At the same time, it is often possible to observe how in the organism a new (decreased or increased) constant level of E. is established for a longer or shorter period of time; this indicates that the regulatory mechanism still possesses a certain reserve of possibilities. Where these possibilities are completely exhausted, we encounter an unstoppable fall or increase in the number of E., leading to the death of the entire organism (see Anemia, Poliglobulia, Polycythemia). Morphological properties of E. Normal mature E. (normocytes) of a healthy person (on E. of other animals see Blood), when examined under a microscope in a fresh state, are anucleate, round or slightly oval, flattened, biconcave cells with an indentation in the middle; they are distinguished by considerable flexibility and ability to deform, thanks to which they pass through narrow capillaries and through their walls, taking on the most bizarre shapes. Due to the hemoglobin contained in them (see Hemoglobin, Hyperchromasia, hypochromasia), when examined individually, they appear colored in a pale greenish-yellow color, slightly darker at the periphery than in the center. In the dark field of view, they appear as homogeneous dark disks with a shiny rim. When fixed smears are stained with a mixture of acidic and basic dyes (eosin-methylene blue, azure-eosin according to Romanowsky-Giemsa), normocytes are selectively stained by acidic eosin (due to the acidophilia of hemoglobin) in a uniformly pink color, the more intense the higher the content of Hb in E. This phenomenon is called orthochromasia, and E. are orthochromatic.-Neither on fixed preparations nor with various methods of vital and supravital staining nor when examined in the dark field of view, any signs of internal structure can be detected in normocytes. In those cases where they are observed, they are either extremely inconsistent or belong to immature E. (see below). On the other hand, a number of authors, starting with Hayem, speak in favor of the existence of such an internal structure based on observed changes in the volume and shape of E. under the influence of hypotonic solutions, heating, electric discharge, etc. (Hamburger, Bechhold), as well as in pathological conditions (Naegeli). Finally, Schilling, partly on theoretical grounds, partly on morphological research, constructed a very complex scheme of the structure of E. Nevertheless, the question remains controversial to this day. The question of the existence of a membrane around E. also remains controversial; despite the histological evidence brought in its favor (Weidenreich, Loewit, Lepeshinskaya, etc.), research on the penetration of various substances into E., cinematographic recording of hemolysis by Comandon, etc., a number of authors deny the existence of a histological membrane, considering it incompatible with phenomena such as the diffusion of Hb from E. discovered by Dietrich, the reversibility of hemolysis studied by Starlinger and others, the fragmentation of E., etc. Depending on these disagreements, the general concept of E. naturally also changes: some imagine it as a bubble filled with a solution of Hb, others as a mesh stroma surrounded by a membrane, the cells of which are filled with Hb, others as a clump of protoplasm adsorbing hemoglobin and devoid of a histological membrane, etc. In various diseases, primarily in anemic states of different origins (see Anemia), E. are found in the blood, differing by a number of features both in relation to their shape and to their color and structure. In pernicious anemia and in sprue, E. sometimes take on a clear oval shape; in Negroes and persons of Negro descent, anemia with sickle-shaped cells has been described; but most often, especially in severe anemias, E. of various irregular shapes are encountered: pear-shaped, star-shaped, etc.-so-called poikilocytes (see Poikilocytosis). In familial hereditary hemolyic jaundice, instead of the usual flattened E., they have a spherical shape. On smears, fixed and stained according to Giemsa-Romanowsky, E. are found that do not take up acidic dye but only basic (basophilic E.), which occurs very rarely, or-much more often-both acidic and basic and are consequently stained not pink but more or less violet-polychromatophilic E. (see Polychromasia). In cases where E. are very poor in Hb, they may remain almost completely unstained except for the rim (hypochromia, oligochromia). With this same staining, and even better with staining with only basic dyes, basophilic punctation is sometimes found in E. on fixed smears. It consists of small speckles scattered throughout the E., stained dark violet by Giemsa. This granularity was first described by Ehrlich in 1885. The question of whether basophilic punctation represents a manifestation of degeneration or regeneration of E. and whether it originates from nuclear substance or from protoplasm has caused many disagreements. Although the views of individual authors still differ at present, the majority of them consider basophilic punctation a sign of 'youth', immaturity of E., and in particular of its primarily basophilic protoplasm, which does not mature to oxyphilia and clumps under the influence of pathological factors. E. with basophilic punctation are occasionally found in healthy people (not more than 1 in 10,000-20,000), but mainly in lead poisoning, and to a lesser extent in other poisonings-mercury, benzene, etc. (Freifeld). In patients with three-day malaria, azurophilic, violet-red granularity, scattered throughout the E., appears in the infected E.-Schüffner's stippling. In tropical malaria, Maurer's stippling is observed in E.-coarse dark violet spots, indicating the severity and toxicity of the disease. In poisoning with many hemolyic poisons, especially potassium chlorate, eosinophilic bright red spherical bodies-Heinz bodies-are found in E., well revealed by Giemsa staining. With these same poisonings, supravital staining with Brilliant Cresyl Blue reveals blue spherical inclusions in the erythrocyte-Heinz bodies, apparently identical with the previous ones. Finally, with the help of supravital staining, a basophilic reticular structure consisting of the smallest granules and delicate threads (vital granularity, substantia granulo-filamentosa, substantia reticulo-filamentosa) is found in E. According to Seyfarth, the reticulo-filamentous substance appears in E. simultaneously with Hb, therefore the earliest stages of E. with still basophilic protoplasm (proerythroblasts, hematogonia) do not contain it; when Hb appears, the reticulo-filamentous substance is first found in the form of fine granules near the nucleus of the erythroblast; as the cell matures and the nucleus decreases, the network around it first becomes denser, and then, starting from the center, becomes thinner until it completely disappears. Thus, the reticulo-filamentous substance is a recognized and indisputable sign of 'youth', immaturity of E., and the density of the network also allows judging the degree of maturity. Naegeli distinguishes two degrees: weak, when the reticulo-filamentous substance appears in the form of fine granules at the periphery of E., and sharply expressed-with numerous and coarse granules. According to Engel, 4 degrees should be distinguished: 1) a dense central coil occupying 1/2-2/3 of E., and a rhomboid network in the rest; 2) the coil occupies 1/4-1/3 of E., the network does not fill it; 3) there is no coil, a rare network, individual threads are encountered; 4) individual threads and their fragments at the edges of E.

Among the loops of the reticulo-filamentous substance, one can sometimes detect nucleoli stained metachromatically in a violet-red color (substance B according to Engel, who considers it identical with Jolly bodies). As for the origin of the reticulo-filamentous substance, all researchers agree on its protoplasmic nature (according to Pappenheim—remains of spongioplasm) and consider it identical with the substance responsible for polychromasia in staining according to Giemsa. Erythrocytes containing reticulo-filamentous substance are often abbreviated as reticulocytes. In the blood of the embryo, reticulocytes account for up to 40% of all erythrocytes (Ferrata, Maximov, etc.), while in the blood of a newborn, Hertz found up to 11%. Regarding healthy adults, opinions somewhat differ: according to most authors who worked by the Schilling-Seifert method, the number of reticulocytes ranges from 0.1% to 0.5%. Trachtenberg obtained higher figures (0.3-1.45%). The content of reticulocytes in the blood can be more or less sharply increased under the influence of various processes leading to enhanced entry of erythrocytes into the blood from the bone marrow and irritation of the latter: after hemorrhages, when in rarefied air, during pregnancy, in poisoning with hemolytic poisons (lead, mercury), in infections (malaria, pneumonia) and in diseases (bronze diabetes, splenomegalic cirrhosis of the liver) accompanied by enhanced hemolysis, under the influence of X-ray rays, after sun baths. An indispensable condition for an increase in the percentage of reticulocytes in all these conditions is the functional completeness of the bone marrow; for example, after hemorrhoidal bleeding in a young, strong subject, the percentage of reticulocytes in the blood can reach 20 and even 40, while in an elderly person or one exhausted by a prolonged illness or repeated hemorrhages, no increase in the number of reticulocytes may occur. Thus, an increase in reticulocytes in the blood simultaneously proves to be a very sensitive indicator of the functional capacity of the bone marrow on the one hand, and the presence in the body or environment of pathological irritants of blood formation on the other. However, before making corresponding conclusions, it is necessary to take into account that an increase in the percentage of reticulocytes can also be caused by factors such as constitutional changes in the structure of erythrocytes (in hereditary familial hemolytic jaundice, where reticulocytes constitute 5-25% or more) or disruption of the maturation processes of erythrocytes due to disturbance of endocrine correlation (Moldavsky, Sherman); toxic effects (arsenic) are also of importance, as well as changes in the type of blood formation itself (increase in reticulocytes in the initial phases of treatment of pernicious anemia with liver) and finally disruption of the functions of the barrier mechanism ensuring the entry from the bone marrow into the blood only of sufficiently mature erythrocytes (Denke and others). Only by taking into account all these possibilities can one correctly evaluate the significance of reticulocytosis in the picture of individual diseases when using various medicinal substances, when analyzing an individual patient. The same should be said about cases of absence of reaction on the part of reticulocytes, when too often the conclusion is drawn about exhaustion or aplasia of the bone marrow (Denke). In addition to the listed forms of erythrocytes associated with changes in their protoplasm, in various diseases, erythrocytes containing nuclei of varying degrees of maturity and normally found only in the bone marrow or even only in the embryo enter the blood (see Hematopoiesis). These include: proerythroblasts (syn. hemagonia)—large cells with basophilic protoplasm and a large reticular nucleus; erythroblasts (syn. erythrocytocytes), differing from the previous ones by the presence of Hb and polychromatophilic staining; normoblasts (see), megablasts (see); karyokinetic forms of these cells are also often found. Along with these nucleated erythrocytes, cells containing only remnants of nuclei in the form of azurophilic granules, Jolly bodies (see Jolly bodies), Cabot rings (see Cabot rings) are also found. All these manifestations of 'immaturity' of the nuclear substance of erythrocytes can be combined with an immature state of the protoplasm (see above), but this parallelism is by no means obligatory. In the words of Pappenheim, 'maturation processes do not all proceed in the cell at the same pace.' Therefore, polychromatophilic erythrocytes without traces of nucleus and normoblasts with completely orthochromatic staining, Jolly bodies in both polychromatophilic and orthochromatic erythrocytes, etc., are encountered. As for the significance of all these forms, they, like reticulocytes, indicate enhanced regenerative activity of the bone marrow and the presence of corresponding pathological irritations (hemorrhage, hemolysis). The difference lies in that for the appearance of nucleated erythrocytes, much more intense or prolonged irritation is required than for the appearance of reticulocytes, and the presence of such erythrocytes can be regarded as a sign of beginning exhaustion of the blood-forming system. In some cases, the appearance of nuclear forms may depend on direct irritation of the bone marrow (carcinosis, leukemia), on changes in the maturation processes of erythrocytes (splenectomy, intoxication), on disruption of the functions of the barrier mechanism (intoxication, endocrine and vegetative dysfunction). Size of erythrocytes. The peculiarities in the shape of erythrocytes allow, at the current level of technology, individual measurement only of their diameter; determination of the volume of erythrocytes is possible only as determination of the average volume; determination of the thickness of erythrocytes—only as the quotient from dividing the volume by the surface calculated from the diameter. The data of various researchers on the diameter of erythrocytes differ from each other to a rather significant degree. Thus, the average diameter is: according to Boros-7.5μ, according to Holler and Kudelka-7.63μ, according to Gram-7.8μ, according to Wiechmann and Schurmeyer-7.9μ, according to Crosetti-8.0μ, according to Gorneffer-8.15μ, according to Millar-8.8μ. It is possible that in these discrepancies not only inaccuracies in the research technique but also racial and especially climatic conditions are to blame. Individual diameters of erythrocytes in each separate person present a rather motley picture and vary within wide limits. The limits of variation are, for example, according to Holler and Kudelka 5.25-9.75μ, according to Boros-5.9-9.3μ, according to Crosetti-6.56-9.84μ, according to Ono and Gisevius, Burker-6.48-9.63μ. At the same time, the main mass of erythrocytes (70-80%) falls on diameters of 7-8μ, and the remaining 20-30% are distributed approximately equally between bodies of larger and smaller sizes. This variation picture can be considered physiological and depending mainly on the individual fluctuations observed in any mass phenomenon (see Variational statistics); Bruce proposes to call it physiological anisocytosis. In various pathological conditions, either changes occur in the ratios between erythrocytes of different sizes toward an increase in the percentage of smaller (microcytes) or larger (macrocytes) bodies, or erythrocytes with abnormally small (4-5μ) or abnormally large (10-12μ) diameter also appear in the blood, or both. In the corresponding cases, one speaks of microcytosis, macrocytosis or simply of anisocytosis. Special mention should be made of the megalocytes occurring in pernicious and some other most severe anemias, the diameter of which can reach 20μ, and schizocytes (fragments of erythrocytes), the diameter of which may fall to 1-2μ. The question of whether certain changes in the diameter of erythrocytes should be regarded as phenomena of degeneration or as signs of 'youth', immaturity has caused quite a few disputes. There is still no unanimity, but the most plausible viewpoint is the one according to which changes in the diameter of erythrocytes in some cases are the result of intoxication of the bone marrow by bacterial toxins or abnormal products of metabolism (microcytosis in tuberculosis, macrocytosis in liver diseases), in some cases—the result of the influence on the bone marrow of pathological changes in internal secretion (microcytosis in chlorosis) or genotypic peculiarities of the bone marrow itself (microcytosis in hereditary hemolytic jaundice), finally the result of irritation of the bone marrow due to blood loss or enhanced hemolysis. Along with this, it is necessary to note that in the bloodstream, various physicochemical factors also influence the diameter of erythrocytes. Thus, we see an increase in diameter in venous blood (influence of CO₂) under the influence of work, during sleep, in conditions of acidosis, and conversely, a decrease in it during alkaline load, hyperventilation, etc. Therefore, determination of the diameter of erythrocytes should also be carried out under usual standard conditions (see above). The volume of an individual erythrocyte is not amenable to determination, and one can only judge the average individual volume, calculated on the basis of data on the ratio between erythrocytes and blood plasma, obtained with the help of a hematocrit, and by counting the number of erythrocytes in 1 mm³ or by other means (see Blood, ratio between formed elements and plasma).

The figure indicating the percentage ratio of the volume of E. to the volume of all blood, divided by the number of E. in 1 mm³ and multiplied by 10⁷, shows the average individual volume of E. in /I³. In practice, it is sufficient to divide the hematocrit reading by the first three digits of the number of E. and multiply by 1,000. Thus, for example, if according to the hematocrit E. constitute 45% of the volume of a given blood and there are 5 million of them in 1mm³, then the average volume of E. is 90/μ³. The opinions of various authors regarding the normal average volume differ considerably, ranging for men between 80 μ³ (Wintrobe and Millar) and 97 μ³ (Simagina) and for women between 81 μ³ (Wintrobe) and 92 μ³ (Haden), with most authors finding the volume of E. in ? women to be 2-3% larger than in men. It is very probable that the discrepancies between the figures of individual authors depend not only on this or that peculiarities or shortcomings of the research methodology and on an insufficiently large number of studies, but also on differences in the living conditions and nutrition of the subjects, climatic conditions, and perhaps also on racial peculiarities. In various pathological conditions, both a decrease in the average volume of E. - microvolumia (tuberculosis of the lungs, posthemorrhagic anemias, chlorotic type anemias, malignant tumors; Simagina) and its increase - macrovolumia (pernicious anemia, hemolytic jaundice, malaria, various chronic infections, liver diseases) are found, and the change in volume may be in contradiction with the change in diameter and depend on changes in the thickness of E., on the flattening or rounding of the latter. Finally, the volume of E. is influenced by the concentration of CO₂ in the blood, blood pH, food intake, sleep, work, etc. The chemistry of E. - see Blood. The physicochemical properties of E. - see Blood. Resistance, or stability of E. - their ability to resist various hemolytic effects. It has been most studied in relation to hypotonic salt solutions (osmotic resistance of E.); in addition, it has been studied in relation to various hemolytic substances (acids, alkalis, hemolysins, saponins, etc.) and thermal effects (thermoreistance). Duncan was the first to pay attention to osmotic resistance in 1867, but its research became more widespread only in the 1880s in connection with the work of the Dutch physiologist Hamburger and a number of clinicians, among whom M. Yanovsky and his collaborators can be mentioned; a further impetus to these studies was given by Chauffard's (1907) discovery of reduced osmotic resistance of E. in hereditary hemolytic jaundice. In physiology, Hamburger, Heber (Höber) and many others achieved considerable success through these studies on the influence of various salts and ions on living cells, on the permeability of cells to various substances, on the structure of cell membranes, etc. In clinical medicine, the study of osmotic resistance of E. has given significantly less, and almost all problems associated with it remain highly controversial. The reason for this is, on the one hand, the lack of uniformity in the research technique, and on the other hand, the complexity of the phenomena on which the osmotic resistance of E. depends. The most common methods for studying the osmotic resistance of E. are various modifications of the method proposed by Hamburger in 1883 and then carefully developed and simplified by Limbeck. Its principle is as follows: in a series of test tubes containing 2 cm³ each of NaCl solution in decreasing concentration from 0.9% to 0.2% (the usual difference in concentration between test tubes = 0.02%), a drop of blood from the finger is placed; the test tubes are shaken and left standing for a time from 15 minutes to several hours, after which they are centrifuged (if sedimentation has not occurred spontaneously); macroscopically determine in which of the test tubes the first traces of yellowing appeared in the fluid standing above the sediment from the released hemoglobin, and in which all corpuscles have undergone complete hemolysis (absence of red sediment). The concentration of the solution in which the first traces of hemolysis appear indicates the minimum osmotic resistance of E. (R_min); the concentration at which complete hemolysis occurs - the maximum osmotic resistance of E. (R_max). In healthy people, Rmin corresponds to concentrations of 0.46-0.44% NaCl, Rmax - 0.34-0.32%. The appearance of traces of hemolysis at a higher concentration is designated as a decrease in Rmin, the appearance of the first traces of hemolysis at lower concentrations - as an increase in Rmin. Similarly, the occurrence of complete hemolysis at a higher or lower concentration is designated respectively as a decrease or increase in Rmax. In various pathological conditions, Rmin and Rmax can change in the same direction (both decrease or both increase), but they can also change independently of each other or in opposite directions. Depending on this, the amplitude of osmotic resistance of E., i.e., the difference between Rmin and Rmax, either remains constant or narrows or expands. Arrhenius and Madsen proposed a colorimetric study of the degree of hemolysis in each test tube (the standard is the laked blood of the subject). The figures obtained are presented in the form of a table or curve and show what percentage of erythrocytes disintegrates in each solution (partial hemolysis). According to Meulengracht, the course of hemolysis in healthy people is as follows: NaCl concentration (in%) . 0.46 0.44 0.42 0.40 0.38 0.36 0.34 0.32 Hemolysis (in%) . 0 0 10 25 55 85 95 100 Colorimetric determination of partial hemolysis gives a much more accurate picture of osmotic resistance than its determination according to Hamburger. Other authors determine the osmotic resistance of E. by counting the number of E. in solutions of different concentrations (microscope method). This method was previously used by Yanovsky and his school, Chanel, Malassez, etc., and more recently by Simmel, Chernyak, Myasnikov. According to Lang (1902), with the help of this method, the following course of hemolysis is found in healthy people: NaCl concentration (in%) .... 0.55 0.5 0.45 0.4 0.35 0.3 Hemolysis (in%) .... 1

6 30

80 The disadvantage of this method is the greater expenditure of labor and time. Depending on various considerations, salt solutions have been changed repeatedly. At present, most researchers use NaCl solutions, Hamburger and Simmel-so-called equilibrated solutions, Chernyak-Na₂SO₄ solutions. It should be noted, however, that the difference in the salt solutions used apparently has significance only in the case where the corpuscles are previously washed; when working with whole blood and within 1-2 hours, the results obtained with all these salts are almost identical.-Resistance in various animals varies within considerable limits. According to Riwosch, they can be arranged in the following series: guinea pig>white rat>dog>gray rat>rabbit>pig>mouse>cat>bull>goat>sheep. It also changes in humans in pathological conditions, especially under the influence of hemorrhages and hemolyzing effects.-The cause of species and pathological differences in the resistance of E. is rooted primarily in the peculiarities of the chemical composition of their stroma and contents. In particular, the works of Riwosch, Masing, Port, and Ge-ber (Masing, Port) can be considered to have established an increase in resistance associated with an increase in the content of phosphoric acid in E., and Meyer and Schaefer (Meyer, Schaeifer) identified the dependence of resistance on the lipocytic coefficient (the ratio of cholesterol to phosphatides). The question of the relationship between the resistance of E. and their degree of maturity has caused quite significant controversy. Most authors come to the conclusion that increased osmotic resistance is inherent in 'young' E. and decreases as they 'age' or mature; among modern authors holding this view, one can note Hamburger, Brinkman, Simmel, Myasnikov, and Svyatskaya. Shustrov developed the opposite view, according to which 'young' E' have reduced osmotic resistance, and the increase in the latter in various pathological conditions is the result of the adsorption of E. of products of hemolysis. According to Meilengracht, mature and immature corpuscles have the same osmotic resistance, and changes in it in hemolytic diseases are due to the fact that enhanced regeneration of E. leads to an expansion of the limits of their fluctuations inherent even in normal conditions. Chernyak believes that 'young' E. can have both increased and decreased resistance depending on the content of immature nuclear or protoplasmic substance in them: the resistance of reticulocytes is as a rule below normal, the resistance of nuclear E. is increased. According to his data, decreased resistance is associated with enhanced regenerative activity of the bone marrow, increased-with its exhaustion. Along with internal factors determining the osmotic resistance of erythrocytes, the substances surrounding them in the plasma also play a significant role. Brinkman and van-Dam found that E. adsorb from the plasma a 'hemolytic complex', in which lecithin plays the main role. Washing E. with equilibrated solutions, which themselves do not change the osmotic resistance of E., removes this 'complex', and the osmotic resistance of E. increases. Unlike the 'secondary' osmotic resistance of E., provided with this complex, Brinkman and van-Dam called the increased osmotic resistance of washed corpuscles 'primary' and believe that it corresponds to the osmotic resistance that red corpuscles have at the moment of their exit from the bone marrow. Hamburger also joined this hypothesis, however, it also met with a number of objections. The influence of electrolytes and their ions dissolved in the plasma on the osmotic resistance of E., changing the colloidal state of the E. shell (Na+, K+ and Cl- ions decrease the osmotic resistance of E., while Ca2+ and HPO42+ increase it), as well as CO₂ of the blood, under the influence of which Cl- ions move into E., their swelling and decrease in their osmotic resistance (Hamburger, Geber), is quite certain. Finally, hemolysins circulating in the plasma and bile acids that reduce surface tension also have an influence on the osmotic resistance of erythrocytes; the effect of bilirubin on osmotic resistance is debatable. In view of such a multitude and complexity of factors determining the osmotic resistance of E., it is not surprising that the assessment of its changes in clinical practice is very difficult. As a result of all possible combinations of factors, the most various diseases are accompanied by the same changes in the osmotic resistance of E., and conversely, it varies in similar diseases and in the same patients at different periods of the disease. In view of this, its diagnostic value is very limited and generally amounts to distinguishing hereditary hemolyic jaundice, in which the osmotic resistance of E. is reduced, from jaundice of other origin, in which it is usually increased; it may have some significance, apparently, also in the recognition of cancerous diseases (Yanovsky, Lang). Provided that secondary factors are taken into account, the study of the osmotic resistance of E. can provide some indications regarding changes in the process of blood formation and maturation of E. and the functional ability of the bone marrow; in combination with the study of pigment metabolism, it gives some possibility to judge the intensity of hemolysis, toxic or endocrinologically caused changes in blood formation, the intensity of regeneration of E. and the degree of exhaustion of the blood-forming system in anemic and hyperglobulinemic conditions. In the same way, the osmotic resistance of E. can be used to study the effect on blood formation of various medicinal and toxic substances, infections, etc. The resistance of E. to hemolytic substances was more thoroughly studied in relation to saponin. Comparing E. of various animals, Riwosch found that their resistance to saponin is the less, the greater their osmotic resistance; a number of authors further established that the saponin resistance of E. is directly dependent on their cholesterol content, and Port drew attention to the inverse relationship between the saponin resistance of E. and their HPO₄2- content. Geber and Nast explained this opposition of saponin resistance of E. and their osmotic resistance by the fact that under the influence of saponin the electric charge of colloids in E. changes, as a result of which HPO₄2- no longer increases their resistance, but, on the contrary, decreases it. Studies of saponin resistance of erythrocytes in patients showed that it remains unchanged in the most various diseases, including hemolytic jaundice, so for clinical practice this study has no significance. The study of the resistance of E. to bile acids, various hemolysins, alkalis and acids also has no clinical significance.-Thermoresistance of E. is expressed in the difference in temperature at which they hemolyze. A. Egorov (1928) first drew attention to it and developed the methodology for its determination. In normal conditions, the beginning of hemolysis occurs at 40-50°, complete hemolysis-at 46-56°. After sunbaths in healthy individuals, the range of thermoresistance of E. narrows and shifts upward, in patients it also narrows, but shifts downward.-Reversal of hemolysis consists in the fact that completely hemolyzed, transparent lacquered blood, when a hypertonic solution of NaCl is added to it, again acquires the usual cloudy appearance, and under the microscope a more or less significant number of Hb-containing E. is found in it. This phenomenon was first described by Brinkman and F. von Szent-Gyorgyi and especially thoroughly studied by Starlinger. The mechanism of reversal remains to this day very controversial; clinical significance of its study has not been acquired. Functions of erythrocytes. E. perform a number of the most important functions in the body. The main one is the gas exchange function, consisting of the absorption of oxygen in the lungs and its delivery to tissue capillaries, on the one hand, and the perception of carbon dioxide in the capillaries and its delivery to the lungs-on the other. Both of these processes are carried out due to hemoglobin, with O₂ being bound by its pigment part, and CO₂ by globin (see Hemoglobin). Therefore, the Hb content in E. is of decisive importance for normal gas exchange. However, along with this, the peculiarities of the shape of the latter also play a significant role, thanks to which the surface of gas absorption and release reaches enormous dimensions. According to Buerker's calculations, taking the surface of one E. as averaging 128 μm², the total surface of E. in 1 mm³ of blood is 640 mm², and in the entire blood 3,200 μm², of which 129 μm² falls to the share of E. located at any given moment in the tissue capillaries, and approximately the same amount to the share of those E. that are in the pulmonary capillaries. In addition, the coincidence of the diameter of E.

(8 µ) with the diameter of capillaries (10 µ) forces E. to pass through the latter one by one and to be in close contact with their walls, thereby significantly facilitating the diffusion of gases from E. into the tissues and back, and increasing the efficiency of the use of the E. surface. Finally, in the same direction acts the continuous rotation experienced by E. in the blood flow, and the sharp deceleration of the blood flow speed (to 0.5 mm in 1 sec.) in the capillaries (see Blood Circulation). The second most important function of E. is the enhancement of the buffer properties of the blood, carried out, on the one hand, due to the buffer properties inherent in hemochromogen and globin, and on the other hand, due to the permeability of the E. shell to anions and its impermeability to cations and Hb. As a result, in tissue capillaries, when the CO₂ pressure in the blood increases, anions from the plasma (especially Cl) move into E. (in accordance with the laws of Donnan equilibrium); at the same time, in the plasma, part of the bases is freed and binds the incoming CO₂, and in E., the anions that have penetrated there detach part of the cations from Hb and form neutral salts with it. In the lungs, however, conversely, the formed oxyhemoglobin, being a stronger acid inside E., attracts cations to itself, and the freed anions pass into the plasma and combine with the cations remaining after the removal of CO₂ (see Buffer properties, Hemoglobin, Blood, physicochemical properties). Similar phenomena also occur when other acidic substances enter the blood from tissues, with pathological disturbances of acid-base balance, with experimental introduction of acids or alkalis into the blood. In the general result, E. account for about 30% of the total buffer capacity of the blood. It is these migrations of ions that determine the third function of E.-their participation in the regulation of the ionic composition of the plasma and in all processes associated with it, in particular in the processes of filtration and reabsorption of salts in the kidneys. The fourth function of E. is their participation in water and salt exchange. This includes phenomena such as the swelling of E. in venous blood due to water coming from the tissues, and the decrease in their volume in the pulmonary capillaries and in the arteries, with the water released partly evaporating in the lungs, partly excreted by the kidneys and sweat glands, but mainly returning from the capillaries back into the tissues. This also includes the absorption and release of water by E. during diuresis, observed by Zyukov, and the fluctuations in the volume of E. with the onset and subsidence of edema and under the influence of water load, studied by Chernyak and Simagina. One can form an idea of the quantitative magnitude of this function of E. if one takes into account that under normal conditions the difference in the volume of E. in venous and arterial blood is about 10%, and that at complete rest about 1,800 cm³ of E. pass through the lungs per minute, and during work-up to 12-15 thousand cm³; thus, in one minute E. give off (resp. absorb) from 200 to 1,500 cm³ of water, and in a day through them passes from 300 to 2,000 liters of water with the corresponding amount of substances dissolved in it. In addition to the listed functions, E. also participate in many other processes: the presence of various enzymes in them (see Blood, blood enzymes), their glycolytic properties (see Glycolysis), the ability of them to adsorb toxins and protein breakdown products, discovered by Zbarsky, the distribution of lipoids in the blood (see Blood) and many others. There can hardly be any doubt that a detailed study of these facts will help to shed new light on the mechanism of many physiological and pathological processes.