HAEMOTRANSFUSION

Blood is a fluid tissue of a human organism which performs important functions. It’s known from the course of physiology. However, in the clinic we face with this tissue in special aspect because from old time and to nowadays it is a riddle, and the attempts to apply it for treatment remain inviting because in some cases they are magically effective.

It was considered that blood is the source of life, there is a soul in it and consequently with blood loss the soul is lost also. Many researchers associated the action of blood with a person’s character, force and physical ability. That is why the blood baths in ancient Egypt and drinking of lost gladiator’s blood in Rome were recommended.

The magical action of blood was named the Medea’s method of treatment. Ovidios Nazon describes the attempts of Medea to rejuvenate an old man with young blood: “Medea with a sword out the throat of the old man and let the old blood pour out, she poured juice in him. After having drunk, with his mouth or wound, Ezon’s gray hair removed and his beard and hair became black. The leanness, as well as both paleness and decrepitude disappeared. The body became plump, the wrinkles became smooth. Ezon was surprised; he remembered himself like this forty years ago”.

Certainly, there are a lot of exaggerations in this legend, but the fact that it underlines the important role of blood in the human life is doubtless. In 1492 blood was transfused from two young men to Pope Innokenti VІІІ, aiming to rejuvenate him, but the miracle did not take place — the pope died from old age and the young men died too.

Attempts to apply blood for treatment were going on. In 1667, the blood of a lamb was successfully transfused to a person by doctor Deni. Then a period of failures came, which for a long time hampered the use of blood. The opponents of haemotransfusion considered that “lamb’s blood, which was transfused into the vein of a person, can give him the features of cattle: dullness and cattle inclinations”. From the first haemotransfusion and to our days haemotransfusiology passed a difficult, sometimes tragic path. It has been caused by the absence of knowledge in anatomy, physiology and the knowledge of the role of blood in a human organism.

Harvey’s discovery (1682) gave a representation of the blood system; the analysis of the reasons of unsuccessful transfusions led at last to the opening of agglutination that is the process of conglutination of erythrocytes, and, finally, to the discovery of the blood groups.

But from time to time doctors transfused blood in cases of emergency, despite of prohibition and punishment. J. Blundell (1820) in England, Volf (1832) and O. M. Filomafitsky (1848) in Russia successfully transfused blood from person to person, aiming to explain the mechanism of its action. The beginning of the previous century was marked by discovery of the blood groups: 1901 — K. Landsteiner (three groups) and 1907 — Ya. Yansky (the fourth group). Mechanisms of action of transfused blood were studied (O. M. Filomafitsky), conditions of its preservation (in 1914, V. Yurevich and M. Rozengard suggested the application of sodium citrate for the prevention of blood coagulation), source of receiving blood (1919, V. Shamov suggested the application of the cadaveric blood).

The system of organization of blood service (1926, due to O. O. Bogdanov’s efforts the Institute of Haemotransfusion was opened in Moscow for the first time in the world) was created and became stronger.

Today the work concerning the study of the specificity of blood, application of blood or its certain components is on.

Now in the clinical practice while solving the question of haemotransfusion doctors take into account the features of blood, opened by immunologists. It is necessary to always remember that blood is a fluid tissue and its transfusion is similar to tissue transplantation, in which over 300 various antigens, which form a number of systems, are revealed. But the clinic takes into account not all of them but only the system which determines the groups of blood and Rhfactor. The blood group is a genetically caused sign, which provides the association of antigens which are in uniform elements and blood plasma. In 1901, K. Landsteiner — a doctor from Vienna — opened a phenomenon of agglutination, having distributed people into three groups of blood. He had found that the serum of the first group agglutinates with erythrocytes of the second and third groups. The serum of the third group agglutinates with erythrocytes of the second group. In a due course Ya. Yansky discovered also the fourth blood group. The similar phenomenon was explained by the existence of the AB0 system, in other words — this is a set of agglutinins and agglutinogens in a certain individual.

According to the statistic data, 42–44% of people in Europe have the second blood group, 38–39% — the first, 12–14% — the third, 4– 6% — the fourth. Each blood group is determined by the association of agglutinins and agglutinogens. So, in the first group there are agglutinins α, β and agglutinogen 0; in the second — accordingly β and A, in the third — α and B and in the fourth — АВ0. The blood groups are designated with the presence of agglutinogens: 0 (І); A (ІІ), B (ІІІ), АВ (ІV). Agglutinogens A, B, and 0 are thermolabile organic compounds (glycoprotein). They are situated in erythrocytes and make the antibodies formation. These antigens already appear in a 3-month fetus and do not change during life. It was considered before that antigen is not present, therefore it was designated as 0. However, today it is known that there is an antigen in the first blood group, but it does not produce the agglutination reactions, therefore it is designated as 0 and it is not taken into account during haemotransfusion.

Agglutinins are thermolabile globulins. They exist in plasma and also in tissue fluid. Their accumulation in plasma occurs gradually, reaching such level (titer) when they are capable of serving as the reason of agglutination reaction. In newborns, the titer is low, but it gradually increases, reaching a maximum at 10–20 years. The titer of agglutinin usually is in adults from 1:32 up to 1:128, β — from 1:16 to 1:64. The higher the titer the more the opportunity of agglutination reaction in interference of the same name agglutinogens of erythrocytes.

During haemotransfusion the Ottenberg’s rule was kept: “During haemotransfusion the erythrocytes which are introducted (agglutinogens) agglutinate because the entered plasma (agglutinins) is diluted in the total amount of fluid and their titer becomes insufficient for patient’s erythrocytes agglutination”. Indeed, clinical physicians warned that in the case of acute anemia during the attempt of fast compensation of blood loss with donor blood of the first group the patient erythrocytes agglutinated because of the high concentration of agglutinins which were entered. This is called opposite agglutination. Therefore, big amount of compatible blood was not recommended to transfuse but to add the fluid into the system. Eventually, the agglutination during the transfusion of the same blood group, in particular the second group was observed.

Last years researches of immunologists and haemotransfusiologists allow to consider the reasons of these manifestations. It was appeared that there are some subgroups of agglutinogens: А₁, А₂, А₃, А₄, A_x, А_z, etc. The strongest antigen is А₁, it exists in approximately 88% of people with the second blood group. This antigen serves as the reason for fast agglutination reaction. A little bit weaker is the action of А₂, which is observed in 12% of people with the second blood group. Other subgroups of antigen A — weak and are not practically taken into account clinically.

Agglutinogen B has also some subgroups (В₁, В₂, В₃), but their titer and antigenic action is insignificant, therefore they are not considered clinically too.

Antigens А₁ and А₂ are capable to produce antibodies against themselves, which called extra-agglutinins α1 and α2. Their association and reactions during haemotransfusion are unforeseen within the limits of one (second) blood group. So, if the patient has А₁ (II) βα₂ blood group, and the donor has А₂ (ІІ) β, the agglutination may begin because of reaction of extra-agglutinins α₂ of the recipients with the donor’s agglutinogen А₂.

During lifetime, when antigens A and B enter an organism, the immune agglutinins α and β are formed. They increase the titer of natural agglutinins sometimes up to 1:512 and then there is no opportunity for it to weaken in a cultivation of liquid of an organism which can lead to agglutination. That is why the Ottenberg’s rule begins to loose its force in practice, and more often only the same blood group transfusion is discussed. It is already become a law in children.

During haemotransfusion, not only the AB0 system but also the Rh-factor system is taken into account. In 1940 K. Landshteiner and A. Wiener, during introduction of erythrocytes to a rabbit from an anthropoid ape, revealed that it produces antibodies, which are capable of agglutinating erythrocytes. The factor capable to produce antibodies was found in human blood erythrocytes and recently in its fluid part. It received the name of Rh-factor. The structure of this antigen is not completely known, but it is considered that it is a polymucoprotein compound. 85% of people are Rh-positive (their erythrocytes contain the Rh-factor), and 15% — Rh-negative. So, during haemotransfusion there is always a danger of the Rh-conflict: whether during a repeated transfusion to the Rh-negative patient of Rh-positive blood, whether during the transfusion of Rh-negative blood which contains antibodies to a Rh-positive patient. Such a simple system in modern medicine is not accurate because it has been revealed that a person has 6 antigens Rh-Hr (D, C, E, d, c, e). The first three antigens are a version of the Rh-factor: D (Rh₀), C (rh’), E (rh”). D is the strongest antigen and E is the weakest one.

As a result, the presence of antigen D (rh₀) is taken into account, 2–3% of Rh-negative donors have antigens C (rh’) and E (rh”). That is why the group of donors with Rh-negative blood can treat those that have no antigens D (rh₀), C (rh’), E (rh”), and the recipient should be considered Rh-negative only if he has no antigen D. Thus, in a human blood there can be one type of Rh-factor or a combination of several types and specific antibodies develop to each of them.

The atigens of H_r–H_r0, hr’, hr” system are in the erythrocytes, but their antigenic properties are weak. At the same time, in case of their presence in a Rh-negative donor during haemotransfusion to a Rh-positive recipient, posttransfusion reaction can occur. It is necessary to remember about these features of the immune system and during haemotransfusion it is necessary to know the Rh-factor and to conduct tests on Rh-compatibility, and, in general, it is better to transfuse the blood of the same group and the same Rh-factor.

Definition of Blood Group

There are some ways of blood group definition: а) with the help of standard serums (known agglutinins); with the help of standard erythrocytes (known agglutinogens); the modern method, which is widely practiced, is the application of anti-A and anti-B monoclonal antibodies.

It’s necessary to estimate the validity of standard serums in definition of the blood group — serum should be transparent, have no additional inclusions and correspond to the terms of preservation. Definition is conducted at an ambient temperature (17–20°С), in Petri’s dishes or special plates. As a rule, serums of first, second and third groups are taken. The serum from the fourth group is applied as a control. Always they use serums of two titers; therefore two drops of standard serum of every of three groups are put on the dish. The blood which must be examined is added there (5–10 times less volume). The serums are mixed with the drop of blood and the answer is determined by the agglutination reaction. If the investigated blood is of the first group, the agglutination is not present in any of the serums, if of the second group — agglutination appears with the serum of the first and third groups, if the blood is of the third group, the agglutination appears in the first and second serum, if agglutination occurs in all of the serums it is the fourth group.

Definition of the blood group with the help of standard erythrocytes is carried out, as a rule, at haemotransfusion stations, because erythrocytes preservation and transportation is difficult. It is necessary to remember that in this case the agglutinogens are known, therefore, the investigated serum, which is received by precipitation or centrifugation of patient’s blood, is added to the drop of standard erythrocytes.

The results are determined 5 min after mixing the serum and standard erythrocytes. If there is agglutination with the erythrocytes of the second and third groups, it is the first blood group. If there is agglutination of the erythrocytes only in the third group, the patient has the second blood group, if agglutination of the erythrocytes of the second group — the third blood group. In case of the absence of agglutination in all the kinds of standard erythrocytes, the fourth blood group is determined. While registering the reaction, it is possible to determine agglutination there where it is actually absent. The reasons for this phenomenon (pseudo-agglutination) can be:

Low temperature in the room (under 15°С).

The registration of the reaction took place later than 5 min —the serum dries out and stimulates agglutination.

At low temperature erythrocytes develop into “monetary columns”, stimulating agglutination; it easily disappears while rocking the plate or by adding a physiological solution.

Concentrated standard serum.

With presence of immune antibodies in the investigated blood,if the patient suffers from sepsis, systemic blood diseases.

Sometimes agglutination can not be noticed, it happens under the following circumstances:

а) definition of the blood groups is carried out at the temperature higher than 25–30°С;

if the reaction is taken into account earlier than 5 min.

if the titer of agglutinins is low (lower than 1:32) and thus theblood group is determined with the help of one series of serums;

if the ratio of serum-blood is not correct and the quantity of blood is too big.

The most reliable method of definition of blood group is using cyoclones anti-A and anti-B. They are the product of hybridome cellular lines, received as a result of the merge of mouse antibodyproducing B-lymphocytes with cells of mouse myeloma. Cyoclones are not the products of vital activity of human cells, it is ascitic diluted liquid from mice — carriers corresponding with the hybridome, which contains specific antibodies of class M, which are directed against antigen A and B.

Cyoclones anti-A and B are produced as a liquid in bottles or ampoules of 20, 50, 100 and 200 dozes. The liquid contains preservatives — 0.1% sodium azide, colored blue (anti-A) or pink (anti-B). The bottles are kept for two years in a refrigerator at the temperature of +2 to +8°С. In definition of the blood group with native blood with and without preservatives the best results happen at high concentration of erythrocytes. On the plate, at the room temperature of 15–20°С, 1 drop (0.1 ml) of the anti-A and anti-B cyoclones is put, then the examined blood (the drop should be 10 times less) is mixed for 3 min long, watching for the reaction. If agglutination is not present in both cyoclones, it is the I blood group; if agglutination occurs with anti-A cyoclones, it is the II group; if with anti-B, it is the third group, and if agglutination is present in both cyoclones — IV blood group.

Definition of Rh-factor

There are some methods of Rh-factor definition in human blood. In emergency situations an express-method is used. To the standard anti-Rhesus-serum of the same with the investigated blood group the blood is added for definition of Rh-factor. A standard serum АВ (ІV), which does not contain any antibodies, is used as a control. The reaction is determined during 3–5 min. The presence of agglutination of erythrocytes with anti-Rhesus-serum testifies that the blood is Rh-positive, the absence of agglutination — Rh-negative. This method is allowed only in special emergency cases and further it is necessary to determine Rh-factor by laboratory methods.

The most reliable method of Rh-factor definition is the test-tube: the reaction is conducted in suspension of gelatin or polyglukin (conagglutination). Two lines of test-tubes (from two series of anti-Rhesus-serum) 3 test tubes in each one are taken for investigation. In each test tube 0.1 ml anti-Rhesus-serum of the same group with the researched blood is poured. Then erythrocytes from the examined blood are added to the first test tube, standard Rh-positive erythrocytes — to the second, standard Rh-negative erythrocytes — to the third. After that, 2–3 ml of 10% solution of gelatin are added to all test tubes and kept in the thermostat for 1 h at the temperature of 37°С. Determination of the reaction is carried out after shaking the test tubes. The presence of agglutination reaction in the examined blood testifies that the blood is Rh-positive, the second and third test tubes serve as a control.

Sources of Blood Obtaining

The basic source for receiving blood is healthy donors. It is legal to take blood from adult people who have not suffered from tuberculosis, syphilis, malaria, are not in the recovery period after the infectious disease, virus hepatitis. It is prohibited to take blood from exhausted people, with metabolism disorder, oncologic patients and also those who suffer from suppuration processes. The single doze of taken blood should not exceed 450 ml. The break between blood taking is 2 months.

The attitude towards a donor is different in different countries. So, in Italy the donorship is considered as an act of “Christian mercy, love and brotherhood”, and the donor who gives blood receives forgiveness of sins. In Holland the person who gives blood 10 times receives a medal with K. Landsteiner’s image. In our country the donor service can be paid if the donor receives material compensation, and also gratuitous if people give blood once a year disinterestedly.

Blood taken from a donor should be kept under special conditions. The history of preservation of blood has begun since 1865, when Doctor V. Sutugin purposed to defibrinate and preserve blood at the temperature of 0°С. In 1867 Rautenberg suggested to mix blood with a solution of carbonic sodium. It prevented it from coagulation. In 1914 Yurevich and Rozengrad suggested the application of sodium citrate in the ratio of 1:10 for preventing blood from coagulation. Nowadays blood is preserved with a solution of glucicir (sodium of hydrocitrate, glucose and distilled water) according to the calculation of 25 ml for 100 ml of blood. Before preservatives of various structure were applied. Besides of preservatives, they contained glucose and chloramphenicol. The blood prepared by such a method is kept in sterile bottles at the temperature of +4–+6°С in the refrigerator. The term of preservation is 10–11 days, but the optimum term is 7 days.

Blood can be preserved by adding heparin (50–60 mg for 1 l of blood). This blood is applied in devices of artificial blood circulation, term of its preservation — 24 h. The preservation of blood is done at low temperatures (–196°С). Such frozen blood is suitable for transfusion in 2 to 10 years. However, because of complex specifications on preserving such blood, this method did not have wide spreading.

There are also other sources of blood obtaining:

Utilized blood is the blood received during bloodletting concerning different diseases, which do not influence its structure. For example, hypertonic disease.

Auto-blood is the blood taken from a patient before an operation or during an operation if it poured into cavities as a result of vessels rupturing. This blood should not contain any impurities (bile, intestinal and gastric contents, urine).

Umbilical-placental blood. Its application was suggested byprof. M. S. Malinovsky. It contains an increased amount of enzymes, proteins, hormones (folliculinum). The amount of blood which is taken changes from 100 up to 400 ml. Today it is widely used for preparing blood components.

Cadaveric blood. In his works V. Shamov proved that even10–12 h after the death the blood keeps its properties. Therefore he suggested taking blood from cadavers. They should be people who died in the result of sudden, more often traumatic, injuries. The blood is prepared under sterile conditions no later than 6–8 h after the death.

Thus, all the above stated proves that haemotransfusion has passed through clinical to scientifically-grounded methods of preparations, preservation and application as a powerful medical factor. The system of preparation, preservation and application of blood was established. In big hospitals there are stations for blood purveyance. No matter what kind of blood preparation should be used, it is necessary to remember that the donor’s blood is subject for investigation in order to prevent from making harm to the patient.

Finding the mechanism of transfused blood action eventually determines the indications for haemotransfusion.

Mechanism of Transfused Blood Action

The effect of transfused blood depends upon the amount, method and speed of transfusion. The entered blood has a substitutive, haemostatic action, increases metabolism, shows immunobiological, desintoxicative and nutritious effect. The basic mechanism of action transfusing blood is transport of oxygen.

In surgical practice the substitutive action is the most important, blood replacement is necessary, because it threatens the life of the patient. Replacement can be carried out rationally, it is impossible to compensate 100% of the blood loss. Usually, 1/3 of the lost blood is transfused, then salty solutions and again blood is transfused. The transfused blood irritates the baroreceptors, assists in the tone of the vascular wall, increases the volume of the circulating blood, respiratory surface of erythrocytes and make influence upon the organs of haemocirculation.

The haemostatic effect is achieved due to the introduction of coagulation factors of the blood — thrombocytes. In particular, it’s found while transfusing fresh blood (direct or 1–3 days after preservation). Together with the donor’s blood, antibodies, globulins, which make immune correction on the organism of the patient, are entered. The entered blood replaces protein, carbohydrates and fats. Besides, protein of the blood adsorbs harmful substances (poison, bacterial toxins), strengthens metabolism oxidizing processes, excretion of urine, i.e. performs detoxication action.