How to determine blood group using standard sera, methods and features of the procedure. Technique for determining blood group using standard sera Determination of blood using standard sera

AB0 blood groups

The blood groups of the AB0 system were discovered in 1900 by K. Landsteiner, who, by mixing the red blood cells of some individuals with the blood serum of other individuals, discovered that with some combinations the blood coagulates, forming flakes (agglutination reaction), but with others it does not. Based on these studies, Landsteiner divided the blood of all people into three groups: A, B and C. In 1907, another blood group was discovered.

It was found that the agglutination reaction occurs when antigens of one blood group (they are called agglutinogens), which are found in red blood cells - erythrocytes, stick together with antibodies of another group (they are called agglutinins) that are found in plasma - the liquid part of the blood. The division of blood according to the AB0 system into four groups is based on the fact that the blood may or may not contain antigens (agglutinogens) A and B, as well as antibodies (agglutinins) α (alpha or anti-A) and β (beta or anti-B) .

First blood group - 0 (I)

Group I - does not contain agglutinogens (antigens), but contains agglutinins (antibodies) α and β. It is designated 0 (I). Since this group does not contain foreign particles (antigens), it can be transfused to all people. A person with this blood type is a universal donor.

Second blood group A β (II)

Group II contains agglutinogen (antigen) A and agglutinin β (antibodies to agglutinogen B). Therefore, it can be transfused only to those groups that do not contain antigen B - these are groups I and II.

Third blood group Bα (III)

Group III contains agglutinogen (antigen) B and agglutinin α (antibodies to agglutinogen A). Therefore, it can be transfused only to those groups that do not contain antigen A - these are groups I and III.

Fourth blood group AB0 (IV)

Blood group IV contains agglutinogens (antigens) A and B, but contains agglutinins (antibodies). Therefore, it can only be transfused to those who have the same, fourth blood group. But, since there are no antibodies in the blood of such people that can stick together with antibodies introduced from outside, they can be transfused with blood of any group. People with blood group IV are universal recipients.

Blood groups according to the ABO system

Method for determining blood groups

The blood group according to the ABO system is determined using the agglutination reaction. Currently, there are three ways to determine blood groups using the ABO system:

According to standard isohemagglutinating sera;

Using monoclonal antibodies (coliclones anti-A and anti-B).

Determination of blood groups using standard isohemagglutinating sera

The essence of the method is to detect group A and B antigens in the test blood using standard sera. For these purposes, the agglutination reaction is used. The test should be done in a room with good lighting at a temperature of 15-25 ° C.

To carry out the test you need: - Standard isohemagglutinating sera of groups O (I), A (II), B (III) and AB (IV) of two different series. Serums for determining blood groups are prepared from donor blood in special laboratories. Serums are stored in the refrigerator at a temperature of 4 - 8° C. The expiration date of the serum is indicated on the label. The label also indicates the titer (the maximum dilution of the serum at which an agglutination reaction can occur), which must be no lower than 1: 32 (for serum B (III) - no lower than 1:16 / 32). The whey should be transparent, without signs of rotting. For convenience, standard serums are tinted in a certain color: O (I) - colorless (gray), A (II) - blue, B (III) - red, AB (IV) - bright yellow. These colors accompany all labels on blood products that have a group affiliation (blood, red blood cells, plasma, etc.).

White porcelain or enamel plates or other plates with a wettable surface, marked according to blood types.

Isotonic sodium chloride solution.

Needles, pipettes, glass rods (slides).

Reaction technique.

1. Under the appropriate designations of the blood group, serum of groups I, II, III is applied to a plate (plate) in a volume of 0.1 ml (one large drop with a diameter of about 1 cm). To avoid mistakes, two series of sera are applied, since one of the series may have low activity and not give clear agglutination. Thus, 6 drops are obtained, forming two rows of three drops in the following order from left to right: 0 (I), A (II), B (III).

2. Blood for research is taken from a finger or from a vein. Using a dry glass rod, 6 drops of the test blood, approximately the size of a pinhead of 0.01 ml (small drop), are successively transferred to the plate at 6 points, each next to a drop of standard serum. Moreover, the amount of serum should be 10 times greater than the amount of blood being tested. Then they are carefully mixed together using glass rods with rounded edges.

A simpler technique is also possible: one large drop of blood is applied to a plate, then it is taken from there with the corner of a glass slide and each drop of serum is transferred, carefully mixing it with the last. In this case, the blood is taken each time with a clean corner of the glass, making sure that the drops do not mix.

3. After mixing the drops, shake the plate periodically. Agglutination begins within the first 10-30 seconds. But observation should be carried out for at least 5 minutes, since later agglutination is possible, for example with red blood cells of group A 2 (II).

4. Add one drop of isotonic sodium chloride solution to those drops where the reaction has occurred, after which the results of the reaction are assessed.

The agglutination reaction can be positive or negative

If the reaction is positive, within the first 10-30 seconds, small red grains (agglutinates), consisting of glued red blood cells, visible to the naked eye, are observed in the mixture. Small grains gradually merge into larger grains or even irregularly shaped flakes. In a negative reaction, the drop remains uniformly colored red.

The results of the reactions of two series in drops with serum of the same group must coincide.

The belonging of the blood being tested to the corresponding group is determined by the presence or absence of agglutination when reacting with the corresponding sera.

If all the sera gave a positive reaction, then the test blood contains both agglutinogens - A and B. But in such cases, in order to exclude a nonspecific agglutination reaction, an additional control test of the test blood should be carried out with standard serum of group AB (IV).

Determination of blood groups using monoclonal antibodies

To determine blood groups using this method, monoclonal antibodies are used, which are obtained using hybridoma biotechnology.

Hybridoma is a cell hybrid formed by the fusion of a bone marrow cell (myeloma) with an immune lymphocyte that synthesizes specific monoclonal antibodies. The hybridoma has the ability to grow unlimitedly, is characteristic of a tumor cell, and has the inherent ability of a lymphocyte to synthesize antibodies.

Standard reagents have been developed: monoclonal antibodies (mAbs): anti-A and anti-B coliclones, used for the determination of erythrocyte agglutinogens. Zoliclones are lyophilized powder of red (anti-A) and blue (anti-B) color, which is diluted with isotonic sodium chloride solution immediately before the sample.

Technique.

Anti-A and anti-B zoliclones are applied to a white tablet, one large drop (0.1 ml) under the appropriate inscriptions: anti-A or anti-B. One small drop of the test blood should be applied next to the drops of antibodies. After mixing the components, observe the agglutination reaction for 2-3 minutes. Evaluating the results is very simple.

Determining blood grouping may be accompanied by errors that lead to incorrect interpretation of the results. Three main groups of errors can be distinguished: errors associated with low quality reagents; technical errors; errors associated with features Scheme for assessing the results of determining blood groups using monoclonal antibodies (coliclones anti-A and anti-B) of the blood being tested. The first two can be avoided by strictly observing the above-described requirements for serum, reaction conditions, etc. The third group is associated with the phenomenon of nonspecific panagglutination (Thomsen phenomenon), the essence of which is that serum at room temperature agglutinates with all red blood cells, even with their own (autoagglutination), and erythrocytes at the same time give agglutination with all sera, even with serum of group AB. A similar phenomenon has been described in a number of diseases: blood diseases, splenomegaly, liver cirrhosis, infectious diseases, etc. Panagglutination and autoagglutination in healthy people have also been described.

The phenomenon of panagglutination and autoagglutination is observed only at room temperature. If the determination of group membership is carried out at a temperature of 37 ° C, they disappear.

It must be strictly remembered that in all cases of unclear or questionable results, blood groups should be re-determination using standard sera from other series, as well as in a cross-sectional manner.

Numerous studies have shown that the blood can contain various proteins (agglutinogens and agglutinins), the combination (presence or absence) of which forms four blood groups.
Each group is given a symbol: 0 ( I), A (II), IN (III),AB(IV).
It has been established that only blood of the same type can be transfused. In exceptional cases, when there is no blood of the same group, and transfusion is vital, transfusion of blood of a different group is permissible.Under these conditions, blood is 0 ( I) group can be transfused to patients with any blood group, and to patients with blood AB(IV) group, donor blood of any group can be transfused.

Therefore, before starting a blood transfusion, it is necessary to accurately determine the patient’s blood type and the type of blood being transfused.

Determination of blood group.

To determine blood group, standard group 0 sera are used ( I), A (II), IN (III), which are specially prepared in the laboratories of blood transfusion stations.
Numbers are placed on a white plate at a distance of 3-4 cm from left to right. I, II, III, denoting standard serums. A drop of standard serum 0( I) groups are applied with a pipette to the sector of the plate indicated by the number I; then apply a drop of serum with a second pipette A (II) groups by number II; they also take whey IN (III) group and the third pipette - applied under the number III.

Then the person being examined is pricked on the finger and, with a glass rod, the flowing blood is transferred to a drop of serum located on a plate and mixed until the color is uniform. A new stick is transferred to each blood serum. After 5 minutes from the moment of staining (by the clock!) the blood type is determined by the change in the mixture. In the serum where agglutination (gluing of red blood cells) occurs, clearly visible red grains and clumps appear; in serum, where agglutination does not occur, the drop of blood will remain homogeneous, uniformly colored pink.

Depending on the blood type of the person being examined, agglutination will occur in certain samples. If the subject has 0 ( I) blood group, then red blood cells will not stick together with any serum.
If the subject has A (II) blood group, then agglutination will not occur only with serum of the group A (II), and if the subject has B (III) group, then there will be no agglutination with the serum B (III). Agglutination is observed with all sera if the blood being tested is AB(IV) groups.

Rh factor.

Sometimes, even with a blood transfusion of the same group, severe reactions are observed. Studies have established that approximately 15% of people do not have a special protein in their blood, the so-called Rh factor.

If these individuals are given a repeated transfusion of blood containing this factor, a severe complication called Rh conflict will occur and shock will develop. Therefore, at present, all patients are required to have their Rh factor determined, since a recipient with a negative Rh factor can only be transfused with Rh-negative blood.

An accelerated method for determining Rh status.Apply 5 drops of anti-Rh serum of the same group as the recipient to a glass Petri dish. A drop of the subject’s blood is added to the serum and mixed thoroughly. The Petri dish is placed in a water bath at a temperature of 42-45°C. The reaction results are assessed after 10 minutes. If blood agglutination occurs, then the subject has Rh-positive (Rh+) blood; if there is no agglutination, then the blood being tested is Rh-negative (Rh-).
A number of other methods for determining the Rh factor have been developed, in particular using the universal anti-Rhesus reagent D.

It is mandatory to determine the blood type and Rh status of all patients in the hospital. The results of the study must be included in the patient’s passport.

How is blood type determined using standard serum? Probably, watching the actions of the laboratory assistant and the chemical reaction taking place on a special tablet, many people asked themselves a similar question. There is nothing mysterious in this process, and the mechanism for establishing group membership is based on the agglutination reaction, which occurs during the interaction of blood components, agglutinogens, and serum elements.

Main characteristics of groups

The definition of a group based on standard sera is based on the fact that human blood contains agglutinogens (A and B) and agglutinins (a and b) in various combinations. When the same agglutinin and agglutinogen meet, rapid agglutination occurs, and on a tablet this process will look like the disintegration of a homogeneous blood stain into many small specks.

• 0 (I) - contains only agglutinins a and b;
• A (II) - there is antigen A and agglutinin b;
• In (III) - there is agglutinogen B and antibody a;
• AB (IV) - there are no antibodies in the serum, and the agglutenogenic complex AB is located on the surface of the erythrocyte.

The blood type is determined using standard serum solutions within 5-10 minutes and does not require additional preparation of the patient for the study. This method can examine both venous and peripheral blood, obtaining the same result.

In emergency cases (preparation for an emergency operation or the need to immediately replace blood loss), mixing is carried out by dripping blood from a puncture on the patient’s finger directly onto a tablet with standard serums poured onto it.

Analysis technique

For research, a flat tablet with recesses in it is used. The recesses are arranged 3 in a row (there are 2 rows) and there is one at the bottom.

Above each pair of recesses, for the convenience of the laboratory technician, I, II or III is written and for applying the corresponding standard sera to the cells.

1. In the first row, a few drops of serum solution of types I, II and III (in accordance with the name of the recess) are poured into the corresponding cells.
2. In the second row, similar solutions are duplicated, but from a different production series (necessary control to avoid false agglutination due to low-quality serum).
3. A drop of venous or peripheral blood is added to the solutions using a glass rod.
4. To avoid accidental damage to red blood cells, mixing is done by gently rocking the tablet.
5. Next, the material is left for 5 minutes, after which the result is assessed.

The type and structure of liquids is assessed based on the type of solution, and compliance with a similar solution in a nearby cell is also checked.

If, for example, agglutination occurred in one well with the number II, but not in another with a similar reagent, then determining the blood group using serum compounds is considered incorrect. It must be repeated using serums from 2 other series.

Evaluation of results

Blood type is determined by standard serum reagents based on the visible agglutination reaction, and the result may be as follows:

1. I - the main and control drops on the laboratory plate remained unchanged.
2. II - a chemical reaction occurred in cells I and III.
3. III - agglutination is observed in depressions I and II.
4. IV - there are changes in all containers of the laboratory plate.

When determining type IV, a control with IV serum is always performed to prevent a false-positive result. There should be no change in the control mix after 5 minutes.

The method of determining blood type using standard serum reagents is used in almost all laboratories. A quick and affordable method allows you to identify a group within a few minutes.

The data obtained are taken into account only when the agglutination is clearly pronounced. A weak chemical reaction may be due to old reagents or due to incorrect analysis. The result cannot be considered reliable. In doubtful cases, in addition to testing with standard serum compounds, tests with coliclones or “erythrotest” can be done to clarify the group.

Using standard serum formulations, group determination is possible in a few minutes. The method is very simple and does not require special skills from the laboratory assistant.

There are two methods: by standard sera (direct reaction) and standard erythrocytes (reverse reaction).

Determination of blood group using standard sera

To determine the blood group using this method, the blood being determined is mixed with serum of already known groups and the blood group is judged by the presence or absence of agglutination. For determination, pre-prepared “standard sera” of blood O (I), A (II), B (III) of two different series for each group are used. Apply 2 drops of standard sera to a glass slide or porcelain plate (the serum group is first marked on the glass with a pencil) in pairs from each series.

Serums must be taken with different pipettes. The patient's finger is treated with alcohol and injected with a special scarifier needle. Take 6 drops of blood (no more than a pinhead each) and place them next to a plate with standard serums. Separate glass rods mix blood and serum. After 5 minutes, 1 drop of physiological solution is added to the sera (to exclude false agglutination).

Research results:

if agglutination is absent in all three paired drops, then the blood being tested belongs to the first blood group O (I);

the absence of agglutination with O (I) and B (P) sera indicates that the blood type is A (II);

in the absence of agglutination with serum of blood group B (III) and in case of agglutination with sera of O (I) and A (II), it should be assumed that blood group is B (III);

agglutination with all three sera indicates that the blood belongs to the fourth group - AB (IV).

Determination of blood group using standard sera from two series is carried out to avoid errors. In case of agglutination with three sera - AB (IV) blood group - it is necessary to carry out an additional determination with AB (IV) blood group serum, which is stored in a special sealed ampoule.

In doubtful cases, the test is repeated with other series of sera or determination is made using standard erythrocytes.

Determination of blood group using standard red blood cells

3 drops of serum prepared from the recipient's blood are applied to a porcelain plate. A pinhead of standard erythrocytes O (I), A (II) and B (III) are added to the serum. After 5 minutes, add one drop of saline solution.

Research results:

• if the reaction is negative with standard erythrocytes O (I) and positive with standard erythrocytes A (II) and B (III), blood group is first;
• in the absence of an agglutination reaction with standard erythrocytes O (I) and A (II) and positive with erythrocytes B (III), blood group is second;
• in the absence of an agglutination reaction with standard erythrocytes O (I) and B (III) and positive with erythrocytes A (II), blood group is third;
• in the absence of an agglutination reaction with all standard red blood cells, blood group is fourth.

“Surgical diseases”, S.N. Muratov

It is useful for every person to remember their blood type. This can unexpectedly come in handy in emergency situations. It’s not for nothing that military personnel have their blood type marked on their tunic or badges. There is no problem with how to determine your blood type. In large medical institutions, laboratories do this. But even in small rural outpatient clinics there are standard kits for determining the group, and all doctors and paramedics are able to carry out the analysis. There is no need to do this on your own when there are specialists.

What blood type a child has can be guessed from his parents. For example, knowing that the parents have the second, third or fourth, you should not hope that the child will have the first.

Similar advice is given by people who “live” on the Internet and collect any information indiscriminately. Sometimes they reinterpret it in their own way.

Indeed, there is a theory of nutrition depending on blood type, and attempts have been made to connect personality with it. But this was developed to select the most suitable diet and prevent diseases. Even psychologists have stopped talking about the connection between personality type and blood number.

Moreover, you cannot judge your group by your favorite dishes or inclination towards leadership.

It is better to learn anything at home from full-fledged articles and consultations with specialists. There may be medical documents (outpatient records, hospital records) that contain information about your blood.

At the request of donors and hospital patients, the passport can be stamped with this information.

Similar stripes are used on army uniforms.

Who invented blood groups

The discoverer of the four blood groups is the Austrian scientist and physician K. Landsteiner, who received the Nobel Prize in Medicine for this in 1930. The discovery made it possible to prevent deaths due to transfusion and to study the compatibility of donor characteristics and the recipient in advance.

The essence of the proposed AB0 system is the presence of antigenic structures on erythrocytes in humans and animals. There are no typical antibodies (gammaglobulins) to them in the plasma. Therefore, the “antigen + antibody” reaction can be used for determination.

Prevalence of blood groups by nationality

The adhesion of red blood cells occurs when an antigen meets its antibody. This reaction is called hemagglutination. It is visible during analysis in the form of small flakes. Determination of blood group is based on obtaining an agglutination pattern with standard sera.

Antigens of erythrocytes of type “A” bind to antibodies “ά”, respectively “B” to “β”. Based on blood composition, the following are distinguished:

• I, or 0 (ά, β) - there are no antigens at all on the surface of red blood cells;
• II, or A (β) - contain antigen A with antibody β;
• III, or B (ά) - there is a type B antigen with antibody ά;
• IV, or AB(00) - has both antigens, but does not contain antibodies.

Antigens are present in the human embryo already in the embryonic period, and antibodies (agglutinins) appear in the serum of newborns during the first month of life.

Standard blood typing (simple method)

To perform a blood group test, the patient's red blood cells (taken from a drop of blood) and standard serum containing known antigens are needed.

Serums are made from donor blood at “Transfusion Stations”, have expiration dates, and require compliance with storage conditions. The series number is indicated on the ampoules. For accurate analysis, two sets of sera of different series are taken. This is done to eliminate errors.

A large drop of four sera is placed in two rows on a flat plate (only III and II are sufficient, but 1 and 1V are also taken for control). Using different glass rods (it is convenient to use eye pipettes), add the blood to be tested into the serum drops (the ratio should be approximately 1:10) and stir gently.

The plate is rocked for five minutes, allowing the serum to mix well with the blood.

Decoding the results

5 minutes pass and the analysis results can be assessed. In large drops of serum, clearing occurs, in some small flakes are formed (agglutination reaction), in others they are not. Here are the possible options:

• if there is no agglutination in both samples with sera of groups III and II (+ control 1 and 1V) - this is the first group;
• if coagulation is noted in all except II, this indicates the second group;
• in the absence of agglutination only with III serum, the third blood group is established;
• if coagulation is observed in all samples, including the 1V-control - group four.

When the sera are arranged in the correct order, with signatures on the plate, it is easy to navigate: where there is no agglutination, there is a group.

There are times when the bonding is not clearly visible. Then the analysis is redone, fine agglutination is examined under a microscope.

Cross reaction method

To clarify the group in case of unexpressed agglutination, the method of double cross-reaction with standard erythrocytes is used. In this case, it is not the serum that is known, as in the simple method, but the erythrocytes. The patient's blood is drawn into a test tube, centrifuged, and the serum is pumped out from above with a pipette for testing.

Standard antisera

2 large drops of serum from the patient are dropped onto a flat white plate. They add standard red blood cells A (II) and B (III) blood groups. Stir gradually and shake the plate.

The result is assessed after 5 minutes:

• if agglutination occurred in both drops, group one;
• if not in any sample, group four;
• if in one of the known red blood cells used, then the group is determined by the presence or absence of coagulation in the drop.

Definition of a group by zoliclones

Zoliclones are synthetic serum substitutes. They contain artificial substitutes for agglutinins ά and β. They are called erythrotests “Tsoliklon anti-A” (pink) and “anti-B” (blue). The expected agglutination occurs between the erythrocytes of the blood being tested and the agglutinins of the coliclones.

The technique does not require the use of two series and is considered more accurate and reliable. The procedure and evaluation of results are the same as in the simple standard method.

Feature: the fourth group AB (IV) is necessarily confirmed by an agglutination reaction with a specific anti-AB zolicone and the absence of nonspecific adhesion of erythrocytes in an isotonic sodium chloride solution.

Express method using the “Erythrotest-group card” kit

The method allows you to determine the group and Rh factor in the laboratory and field conditions. The set includes a card with holes. Dried reagents are already applied to the bottom of the wells. Here, in addition to “anti-A”, “anti-B” and “anti-AB”, “anti-D” is used, which gives the result of the Rh factor.

You can use blood in any form; a compound with a preservative and taken from your finger will do.

Before the study, the patient’s name is written down on the card, and a drop of water is added to each well to dissolve the ingredients. Then add blood into the wells with reagents using separate sticks and mix lightly. The final result is “read” after three minutes.

The blood type is always rechecked if a transfusion is necessary. At the same time, group and individual compatibility is monitored. Indeed, in fact, much more antigenic properties have been found in human blood than in the AB0 system. They are simply weakly manifested in the majority of the population.

But in a patient with serious diseases that change the properties of the blood and allergize the body, they become decisive and require consideration of the presence and level in the blood. Therefore, the patient's knowledge of his own group adds confidence to the outcome of the study.