VI. Reference basis of action

Determination of blood type is conducted in the apartment with satisfactory illumination at a temperature +15-25^оС. A whole blood, washed red corpuscles, is used, red corpuscles in plasma, to the whey or in a 0,9% solution of sodium of chloride, standard whey O(І), A (ІІ), B (ІІІ) two different series of every group and standard whey of group AB (IV). Standard whey for determination of blood types is made in the special laboratories. Whey are saved in a refrigerator at a temperature + (6±2) ^oC.

Standard red corpuscles prepare donors from a blood. Take away blood from donors in a quantity 2-4 ml in a test tube, that contains to Citrate sodium or heparin. Then in a test tube refill a 0,9% solution of sodium of chloride to 10 ml and maintenance of centrifugation. Sediment and add 0,9% solution of sodium of chloride to the initial volume of blood and use as a standard at determination of blood types by a cross method. Standard red corpuscles are saved in a refrigerator at a temperature + (6±2) . Term of saving 3 days. Standard red corpuscles can be got in the prepared kind in establishments of service of blood, which make these standards.

Monoclonal antibodies of anti-A and anti-B intended for determination blood type by system of ABO in place of standard isohemagglutination whey. The monoclonal antibodies of anti-A and anti-B formation by two different hybrids and are the divorced ascetic liquid of mise-transmitters, which specific immunoglobulin of class of M are found in (are directed against group-specific antigens A or B).

For determination of blood type needed:

1. isotonic solution (0,9%) of sodium of chloride

2. white porcelain or other plates, subject glass with the moistened surface

3. test tubes

4. pipettes

5. glass sticks

6. cotton wool hygroscopic

Technique of determination of blood type by means standard whey.

1. To note on plate of blood type „O” „A” „B” and the last name of recipient or number to the small bottle of donor.

2. Under the proper marks to inflict on one large drop (0,1 ml) of every whey of both series.

3. Next to every whey to inflict on 1 to the little drop (0,01 ml) of the explored blood (red corpuscles).

4. To mix up by separate glass sticks each the drop of blood (red corpuscles) with the proper whey.

5. Mixing up all drops rock a plate, then on 1-2 abandon min and again rock. The supervisions after motion of reaction conduct no less than 5 min, although agglutination begins already during first 10-30 sm. The supervision must be continued farther to 5 min, because possible late agglutination, for example with the red corpuscles of group A.

6. Through 3-4 min to the drops of mixture of whey with red corpuscles, where agglutination happened add on 1 drops (0,05 ml) of a 0,9% solution of sodium of chloride and continue to look after to 5 min at the periodic waggle of plate.

Method of determination of blood type of the system by the ABO monoclonal antibodies.

Determination of blood types of the system by the ABO monoclonal reagents (Coliclon anti-A and anti-B) is conducted on plate. So as have Coliclon high activity, for every determination of blood it is enough to apply on one series of reagents of anti-A and anti-B. Coliclon anti-A and anti-B inflicts on plate on one large drop under the proper inscriptions: anti-A and anti-B and mix up with blood that is explored.

The results of reaction are presented in table in which the included results of determination of agglutinins in plasma of donors with the help of standard red corpuscles.

Reaction of red corpuscles, that are explored with Coliclon Anti-A Anti-B	Reaction of plasma, that is explored with standard red corpuscles A (ІІ) B (ІІ)	Blood type, that is explored
1) – –	+ +	0 (І)
2) + –	– +	A (ІІ)
3) – +	+ –	B (ІІ)
4) + +	– –	AB (IV)

1) Agglutination is not present (–) neither with Coliclon anti-A, nor with Coliclon anti-B. Consequently red corpuscles, that are explored do not carry in the composition of antigens A also B and a blood belongs to O (І) groups. it is confirmed by the presence of agglutinins   in a whey, that is explored on to the results of positive reaction with standard red corpuscles A (II) also B (ІІІ).

2) Agglutination (+) is observed with Coliclon anti-A only. Consequently, red corpuscles, that are explored, carry in the composition only an antigen A. Blood behaves to the group A (ІІ). It is confirmed by the presence of agglutination of whey, that is explored (plasma) on to the results of positive reaction of agglutination with the standard red corpuscles of group B (ІІІ).

3) Agglutination (+) is observed with Coliclon anti-B only. Consequently, in red corpuscles, that are explored, there is an antigen In and a blood behaves to the group B (ІІІ). It is confirmed by the positive reaction of agglutination of whey (plasma) that is explored, with standard red corpuscles groups A (ІІ).

4) Agglutination (+) is observed both with Coliclon anti-A, and with Coliclon anti-B. Consequently, a blood, that is explored, behaves to the AB (IV) group. It is confirmed by absence of agglutinins   in a whey (to plasma), that is explored, on to the results of absence of agglutination with the standard red corpuscles of groups A (II) also B (ІІІ).

Method of determination to the Rh-factor by an express-method with the use of standard universal reagent anti-Rh D.

The express-method of determination to the Rh-factor is used in the urgent case. Under the proper marks on 1 drops 0.1 ml. test and control whey. Add the half volume of experimental blood and mix. A dish is rocked by 3-5 minutes, then a result is read. At presence of agglutination of red corpuscles with whey anti-Rh blood of Rh-positive, in default of agglutination – Rh-negative.

Method of determination of antigens of the system Rh by means monoclonal antibodies (Coliclon anti-D super).

On a glass plane 2 drops of test-reagent of anti-Rh (0.1 ml) and 1 drop of blood are inflicted, that is explored (0.05 ml) and carefully mix up. After a 20-30 second begin to rock a plane. Clear agglutination is observed after a 60 second already. It follows to take into account the result of reaction through 3 min. Presence of agglutination in a blood, that is explored indicates in the presence of Rh-positive red corpuscles, absence – in the presence of Rh-negative red corpuscles.

Method of conducting of tests on compatibility.

1. Test on individual compatibility on the blood type of the system ABO.

A test on compatibility on the blood types of the system ABO is executed in obedience to a rule Otenberg „The Agglutination red corpuscles of donor by the whey of recipient”. Whey of recipient, which get by centrifugation or defending on plate mix up with the red corpuscles of donor and rock during 5 min. Absence of agglutination of red corpuscles of donor by the whey of recipient testifies to compatibility of blood on the system ABO, presence of agglutination – about its incompatibility. Sure, incompatible blood transfusion will result in agglutination in a bloody river-bed and origin of hemotransfusion shock. Pouring such blood not impossible. The reaction of agglutination indicates on that a blood type is definite wrong and it is needed to get back to determination of blood type. If donor and recipient of one group, such reaction does not arise up, it is possible to pour an same group blood only.

In default of reaction the agglutinations (a blood on the system ABO is compatible) proceed to a next test.

2. Test on compatibility on a Rh -factor.

The test of Solovjova is a classic test on Rh-compatibility.

Drop of whey of recipient mix up with the red corpuscles of donor in correlation 10:1 in a cup Petri, put on an aquatic bath-house on 10 min at a temperature 46-48oS. At presence of agglutination there is a blood incompatible on a Rh -factor, and pouring such blood not impossible, it is possible to pour a blood same Rh blood only, at determination to Rh-factor there was an error. More frequent an error arises up at determination to Rh-factor at a recipient by an express-method. Rh-factor is determined by more exact laboratory methods on the station of blood transfusion. The presence of agglutination at determination of Rh-compatibility requires returning to determination to Rh-factor at a recipient and donor. Absence of agglutination indicates on compatibility of blood on Rh-factor and it is possible to conduct the last test for a recipient.

In our time the compatibility on a Rh-factor is conducted by the mutated tests:

а) Test on compatibility of blood on Rh-factor with the use of a 33% solution of poliglucin.

A test is conducted in a test tube without heating during 5 min. On a bottom test tubes bring in 2 drops of whey of patient, 1 drop of donor blood and 1 drop of a 33% solution of poliglucin. Shaking the table of contents of test tube off mix up, then place a test tube almost horizontally and slowly scroll thus, that maintenance spread on to the walls of test tube. Continue such procedure 5 min. Then in a test tube add a 3-4 ml isotonic solution 0.9% sodium chloride, mix up by the 2-3-multiple inverting of test tube and looking through light with a naked eye. Estimation of results. The presence of agglutination of red corpuscles on a background transparent liquid indicates on that blood of donor and recipient incompatible on a Rh-factor. If maintenance of test tube is evenly painted without the signs of agglutination of red corpuscles, blood of donor compatible from a blood of recipient after Rh-factor.

b) Test on compatibility on a Rh-factor with the use of a 10% solution of gelatin.

Conduct a test in test tubes at the temperature of 46-48^oC during 10 min. On a bottom test tubes enter a 1 drop of red corpuscles of donor, which are washed to isotonic solution 0.9% chloride sodium, then 2 drops of warmed-up solution of gelatin and 2-3 drops of whey of recipient are added. Displace the table of contents of test tube and put on an aquatic bath-house at the temperature of 46-48 ^oC on 10 min Then bring a test tube out of water bath-house and add a 5-8 ml isotonic solution sodium chloride. 2-3 times invert a test tube and estimate the reaction with a naked eye or through magnifying glass. Estimation of reaction. The presence of agglutination on a background the liquid lighted up means that blood incompatible on Rh-factor and it not impossible to pour her. If maintenance of test tube is evenly painted and not determined to agglutination, blood compatible on a Rh-factor and she can be poured.

3. Biological test.

Before transfusion of preparations of blood container or bottle with red corpuscles mass, by plasma it is needed to survive at a room temperature 30-40 min, and in the exigent case to warm up on an aquatic bath-house at a temperature 37 ^oC. A biological test is conducted regardless of speed of introduction (by a trickle or drop) by a next method. By a trickle 10-15 ml preparations of blood are poured, then during 3 min look after on the state the patient. In default of clinical displays of reactions and complications (increase of frequency of pulse, breathing, appearance of shortness of breath, hyperemia of person) a test is repeated three times. Absence of reactions and complications after conducting of biological test is basic for transfusion of basic dose of preparations of blood. In the case of origin of clinical signs of reaction or complication (the conduct of patient is uneasy, the fever appears, compression after a breastbone, pain in back, stomach, main pain, decline of arterial pressure, tachycardia, pallor of skin, with a next cyanosis) it is necessary to halt transfusion, to prepare other non-permanent system for transfusion by salt solution and indoors by a needle with a vein to begin to pour salt solution.