Blood Banking - Module 1.pptx health medical lab

Blood Banking - health medical lab

1. Basic Principles of Blood Banking; Antigen, Antibody, naturally occurring antibody, Complement, ABO & Rh blood group system, Methods of blood group determination, Forward and Reverse grouping, Slide & Tube method, Gel method. By Sujoy Tontubay, Assistant Professor (Medical Lab Technology), iLEAD , Kolkata. B.Sc. (University of Calcutta), M.Sc. in Biomedical Laboratory Science(V.U), Post Graduate Diploma in Epidemiology and Public Health(V.U) Former Guest Lecturer (Medical Laboratory technology) in Guru Nanak Institute of Pharmaceuticals Science and Technology (G.N.I.P.S.T), Lokenath Educational Institute.

2. Basic Principles of Blood Banking Blood banking refers to the process of collecting, separating, and storing blood. The first U.S. blood bank was established in 1936. Today, blood banks collect blood and separate it into its various components so they can be used most effectively according to the needs of the patient. A blood bank is a place where blood is collected and stored before it is used for transfusions. Blood banking takes place in the lab. This is to make sure that donated blood and blood products are safe before they are used. Blood banking also determines the blood type. The blood is also tested for infectious diseases.

3. Facts about blood banking According to the American Red Cross: About 36,000 units of blood are needed every day. The number of blood units donated is about 13.6 million a year. About 6.8 million volunteers are blood donors each year. Each unit of blood is broken down into components. These are red blood cells, plasma, cryoprecipitated AHF ( cryo ), a white blood cell called a granulocyte, and platelets. One unit of whole blood and its parts may be transfused to several people. Each person may have a different need. More than 21 million blood components are transfused each year.

4. Who are the blood donors? Most blood donors are volunteers. But you may also donate blood several weeks before having surgery. This is so that your blood is available in case you need a transfusion. Donating blood for yourself is called an autologous donation. Volunteer blood donors must meet certain criteria. These include: Must be at least 16 years of age, or the minimum age set by state law    Must be in good health Must weigh at least 110 pounds Must pass the physical and health history exam given before donation Some states let people younger than 16 or 17 years to donate blood, with parental consent.

5. What tests are done in blood banking? Certain standard tests are done in the lab once blood is donated. These include: Typing. This is blood type. Blood can be A, B, AB, or O. Rh typing. This can be Rh positive or Rh negative. Screening for any unexpected red blood cell antibodies. These antibodies may cause problems in the person getting the blood. Screening for current or past infections. The list includes: Hepatitis viruses B and C HIV Human T-lymphotropic viruses (HTLV) I and II Syphilis West Nile virus Chagas disease 

6. What are the blood types? According to the AABB, blood types make up the following portions of the U.S. population: O Rh-positive - 39% A Rh-positive - 30% B Rh-positive - 9% O Rh-negative - 9% A Rh-negative - 6% AB Rh-positive - 4% B Rh-negative - 2% AB Rh-negative - 1%

7. What are the components of blood? Blood or 1 of its components may be transfused. Each component serves many functions. Red blood cells. These cells carry oxygen to the tissues in the body. They are commonly used to treat anemia. Platelets. They help the blood clot. They are used to treat leukemia and other forms of cancer. White blood cells. This is a type of white blood cell that helps fight infection. It helps in the immune process. Plasma. This is the watery, liquid part of the blood. Red blood cells, white blood cells, and platelets are a part of this liquid. Plasma is needed to carry the many parts of the blood through the bloodstream. Plasma serves many function. It is generally transfused to replace blood-clotting proteins. Cryoprecipitate AHF. The party of the plasma that contains clotting factors that help control bleeding. Albumin, immune globulins, and clotting-factors may also be separated and processed for transfusions.

8. Antigens Antigens are carbohydrate or proteins found on the surface of all blood cells (red cells, white cells and platelets) but can also be found on body tissue, body fluids and on surfaces of bacteria and viruses. Antigens can trigger the immune system to produce antibodies directed against the donor’s blood group antigens if the recipient does not have that antigen. These antibodies (or alloantibodies) can be clinically benign or cause severe transfusion reactions such as acute or delayed haemolytic reactions and haemolytic disease of the fetus and newborn (HDFN).

9. Antibodies Antibodies are immunoglobulins (Ig); there are five main subtypes and each have a unique role within the immune system: IgG  IgM  IgA  IgD , and   IgE . Almost all antibodies to blood cells are IgG or IgM subtypes. IgM antibodies are usually naturally occurring following exposure to environmental substances that have a similar structure to a red cell antigen. These IgM antibodies are generally non-reactive at 37 °C and are therefore of little clinical significance. However, IgM anti-A and anti-B are potent haemolysins , capable of causing intravascular haemolysis by binding complement and are therefore classified as clinically significant antibodies. IgG antibodies are formed following exposure to foreign red cell antigens through transfusion or during pregnancy. These IgG antibodies are reactive at 37 °C and are usually considered clinically significant as they’re capable of mediating destruction or sequestration of transfused allogeneic red cells IgG antibodies can also cross the placenta and can cause haemolytic disease of the fetus and newborn (HDFN).

10. Naturally Occurring Antibodies Antibodies are immunoglobulin proteins secreted by B-lymphocytes after stimulation by a specific antigen. The antibody formed binds to the specific antigen in order to mark the antigen for destruction. The type of antigenic exposure occurring in the body determines if the antibody is a naturally occurring or immune antibody. Naturally Occurring Antibodies Naturally occurring antibodies can be formed after exposure to environmental agents that are similar to red cell antigens, such as bacteria, dust, or pollen. Sensitization through previous transfusions, pregnancy, or injections is not necessary. Some naturally occurring antibodies are present without known environmental exposure. Antibodies are usually IgM and react best at room temperature or lower. Most of these antibodies are not clinically significant with the exception of ABO antibodies. Most people with healthy immune systems are expected to present with naturally occurring ABO antibodies appropriate for their ABO type. All other naturally occurring antibodies are considered "unexpected". Examples of naturally occurring antibodies include anti-A, anti-B, anti- Cw , anti-M, and antibodies in the, Ii , Lewis, and P systems.

11. Immune Antibodies Immune antibodies occur in the serum of individuals who become sensitized to foreign antigens through pregnancy or transfusion. These antibodies are produced specifically against RBC antigens. IgM predominates in the primary response; IgG in the secondary response. Most react at 37°C and are considered clinically significant. Examples include antibodies in the Rh, K, Duffy, and Kidd systems.

12. Complement Complement is a blood test that measures the activity of certain proteins in the liquid portion of your blood. The complement system is a group of nearly 60 proteins that are in blood plasma or on the surface of some cells. The proteins work with your immune system and play a role to protect the body from infections, and to remove dead cells and foreign material. Rarely, people may inherit deficiency of some complement proteins. These people are prone to certain infections or autoimmune disorders. There are nine major complement proteins. They are labeled C1 through C9. This article describes the test that measures total complement activity. Normal Results The normal results for this test are: Total blood complement level: 41 to 90 hemolytic units C1 level: 14.9 to 22.1 mg/dL C3 levels: 88 to 201 mg/dL C4 levels: 15 to 45 mg/dL

13. ABO and RH Blood Grouping system Blood is a fluid connective tissue and the most crucial component of the circulatory system. In a healthy person, approximately 5 liters (12 pints) of blood circulates throughout their body. It consists of erythrocytes, leukocytes and platelets suspended in plasma along with the millions of different molecules with its own specific roles and functions. Even though components of blood are the same for all humans, there are various blood types. In fact, there are more than 40 blood groups, but all of them are not clinically significant.

14. Blood Group System Karl Landsteiner, an Austrian scientist discovered the ABO blood group system in the year 1900. In his experiments, he mixed different blood types and noted that the plasma from certain blood type produced agglutinates or formed clusters which were caused by the absence of molecules on red blood cells and resulting in antibodies to defeat that molecule. He then made a note of the agglutination and divided the blood types into 4 different groups. For the discovery of ABO blood group, he was awarded the Nobel Prize. The blood grouping system is pivotal in blood transfusion. Our immune system recognizes another blood type as foreign and attacks it if introduced in the body causing a transfusion reaction.  Any inappropriate match with the Rh and ABO blood types, causes the most serious and life-threatening transfusion reactions. Therefore, before blood transfusion, it is suggested to have a blood group checked.

15. What are ABO and Rh blood groups? During the blood transfusion, the two most important group systems examined are the ABO-system and the Rhesus system. The ABO blood group system consists of 4 types of blood group – A, B, AB, and O and is mainly based on the antigens and antibodies on red blood cells and in the plasma. Both antigens and antibodies are protein molecules in which antigens are present on the surface of Red Blood Cells and antibodies are present in the plasma which is involved in defending mechanisms. On the other hand, the Rh blood group system consists of 50 defined blood group antigens. In the Rh system, the most important antigens are D, C, c, E, and e. The ABO and Rh blood systems are discussed in detail below.

16. 1. ABO blood Group system The basis of ABO grouping is of two antigens- Antigen A and Antigen B. The ABO grouping system is classified into four types based on the presence or absence of antigens on the red blood cells surface and plasma antibodies. Group A – contains antigen A and antibody B. Group B –contains antigen B and antibody A. Group AB –contains both A and B antigen and no antibodies (neither A nor B). Group O – contains neither A nor B antigen and both antibodies A and B. The ABO group system is important during blood donation or blood transfusion as mismatching of blood group can lead to clumping of red blood cells with various disorders. It is important for the blood cells to match while transfusing i.e. donor-recipient compatibility is necessary. For example, a person of blood group A can receive blood either from group A or O as there are no antibodies for A and O in blood group A.

17. 2. Rh Blood Group System In addition to the ABO blood grouping system, the other prominent one is the Rh blood group system. About two-thirds of the population contains the third antigen on the surface of their red blood cells known as Rh factor or Rh antigen; this decides whether the blood group is positive or negative. If the Rh factor is present, an individual is rhesus positive ( Rh+ve ); if an Rh factor is absent individual is rhesus negative (Rh- ve ) as they produce Rh antibodies. Therefore, compatibility between donor and individual is crucial in this case as well.

18. Rh blood group The most significant Rh antigen is D.  When the D antigen is present on the red cell surface, the red cells are called D positive.  Approximately 84% of the Australian population are D positive.  The remaining 16% of the population that lack the D antigen are called D negative. D is highly immunogenic. Antibodies (anti-D) may occur after an individual is exposed to D antigens via transfusion, pregnancy or organ transplantation. These antibodies are clinically significant and can cause haemolytic transfusion reactions and result in haemolytic disease of the fetus and newborn during pregnancy.  D negative females of child-bearing age should receive D negative red cells. D positive individuals should receive D positive red cells unless these are not available or are deemed unsuitable (e.g., HSCT or extended phenotype matching in sickle cell disease).  If transfusion of D positive red cells to a D negative recipient, the haematologist should be consulted and consideration given for the administration of RhD immunoglobulin. Note: Platelets contain red cells and therefore if D positive platelets are transfused to a D negative female of childbearing potential, consider RhD immunoglobulin prophylaxis. 

19. Blood compatibility  It is preferable for patients to receive red cells and platelets of the same ABO and D group.  However, if ABO and D identical products are not available, a patient may be offered an alternate compatible product (see below)  Red cell compatibility   Patient ABO group            Best option                                                                                                                                                                             OK to use  Never use   Unknown   O negative  In an emergency when O negative red cells are not available, O positive red cells may be administered to male patients.    -                                                                                  A, B, AB                                                                                                            O  O   -   A, B, AB   A  A  O   B, AB   B  B  O    A, AB   AB  AB  O, A, B     - 

20. In an emergency, when D negative components are not available, transfusion should not be withheld. D negative males may receive D positive red cells but should be monitored for haemolysis and are a risk of developing anti-D antibodies. Suggest DAT and BGAB following transfusion.  D compatibility - red cells and platelets Patient D type                    Best option                                                                                                                                                                              OK to use  Never use     D negative            D negative                                                                                            -                                                                                    D positive*                                                                                                     D positive  D positive   D negative   -

21. Platelet compatibility - Platelets contain red cells and therefore D compatible products are preferred. If D positive platelets are given to a D negative female of child-bearing potential, then consider RhD immunoglobulin.   Patient ABO group          Best option                                                                                                                                                                    OK to use                                                            Avoid if possible                                                                                      Unknown                  O negative (low titre#) or A negative (low titre)                 -                                    B, AB                                                                      O                                O         A   B, AB   A  A   B, O    AB  B  B   A, O   AB  AB  AB (not routinely available)    A, B    O

22. Plasma compatibility (Fresh frozen plasma and Cryoprecipitate)Plasma components should be compatible with the ABO group of the recipient. Plasma components of any D type can be given irrespective of the recipient's D status. RhD immunoglobulin is not required.   Patient ABO group     Best option                                                                                                                                                           OK to use                                                            Avoid if possible                                                                           Unknown   AB  In an emergency when AB plasma is not available, A (low titre #) plasma may be given with caution^.   -  B  O  O  A, B, AB  -  A  A  AB  B  B  B   AB  A   AB                AB  -   A, B 

23. blood group determination Aim The main purpose of conducting this experiment is to understand the basic concept of the ABO blood group system and to know our blood group and type. Materials Required Toothpicks Blood sample Alcohol Swabs Lancet Clean glass slide Sterile cotton balls Biohazard disposal container Monoclonal Antibodies (Anti-A, B, and D) All these equipment will be readily available in a blood test tool kit.

24. Procedure First, take a glass side and mark three circles on it after cleaning the slide. Unpack the Monoclonal Antibodies (MAB) kit. Now with the help of a dropper, add the Anti-A, Anti-B and Anti-D in the first, second and third circle respectively in a sequential order. Keep the slide aside safely without disturbing. Now you need to wipe the ring finger with the alcohol swabs and rub gently near the fingertip, where the blood sample will be collected. You need to prick the ring fingertip with the lancet and wipe off the first drop of the blood. As blood starts flowing out, allow it to fall on the three circles of the glass slide by gently pressing the fingertip. We must apply pressure on the pricked part in order to stop the blood flow.Use the cotton ball if required. Mix the blood sample gently with the help of a toothpick and wait for a minute to observe the result.

25. Forward and Reverse grouping Forward Grouping (testing the patient’s red cells) Label four tubes with the patient/donor’s name and ID, assigning each tube a corresponding reagent label: anti-A, anti-B, anti-A,B, and anti-D. Dispense a drop of commercial anti-A serum into the tube labeled anti-A. Repeat this process for anti-B, anti-A,B, and anti-D, adding each serum to its respective labeled tube. Carefully add one drop of well-mixed prepared red cell suspension to each tube of the antiserum. Thoroughly mix the reagents and red cell suspension by gently flicking the base of each tube. Centrifuge the tubes according to the reagent manufacturer’s instructions. Usually, centrifugation is performed at 1500 rpm for 15-20 seconds using a serofuge . Gently resuspend the cell button in each tube and examine for agglutination. Interpret and record the test results. Compare the test results for the red cell suspension with those obtained from reverse grouping.

26. Forward Grouping (Cell Grouping): - Tube method Label tubes with donor/patient and test identification. Prepare cell suspension for cells being tested (Refer SP 015) Place two drops of anti-A, anti-B and anti-AB reagent in the appropriately labelled tubes. Add to each tube one drop of a 2 – 5% cell suspension (in normal saline, serum or plasma) of the red cells to be tested. Mix the contents of the tubes gently and incubate at room temperature for 15 minutes. Centrifuge at 1000 rpm for 1 minute.

27. Reverse Blood Grouping /Serum Grouping Reverse blood grouping is a procedure to confirm ABO blood group based on the presence or absence of anti-A and anti-B in serum using known A and B red cells. It is cross check for forward typing. Performing both forward and reverse grouping provides a check for accuracy. Because of the lack of synthesized immunoglobulins, anti-A and Anti-B in newborns and very young infants, this procedure is not performed on infants below 4 months of age. Principle The reverse blood grouping procedure is based on the principle of direct hemagglutination. The erythrocytes of a person contain blood group antigens on the surface of the membrane. When these antigens are allowed to treat with corresponding antibodies, antigen-antibody reaction occurs and form agglutination.

28. Requirements Specimen: Serum is specimen for reverse blood grouping. No special preparation of the patient is required prior to collection. The specimen should be tested as soon as possible after collection, but specimens may be stored at 2 to 8?C if there is a delay in testing. Storage may result in weaker-than-normal reactions. Cell Suspension: Although red cell reagents for serum grouping are available commercially, most laboratories prepare their own A and B test red cells from persons known to be group A and group B. Make pooled cell suspension as follows: Label tubes as A and B. Tube A: Place 1 drop of red cells each from 3 of A group samples. Tube B: Place 1 drop of red cells each from 3 of B group samples. Add Normal saline and to suspend the cells. Centrifuge the tubes for at least 1 minute at 1000 rpm. To make 5% red cell suspension, add 1 drop of RBC to 19 drops of saline. Make 20% suspension for slide method. Test the pooled cells prepared by adding the antisera (Anti-A, B) in use.

29. Procedure The reverse blood grouping can be performed in two methods: Tube and Slide method. The Tube method is preferred to slide method. Tube Method Label two test tubes as A and B. Add two drops of serum to be tested in each tube. Add one drop each of A and B cells suspension to the corresponding test tubes. Mix well and centrifuge both tubes at 1000 rpm for 1 minute. Gently remove the tubes and completely resuspend cells and examine macroscopically for agglutination and if negative, microscopically. Record the reactions and interpret the results.

30. Slide Method Mark a clean slide into two halves, labeling the left and right side side as A and B. Add a drop of serum to be tested on both sides. Add one drop each of A and B cells suspension (20%) to the corresponding sides. Using a clean applicator stick, mix the serum and cell suspension on both sides separately and spread into a smooth round circle. Rock the slide gently for 2 minutes and look for agglutination. Record the reactions and interpret the results.

31. Results and Interpretation If agglutination is observed with A cells only, then the patient’s blood group is B If agglutination is observed with B cells only, then the patient’s blood group is A If agglutination is observed with both A and B cells, then the patient’s blood group is O If agglutination is not observed with both A and B cells, then the patient’s blood group is AB

32. Gel method SPECIMEN COLLECTION AND PREPARATION - Blood samples collected in EDTA, sodium citrate or sodium heparin should be used. The collection, separation and handling of the blood should be performed by qualified technical personnel according to current standards, and following the instructions of the manufacturer of the materials used for collecting the sample.

33. PROCEDURE Allow DG Gel 8 ABO/Rh + Kell cards, additional reagents and the samples to reach room temperature (18-25 ºC). Inspect the condition of the cards before use (see Warnings and Precautions). Identify the cards to be used and the samples to be tested. Prepare a 5% red blood cell suspension in Grifols Diluent (50 μL of packed red blood cells in 1 mL of Grifols Diluent). Carefully peel off the aluminum film that covers the microtubes to prevent cross-contamination of the microtube contents among them. Ensure the re-suspension of the red blood cell suspension before use. Add 10 μL of the 5% red blood cell suspension into each of the A/B/AB/D/K/ Ctl microtubes. Note: Carefully dispense the red blood cell suspension and the serum or plasma, avoiding contact of the pipette tip with the wall or the contents of the microtubes to prevent carryover. Thoroughly mix the vials of A1/B Reagent Red Blood Cells 0.8% from Medion Grifols Diagnostics AG to ensure homogeneous suspension. Dispense 50 μL of A1 Reagent Red Blood Cells into the first microtube N, and 50 μL of B Reagent Red Blood Cells into second microtube N. Add 50 μL of serum or plasma in to the corresponding microtubes. Note: Carefully dispense the red blood cell suspension and the serum or plasma, avoiding contact of the pipette tip with the wall or the contents of the microtubes to prevent carryover. Centrifuge the gel card in the DG SPIN centrifuge. 12. After centrifugation, remove the gel card from the centrifuge and read the results.

34. RESULTS Report results as an agglutination grade, absence of agglutination or hemolysis. Negative results: no agglutination and no hemolysis of red blood cells is visible in the microtube. In a negative result the red blood cells are located in the bottom of the gel column. Positive results: agglutination and/or hemolysis of the red blood cells is visible in the microtube. In a positive result the agglutinated red blood cells may remain throughout the gel column showing different reaction grades (see Reaction Grades and Figure 1 for a picture of example of reaction grades). Some positive reactions may also form a pellet in the bottom of the microtube. A very weak reaction may indicate a very weak or partial expression of the ABO, RhD and K antigens