13 ABO grouping: It is one of the most important tests carried out in daily routin of blood banking.it is performed on all potential transfusion recipients and blood donors. The imprtance of the test is due to the nature of the ABO system.un like other blood group system the antibodies (Abs) are present in the serum of all the adults where the corresponding antigen (Ag) is absent. (Ab) (Ag) ABO procedure is devided in to: Serum Cell Forward (Red Cell grouping) Performed in bench1&2 Reveres (serum grouping) Performed in bench 2 Non Rh grouping: The Rh grouping system consists of a number of antigens (D, C, E, c and e), the most important of these is Rh (D) Ag, and it is the prsence or absence of this Ag that defines whether a person is termed Rh (D) negative or Rh (D) positive. "#$%&%!'(% )* % )* P/t s red blood cells (RBC s) tested againest known antisera. -Slide & plate test. -Tube method.
14 Tube test: Anti-A, Anti-B, Anti-AB *(not used), Anti-D, and normal saline tubes), must be prepaired and kept in rack before the sample are received to save time for BG, and X-matching. PROSEDURE: 1- Give the sample seq. number (the same number must be on its request) 2-Wash the blood 3 time to remove unwanted Ag, and other substances which may interfer with agglutination. Wash is important >> Why? -help to remove non specific unwanted Ags, Abs. -Get sure (specific) results. 3- make suspension sus (2-5%), by adding approximatly 2 drops of blood + 20 drops 2 ml of Normal saline (N.S). Suspension is important to: Make the Ag/Ab reaction more clear, and to avoid false ve or +ve results. Very heavy suspension may give false +ve, very light give very weak view and so, false ve or giving doubtful view need microscopical examination more time is wasted. 4- Move 2 drops of suspension to previously prepaired (Anti-A, and Anti-B) tubes 1:1, and 2:1 for Anti-D. Ratio is important to get good, readable, clear and sure results. 5-Centrifuge for 15 sec. High speed (or 20 sec at 3000rpm) 6- Dislodge the cell button and observe aggl. (Macroscopic examination). 7- Give score, and record it. In case of Anti-D tube;if: -Agglutination. Group+ -No aggl Group see under microscope look for any clumps (not just sticking) e.g, A+ = ABO grouping = A (mean the p/t have A Ag on his RBC s) Rh grouping=+ (mean the p/t have rhesus monky Ag on his RBC S), and vice versa.
15 Microscopical examination: It used with Rh ve group befor and after testing for D Ag. -ve D tube microscopic examination if still ve Add albuminincubation 20mins wash 2 time add coomb s reagent centrifuge and read, - If ve gp is ve. In D -ve blood bags they use 2 tube loaded with Anti-C & Anti-E (C & E are weak Rh Ags) centrifuge read agglutinantion. - If +ve gp D +ve (weak Rh Ag). To make suspension N.S Anti-D Anti-A Anti.B (2 drpos of each antisera) Figure: Tubes loaded with anti s and normal saline ready to be uesed for blood grouping during BG and X-matching prosedure. Table: Forward ABO blood grouping. + = agglutination, - = no agglutination. Anti-A Anti-B Anti-D Blood group A B O AB+
16 Table: Grading Table. Grade Score Notation of serological reaction One solid clump, no free cells,clear supernatant Several large clumps,clear supernatant 2+ 8 Many medium-sized clumps,cluody red suepertanten 1+ 5 Many small clumps cluody red supernatant _+ 3 Many very small clumps (+) 1 Apear negative, but visible microscopicslly 0 0 -ve macro-, and microscopically Hs H Hw Strong hemolysis Moderate hemolysis Trace hemolysis Red supernatant, few or no intact RBCs Pink supernatant,some intact RBCs Pink-tinged supernatant,many intact cells mf Mixed field Small, tightly agglutinated clumps, againest a background of free cells microscopically, the clumps refractile Slide test (rapid test): 1. Whole (not susbended blood) can be used. 2. Mix with wooden applicator. 3. Gently rotate the slide for a peroid not exceeding 2 minutes. Agglutination occurs within sec. +#,%% )*%% )* Here the p/t s serum tested againest known A, B, O cells. Test cell A1 A2 B O Blood group A B O AB
17 PROSEDURE: 1-take samples from any bags with known (group A, B and O) 2- Wash the bag cell makes suspension. 3- Centrifuge the p/t sample serum. 4-add 2 drops of serum on blood suspension. 5- Centrifuge (better to incubate15-30 mins at room temp. 1 st ) 6- Dislodge the cell button and observe the agglutination. In case of group O serum continue. 7-Incubate at 37C /1 hr. 8-centrifuge look for haemolysis in tube A & B. if we have agglutination high Anti-A.Anti.B titer dangerous O only can be given to group O p/ts. As shown in table (O) tube (tube with O cells) is always give ve results in normal people. In Bombay Phenotype p/t s, O tube give +ve results because of the presence of Anti-H Abs in his serum (not present in normal people). -agglutination in O tube mean high Antibody titration given to O blood group p/t Only- and the blood bag labeled (only for O), dangerous O group. Actions affect test Quality Control:- 1. No wash before performing the procedure, this may affect the specificity and sensetivity of the test, because washing help to remove many unwanted Ags this step point is missing in all blood bank porsedure. 2. Suspention have no standard concentration, (desired 2-5%), and that make it hard to reading and comparing the tubes. 3. As known anti-a and anti-b,antiab antibodies are IgM Ab s and its activity are reduced delayed- in high temperature like body temperature 37C, with this some time Incubation of the plate in the incubator for 5-10 mins then reading the agglutenation directly are sometimes done!(false ve ).
18 4. Ag/Ab ratio is variable and that may adversly affect their reaction, like adding 1 drop of anti-a with 3 or 4 drops of blood suspension(with antia,and B usually 1:1 is the best zone of equivalence ). 5. Rh grouping: Wash before adding coomb s reagent in detecting (D) some time ignored. -# %#.()* The function of X-match is to prevent ABO incompatibility ( between the p/t and blood bag), particularly when A or B cells are transfused into a group O person, with a high titer of anti-a/anti-b, it is important to note that in addition to agglutination,haemolysis is also a sign of incompatibility. The actual procedure followed will depend on the clinical circumstances, scince emergency situation the testing protocol may have to be modified in order to provide blood as quickly as possible to the patient (P/t). Major cross-match = p/t s serum + blood bag RBC s sus. Minor cross-match = blood bag serum + p/t s RBC s. PROSEDURE: If blood transfusion request form received, -Check the p/t s name and file number. -Check diagnosis, haemoglobin level, blood group if known. -Check blood unit type and number. -In performing Major cross-match prosedure we have 3 steps (phases), ve result of the 1 st moving to the next step: 1). Saline phase 2). LISS or 22% Bovin albumin phase 3). Coomb s (anti-human globulin) phase. -In blood bank we put step 2 and 3 in one step and check for agglutination once. e.g 1 RBC or (whole blood) unit is wanted:
19 1. Give the sample seq. number (the same number must be on its request form). -The 1 st sample in morning shif labeled M1,.. and so on. 2. Lable 2 test tube with (AC1) and (SP1), and 3 rd extra tube to save the remaining serum and seal it with the sample tube with parafilm. 3. Centrifuge the tube sample D5BB, (AC)= Autocontrol (Sp) = Serum protein P/t s serum + p/t s serum Centrifuge machine p/t s Cells + Bag cells Make sus. From the sample and blood bag blood grouping for both of them (even if known). - if the p/t s group unknown wait for BG and then take the same group from the blood bags (or other blood component). 4-Add 1 drop of sus. Into Ac, SP tubes. 5-add 2 drpos of serum in to Ac, Sp tubes. 6-Centrifuge 15 sec/3000 rpm 7-Look for haemolysis +agglu. ((SALINE PHASE)) 8-if ve add 2 drpos albumin 9-incubate (20 mins) 10-wash 2 time at least. 11-add 2 drops of coomb s reagent. 12-centrifuge 13- read -ve microscopical exam.((coomb s PHASE)) 14- Record the result.
20 Serum remainig AC M1 SP M1 serum+rbc sus. Anti-D Anti-A Anti-B Cross-matching - URGENT cases: request for blood transfusion need to give blood directly and do not wait for donation from p/t s relatives has high priority on other requests like: R.T.A ( Road Traffic Accident) CCU (Cardiac Care Unit). CRF (Chronic Renal Failur). Other wise we check the haemogolin (Hb) level to asses whether the crossmatching must be done immediatly or can wait. The doctor ask for blood in EMERGENCY TRANSFUSION REQUEST uncross matched, urgently crossmatched, only ABO & Rh (same as p/t), or group O. other wise we check the haemoglobin level to deside whether the p/t need the blood now or can wait for a donation. Actions affect Cross-matching Quality Control:- 1.Using different suspention concentration make it hard to compare between tubes from the first look, AutoControl(AC) test tube (p/t s cells and serum) used to detect any auto Abs and usually as ve control in normal people for
21 other test tube contains sample from blood bags want to be X-matched with p/t s serum, but if we use different suspension concentrations in these tubes the role of AC tube is reduced because it is hard to compare especially if the reaction is weak. 2. Microscopical evaluation is not always performed on ve results, and rarly used by some tec. while some other use it frequently in X-matching ve tubes. -#/ /)%. 0%/ To detect already sensetized RBCs e.g Haemolyitc disease of new born. 1 st pregnancy Rh ve mother Rh +ve baby PROSEDURE: 1- Take 5 drops of blood sample. 3-make sus (2-5%) 4-add coom s reagent (2drops). 5-centrifuge The mother produce Ab againest Rh Ag. 6-dislodge the cell button and observe agglutinatiom. Actions affect DCT Quality Control:- There are differences between the procedure followed by each technician. Some important steps are missed like: 1-Wash before adding Coomb s reagent. 2 nd pregnancy Rh ve mother Rh +ve baby 2-Coomb s control check cells are not available (CCCC), coomb s control not used, but some staff use tube with Rh+ve O group + anti-d and add
22 coomb s reagent and run it with our DCT sample as +ve control and to insure that coomb;s reagent are working. 3- +ve results of DCT are not followed with other step to identefy the Abs: (elution and then titration). Elution: (wash out material) to remove one substance from another, usually an adsorbed material from an adsorbent surface, by washing it out with a solvent
23 1. Normal Saline (0.9% NaCl): To maintain osmotic balance and prevent the RBC from getting shrinking (if NaCl is less than 0.9%), or swelling up and get burst, which will interfer with aggl. reaction % -- 30% Bovin Albumin: Help to reduce the charge arround the RBC s, which may cuase a repulsion between the cell and the Abs and also between one cell and another. And so, make the agglutination easier to occur, and help it to be more clear. Usage: -ve tubes of incombatibility test (X-matching) before adding coomb s reagent. 3. Coomb s reagent (Anti-Human Globulin) or anti-antibody: IgG anti-body or incomplete antibody, have y shape (2 hands only) make very small clot (not easy to see or detect), unlike the IgM class Abs with 5 hands make visible clot or agglutination. So, if we have weak +ve agglutination (IgG attached to Red cell surface), we need to add coomb s reagent to be attched to that IgG on the cell surface and make a big visible clot.
24 Negative AHG reaction Fig: AHG by binding to IgG Abs on cell surface, bring the cells close to each Usage: In tests detecting IgG class Abs. 1- DCT. other and so the reaction is more visible. 2- IDCT (X-matching, Antibody screening). 3- with ve Rh group to detect Du (weak D or Rh Ag). /.<):-7,1*),.+ 7-/.*10=+ 0*,110,, 4. Store the platelets at RT (Room Temperature): Platelets when stored at refregerator show higher activity but removed quickly from the circulation. 5. Anti-AB; - To be sure of the results obtained with anti-a, Anti-B. It helps to identify group A 2, which give ve reaction with anti-a and anti-b alone. That mean if we get no agglutination with anti-a and anti-b that does not mean necessary that the blood group is O, it may be group A 2 by the using of Anti-AB we can decide.
25 6. Incubator; Anitibody classes are (IgG, IgA, IgM, IgE, and IgD). In blood bank we deal with IgG (e.g Anti-D) and IgM (anti-a and Anti-B). IgG= act better in hight temperatur (37C) incubator. IgM= Cold antibody act well in room temperature (RT), and so if incubated in 37C that may delay the reaction. 7. Anti-H: Major Ag in human RBC s are: A antigen (in A group) B antigen (in B group) H antigen (in all blood group but in different persent) the greatest amount is present on red cells of O group, intermediate amounts are present on red cells of A 1 and A 1 B. Blood with out H antigen is extremely rare (Bombay). And so used to distenguish between normal O group and Oh (Bombay group). No agglutination with anti-h) Fig: Simple chemical strucure of ABO antigens.
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