Blood Bank Procedures PDF

BLOOD BANK PROCEDURES TO GIVE BLOOD SAFELY CONTENTS Process overview: sample requirements, rationale for testing and reasons for administering blood. Blood grouping and antibody screening. Antibody ID, tests and theory for tests. Phenotyping, selecting blood Crossmatching: Electronic a...

BLOOD BANK PROCEDURES TO GIVE BLOOD SAFELY CONTENTS Process overview: sample requirements, rationale for testing and reasons for administering blood. Blood grouping and antibody screening. Antibody ID, tests and theory for tests. Phenotyping, selecting blood Crossmatching: Electronic and serological LEARNING AIMS AND OBJECTIVES PROCESS OVERVIEW: SAMPLE REQUIREMENTS, RATIONALE FOR TESTING AND REASONS FOR ADMINISTERING BLOOD. PROVISION OF BLOOD FOR PATIENTS Blood administered through transfusion must: Not cause a predictable reaction. Correct, the issue the product is being administered for. Be administered to the correct patient Blood administered through transfusion should not: Complicate patient care, long-term.  COMPATIBILITY SCHEMATIC Blood Group Is the Ab Electronically and Antibody No match red cells screen screen Positive Yes Identify Is the antibody Antibody Auto positive Yes Is the antibody No clinically ye significant s Perform No Phenotype red cells IgM DAT confirm antigen Serologically Positive negative crossmatch blood IgG Positive, specific Serologically match and significant antigen negative blood SAMPLE ACCEPTANCE CRITERIA FOR CROSSMATCH 5 points of patient ID Patient First and surname, D.o.B, Hospital number / NHS no., date and time of sample Sample must be handwritten; signed by sampler Collected within 72 hours Since last transfusion. PRINCIPLES OF COMPATIBILITY Compatibility linked to product type Red Cells To correct anaemia Platelets To correct thrombocytopenia Fresh frozen plasma Prevent microvasculature bleeding due to loss of clotting factors Treatment of coagulation deficiencies PRINCIPLES OF COMPATIBILITY Red cells Requirement to match antigen and plasma groups Prevent HTR immediate and delayed types Platelets and plasma Plasma groups must be matched by antibody Or reduce risk of reaction Platelet antigens can cause reactions and prevent therapeutic benefit. BLOOD GROUPING AND ANTIBODY SCREENING. COMPATIBILITY SCHEMATIC Blood Group and Antibody screen ABO BLOOD GROUPING Forward Group Detect antigen on the cells Known antisera tests to detect different A subtypes Reverse Group Detect antibodies in plasma /serum Use known cells across 2 groups A1 and A2 cells. RhD Type Must detect Partial D groups  A1 Cells IgM anti-D ANTIBODY SCREEN Confirm presence or absence of antibody Reagent Screening Cells between them they should express as a minimum the following antigens R2R2 & R1R1 or R1wR1 (C, c, D, E, e) K, k, Fya, Fyb, Jka, JKb, S, s, M, N, P1, Lea, Leb in addition cells showing the following homozygous expression should be included phenotypes Jk(a+b-), Jk(a-b+), S+s-, S-s+, Fy (a+b-), Fy(a-b+)  ANTIBODY-ANTIGEN REACTION Physically (inc. hydrophobic / hydrophilic) and electrostatically complementary. Graph of E.g. shape, charge and interactions with water Hypervariable regions create specificity for both antigen and epitope the interaction between antibody (Ab) and antigen (Ag) is held together by a combination of weak non-covalent forces electrostatic forces (Ionic Bonding) hydrogen bonds hydrophobic bonds van der Waals forces ‘Goodness of Fit’ affects antibody affinity AFFINITY VS. AVIDITY Affinity = Single clone of antibody’s draw to specific epitope. Avidity = Sum of a polyclonal antibody binding strength to a specific antigen. Titre = Amount of antibody available to bind antigen HEMAGGLUTINATION: SENSITISATION AND AGGLUTINATION FACTORS AFFECTING SENSITISATION Electro-repulsion: Electrostatic inference with binding Steric hinderance Physical interference with epitope binding Temperature Incubation temperature to detect antibodies TEST PRINCIPLES LOW IONIC STRENGTH NORMAL IONIC STRENGTH 25μL plasma, 50μL reagent cells 25μL plasma, 50μL reagent cells Inc. 37°C, for 15 mins Inc. 37°C, for 45 mins Centrifuge and wash cells Centrifuge and wash cells Add AHG Add AHG Inc. 37°C, for 10 mins Inc. 37°C, for 10 mins 0.03M NaCl Solution 0.15M NaCl Solution 1-2 vol. of plasma – cells 2-4 vol. of plasma – cells 1-2% cell suspension 2-3% cell suspension ANTIBODY IDENTIFICATION SYMPTOMS OF ANTIBODY MEDIATED HAEMOLYSIS COMPATIBILITY SCHEMATIC Blood Group Is the Ab and Antibody screen screen Positive Yes Identify Antibody Is the antibody Yes clinically significant No Serologically crossmatch blood  CLINICAL SIGNIFICANCE OF RED CELL ANTIBODIES Must be active at or near 37°C (e.g. minimum of 32°C). Intravascular Complement mediated, caused by deposition of huge amount of antibody on red cell surface (usually IgM). Complement activated to MAC Extravascular haemolysis Moderate amount of antibody deposits on red cell membrane Deposition of complement to C3b can be eliminated through RES. C3b can be inactivated to C3c or C3d To exclude antigens: 2 + phenotypes must be negative 2 negative phenotypes must be positive ENZYME TREATMENT OF RED CELLS Reduction of net negative charge on red cell surface glycolax Glycolax composed of sialic acid and sugar residues Papain and Bromelain (used independently) can cleave some of these off including some red cell antigens M,N,S,s (MNS blood group system), Fya, Fyb (Duffy blood group system). Lowers overall negative charge Exposes and enhances reactions to: ABO, Rh, Lewis, Kell, Kidd ENZYMES DEGRADE SOME RED CELL ANTIGENS 28 ANTIBODY DETECTION METHODS - COLUMN AGGLUTINATION TECHNOLOGY (CAT) aka ‘Gel cards’ comprised of six microtubes/columns in a plastic cassette neutral cards used for direct or enzyme techniques AHG impregnated cards used for LISS IAT (and DAT) techniques procedure 1) incubate serum/cell mixture in reaction chamber at 37oC 2) centrifugation to push sensitised cells/agglutinates through the individual columns of the cassette Diamed-ID Micro Typing System principle is to trap agglutinates in a Sephadex gel matrix free cells will pass through the matrix during the centrifugation phase in the LISS IAT cassette, cells sensitised with antibodies will form agglutinates as they pass through the AHG impregnated gel matrix BioVue System uses a slurry of glass beads in the microtube to trap agglutinates 29  COLUMN AGGLUTINATION TECHNOLOGY (CAT) Diamed System BioVue System 30  COLUMN AGGLUTINATION TECHNOLOGY ADVANTAGES AND DISADVANTAGES Advantages: Disadvantages: High cost consumables Better standardisation Old skills can be lost No washing required Less sensitive? (IAT) Easily automated Improved safety Stable reactions Less patient sample required No problems with Rouleaux.  ANTIBODY DETECTION METHODS - SOLID-PHASE TECHNOLOGY principle to capture any IgG antibodies present in sample onto a solid surface normally used to detect antibody by Antiglobulin techniques Solid Phase Technique 1. red cell antigens are bound to surfaces of microtitre wells 2. test serum/plasma is incubated in microplate 1. - any antibody present will firmly adhere to antigen on the microplate well surface 3. excess serum/plasma is washed off 4. IgG coated indicator cells plus anti-IgG AHG are added Positive test - the indicator cells will adhere to the well sides - seen as a ‘mat’ in the well Negative test - if no antibody is bound, the indicator cells fall to the bottom of the well – seen as a button in the well IgG coated control cells are used to validate all negatives 32 SOLID-PHASE TECHNOLOGY 33  SOLID-PHASE TECHNOLOGY Advantages Increased sensitivity as reaction is more stable and not easily washed off or disrupted Reading is less subjective Reading can be performed by automation using a plate reader and results uploaded directly into a computer system Rapid washing Disadvantages Technical expertise – prone to false positives If commercial kit – high cost consumables READING & GRADING AGGLUTINATION REACTIONS for the standard tube technique - a grading system similar to below is used: 5 : complete reaction, all the red cells are agglutinated in one clump 4 : red cells agglutinated in several large clumps, supernatant is clear 3 : many medium sized agglutinates, clear supernatant 2 : numerous small clumps, mildly cloudy supernatant 1 : numerous very small clumps, cloudy red supernatant, very small agglutination best seen microscopically 0 : negative – no agglutination – very red cloudy mixture *other techniques may have their own agglutination grading systems DIAMED - REACTION GRADING CHART 36 MICROSCOPIC EXAMINATION OF AGGLUTINATION 37 PHENOTYPING, SELECTING BLOOD SELECTING BLOOD For all patients For special considerations Blood must be: Antigen negative CMV negative ABO, Rh D compatible (and K Irradiated negative if of child bearing Sickle negative potential). Blood should be: Rh compatible (if results are available). DAT Warm clinically significant IgG positive C3b / C3d Cold antibodies ?clinically significant C3b / C3d positive PHENOTYPING Confirm Antigen negative Presence of antibody minus auto infers antigen negative Test for antigen Test for antithetical pair Positive control Heterozygous Negative control PHENOTYPING Patient cells + anti Jka Heterzygous cells + anti Jkb reagent reagent Patient cells + anti Jkb reagent Homzygous Jkb cells + anti-Jka Heterozygous cells + anti Jka reagent Homozygous Jka cells + anti-Jkb CROSSMATCHING: ELECTRONIC AND SEROLOGICAL CROSSMATCHING Red cells must be: ABO and Rh D compatible (at minimum): Ideally ABO and Rh D identical Women and girls below menopausal age K neg too at minimum Plasma and platelets ABO Identical if possible Anti-A or anti-B compatible transfusions ELECTRONIC CROSSMATCHING To qualify patient must have: Grouped successfully ABO RhD Negative antibody screen Match blood types through the computer (Laboratory Information Management System LIMS) from patient to donor Donor phenotypes known from NHSBT recorded against donor and donation no. Patient and donor information encoded through barcodes. EI (ELECTRONIC ISSUE) Matches ABO and Rh phenotype (minimum) LIMS (laboratory Information Management Systems) must include K search for females. Links donation (G no. + product code) to hospital number and crossmatch label produced Product cannot be assigned to any other patient Blood tracking software BLOOD TRACKING SYSTEM SEROLOGICAL CROSSMATCHING Phenotyped units selected antigen negative LISS IAT React donor red cells against recipient plasma / serum Predicts if reaction possible from existing antibodies. Ideal negative result If negative result not possible (possible auto antibody) give most suitable (medical concession) COMPATIBILITY VS SUITABILITY Compatible No evidence of reaction present Predictive of reactions from present antibodies Suitable Crossmatched result = positive Cannot be positive for clinically significant antibody Interference from auto antibody REACTIONS AND EVENTS Most likely due to: Sampling errors Wrong patient identified, haemodilution (drip arm sampling) Risks: product reaction, over transfusion Labelling errors Bed side checks not completed, Product reactions Documentation errors Incorrect unit or product type identified for patient Product reactions / unnecessary infusion