Bloodbank 2 unit 1

Unit 1

Compatibility testing 

• What is it? 

  • Routinely performed only with donor products that contain red blood cells 

  • Compatible vs incompatible 

• Goals 

  • Not cause harm to the patient 

  • Maximize in vivo survival of the transfused red blood cells 

• Why transfuse red blood cells? 

  • The biggest issue when something is going on with the patient → checking Hgb (low Hgb will require transfusion)

Pre-transfusion testing: specimen collection, handling, and processing - Preanalytical

• Accurate patient ID 

  • Requisition must contain

    • Full patient name 

    • Unique ID number 

  • The requisition must match the patient's info 

  • Label specimen at the bedside 

    • Patient’s full name 

    • Patient’s unique ID number 

    • Date of specimen collected 

    • Initials of phlebotomist 

  • Handling in BB 

    • Test asap 

• Blood bank history review 

  • The patient’s blood bank history needs to be reviewed for 

    • History of ABO → any discrepancies 

    • Previous history of unexpected antibodies 

    • Additional testing → prewarm technique 

    • Special transfusion requirements  

      • Irradiation (prevent GVH)

      • CMV 

    • History of stem cell/bone marrow transplant 

• Sample collection and storage for Blood bank 

  • The patient has been transfused or pregnant within the past 3 months

    • A new specimen must be collected every 3 days for antibody screening and compatibility test 

  • The patient’s blood bank history is unknown 

    • A new specimen must be collected every 3 days

  • The patient has a reliable blood bank history and there is no history or currently identified antibody

    • The sample can be held or reused 

  • The patient is being transfused 

    • More frequent samples may be requested 

  • The patient samples following a transfusion must be held for 7 days 

Pre-transfusion testing: specimen collection, handling, and processing - Analytical

• Donor testing 

  • ABO/Rh 

  • Antibody screen 

  • Infectious diseases

    • HBsAg, Anti-HBc, Anti-HCV, syphilis, Chagas 

  • If the transfusion facility differs from the collection facility, ABO typing must be repeated 

• Positive antibody screen 

  • Do an antibody ID 

• Unit selection

• Blood inventory and use 

  • All blood utilization and disposal is logged 

  • Inventoried daily 

• Protocols for Effective Blood Utilization 

• Compatibility testing (crossmatch)

  • Major crossmatch → Patient’s serum/plasma and donor red blood cells are mixed 

  • Minor crossmatch → donor plasma and patient red blood cells are mixed 

  • Types 

    • Serologic 

      • Immediate spin → ABO compatibility only, tube testing 

      • Antiglobulin → history of Antibody, current ID of antibody, gel or solid phase 

    • Computer crossmatch 

      • Eliminates the need for serologic crossmatch 

      • Requires 

        • Two ABO interpretations on file

        • No clinically significant antibodies detected or in the history

        • LIS is validated 

  • Interpretation of compatibility testing 

    • Compatible 

    • Incompatible 

  • Documentation 

    • Grade reaction → 1+, 2+, 3+, 4+ 

    • Symbols are not appropriate 

  • Causes for incompatible cross-matches 

    • Incorrect ABO → operator error 

    • Alloantibody → low-frequency antibody 

    • Autoantibody → if everything is positive, including autocontrol

    • Abnormalities of the patient’s protein 

    • Contamination

•  Does not guarantee a successful transfusion outcome 

• Limitations to compatibility testing 

  • Does not ensure RBC survival 

  • Patients can still experience some ill effects 

• Special consideration 

  • Medically necessary to transfuse blood products after completing pre-transfusion and compatibility testing 

  • The physician must complete an emergency release form 

• Massive transfusion 

  • Administration of 8-10 packed Red cell units to an adult patient in less than 24 hrs 

  • 1:1:1 ratio RBCs, plasma, platelets

  • Protocol 

    • 4-6 units of packed red cells 

    • 4-6 of Fresh frozen plasma 

    • 1 unit of apheresis platelets 

• Neonatal transfusion 

  • ABO and Rh typing 

  • Antibody screen 

    • Positive requires antibody ID 

  • Aliquots for transfusion 

• Intrauterine transfusions 

  • Severe cases of fetal anemia 

  • Insert a needle into the umbilical vein using sonography 

  • Transfusion 

    • O negative 

    • <7 days old

    • Irradiated 

    • Negative for Hgb S 

Post-transfusion reaction - release of blood and transfusion 

• Blood release

  • Donor units must be labeled 

  • Must have a request form or label with the patient's information

    • Compared with donor unit for accuracy  

  • Unit inspected for 

    • Hemolysis 

    • Clots 

• The transfusion 

  • Nursing staff must check the unit and compare the information before the transfusion 

  • Vital signs are taken 

  • All paperwork returned to the lab 

  • Post hemoglobin/ hematocrit 

ABO discrepancies 

• ABO discrepancies 

  • There is a discrepancy between the forward and reverse → no reverse on babies. 

    • Weak reacting or missing antigens 

    • Weak reacting or missing antibodies 

    • Unexpected antigen reaction 

    • Unexpected antibody reaction 

  • A discrepancy between current results and previous results 

    • Bone marrow transplants 

  • Transfusions need to be delayed until the discrepancy is solved 

  • Emergency → give O neg and the physician needs to sign 

• Resolving ABO discrepancies 

  • First, repeat it 

    • Wash RBCs before testing - may require multiple washes 

  • Clerical check of specimen and requisition 

    • Inadequate ID or mislabeled tubes 

  • Check 

    • Patient’s age 

      • Newborn → wash more times, their cells contain a gelatinous coat 

      • Elderly 

    • Diagnosis 

    • History of transfusions, transplants, or pregnancies 

    • Medication 

  • Technical errors 

    • Failure to add reagents 

    • Failure to follow manufacturer’s instructions 

    • Contaminated reagents 

  • Centrifuge 

    • Under centrifugation 

      • Too slow or too short will give a false negative

    • Over centrifugation 

      • Too fast or too long of a time will give a false positive 

  • Temperature 

    • Too warm may cause false negative 

    • Too cold may cause a false positive 

  • Cell suspension concentration 

    • Prozone → too weak cell suspension. Excess antibody 

    • Postzone → to heavy cell suspension. Excess antigen 

  • Delayed reactions recorded 

    • Reactions need to be recorded immediately following centrifugation 

    • Allowing reactions to sit at room temperature may cause 

      • Weak reactions to disappear 

      • Interference from cold agglutins 

  • Missed observation of hemolysis 

    • Hemolysis can be due to antigen/antibody interaction 

  • Missed observation of weak reactions 

    • Shaking tube too hard 

  • Missed observation of mixed-field reaction 

    • Two cell populations present 

  • Dirty glassware 

  • Fibrin 

    • Small clots mistaken for reactions 

  • Collect new specimen 

    • The wrong patient was drawn 

      • Mislabeled specimen

    • Specimen collected above an IV site 

    • Traumatic draw

Additional testing if ABO discrepancy persists 

• RBC problems - forward typing 

  • Least common discrepancy 

  • Weak reacting or missing antigens 

    • Newborns → antigens not fully developed 

    • Elderly → antigen strength may diminish 

    • Disease 

      • Leukemia, Hodgkins lymphoma 

    • Subgroap of A or B 

      • Number of A antigen sites 

        • 1) A1 - most/strongest 

        • 2) A2 - second strongest 

        • 3) A3 

        • 4) Ax - fewest/weakest

    • Unexpected positive reaction 

      • Rouleaux 

      • Wharton’s jelly 

      • Cold agglutins 

      • Chimerism 

  • Resolution of weak or missing reactions 

    • Check age and diagnosis 

    • Incubate at room temperature for 15-30 minutes 

    • Incubate at 4 degrees Celsius for 15-30 minutes 

      • Include auto control 

    • Different reagent 

    • Treat the patient’s RBC with enzymes 

      • Enhance antigen expression 

  • Resolution of Subgroups of A 

    • Lectins 

      • Anti A1 

    • Anti A, B 

      • More sensitive to A antigen in some instances 

    • A2 cells 

    • Secretor studies 

    • Adsorption/elution studies 

  • Unexpected positive reactions 

    • Rouleaux - always in multiple myeloma 

      • Stack of coins 

      • Resolution → wash RBCs with saline multiple times

    • Cord blood 

      • Protein substances that can trap RBCs and cause them to stick together 

      • Resolution → wash cells a minimum of 2 times before testing 

        • May need to wash 4-6 times before testing 

        • Collect capillary samples 

          • No washing 

    • RBC coated with cold autoantibodies 

      • Positive DAT and auto-control 

      • Resolution → wash RBCs using warm saline 

        • Prewarm technique 

        • Treat RBCs with chloroquine or EGA to remove the autoantibody 

    • Polyagglutination 

      • The patient’s cells react with all antisera including control 

      • Types 

        • Tn activation → abnormality of the red cell membrane. Not bacterial   

        • Other types 

          • Tk, Th, and VA activation → bacterial 

          • Cad and NOR → inherited 

      • Resolution → switch to monoclonal reagents 

        • Secretor studies 

        • adsorption/elution studies 

        • Lectins 

    • Acquired B antigen 

      • Group A1 RBCss acquire B-like antigens due to bacterial enzymes

      • Patients Group A cells absorb B-like bacterial polysaccharide 

      • Bacteria 

        • E. coli, Proteus vulgaris 

      • Resolutions → check diagnosis 

    • Chimerism 

      • Dual population of red cells present in one individual 

        • Mixed field reactions 

      • Resolution → check patient history 

    • The patient has an antibody to yellow dye in the anti-B reagent 

      • Acriflavine or caprylate 

      • Resolution → don't use reagents 

    • RBC membrane had been modified due to drugs 

      • Resolution → check patient history 

• Serum problems - reverse type 

  • Most common form of ABO discrepancy 

  • Weak or missing antibodies 

    • Decreased antibody titers 

      • Newborn 

      • Elder

      • Leukemia 

      • Chimerism 

    • Resolution 

      • Check age 

      • Check diagnosis 

      • Incubate plasma at room temperature for 15-30 mins 

      • Incubate plasma at 4 degrees Celsius for 15-30 mins 

      • Increase plasma/cell ratio 

      • Treat A1 and B cells with enzymes 

  • Unexpected positive reactions 

    • Rouleaux 

      • Resolution → saline replacement technique 

      • True agglutination will stay, and rouleaux will go away

    • Cold autoantibodies 

      • Anti-I is the most common 

      • Anti-H in A1 or A1B patients 

      • Test patient's plasma against screen cells and auto-control 

        • All screen cells and auto-control positive = cold autoantibody 

        • One or more screen cells positive and auto control negative = cold alloantibody 

      • Resolution → 

        • Prewarm technique

        • Cold autoabsroption for cold autoantibody 

          • Incubate-washed RBCs and patient plasma together at 4 degrees Celsius for an hour 

        • Cold alloantibody  

          • anti-M or anti-P1

          • Perform antibody ID 

    • Anti-A1

      • Found in some A2 and A2B people 

      • Found in almost all other subgroups of A

      • Resolution → 

        • Confirm patient is A1 antigen-negative by testing with Anti-A1 lectin 

        • Perform IAT to exclude alloantibody 

Hematopoietic stem cell transplants 

• HSC transplants 

  • Obtained from 

    • Bone marrow 

    • Peripheral blood stem cells 

    • Umbilical cord 

  • Possible treatment for 

    • Aplastic anemia

    • Sickle cell 

    • Leukemia 

  • ABO compatibility is not necessary 

  • Patients receiving HSC switch their blood group 

  • ABO-incompatible transplants can be successful 

Advance methods

• Review of antibody screen 

  • Selectogens and panoscreen

  • Detect as many clinically significant antibodies as possible 

    • Causes HDN, HTR, or decreased RBC survival 

• Potentiators 

  • 22% albumin 

    • Reduces zeta potential 

    • Longer incubation 

  • LISS 

    • Increasing the amount of antibody taken up by the red cells and reducing the zeta potential 

  • PEG 

    • Accelerates antibody red cell binding by removing water 

    • IgM antibodies do not react with PEG

• Antibody ID 

  • History 

    • Transfusion, pregnancy, IV, transplant 

  • Panel 

    • 11 to 20 group O red blood cells 

    • Show homozygous expressions → Rh, Duffy, Kidd, and MNSs 

    • Interpretation 

      • Crossout technique 

      • Phase of reaction 

      • Strength of reaction 

  • Dosage 

    • Weak expression of antigen 

    • Heterozygous expression 

    • Patients with the genotype MM, which is homozygous will have more antigen sites than a patient having the genotype MN, which is heterozygous 

  • Selected panels 

    • Use when the initial antibody panel does not identify clear-cut antibodies 

      • Multiple antibodies 

  • Autocontrol 

    • Not normally performed 

    • Indicate the presence of autoantibody 

    • Positive auto control in the AHG phase 

      • DAT is the next test 

  • Antigen typing 

    • Antibody has been identified, need to check for the corresponding antigen on the patient’s cells and donor unit 

    • May be used to eliminate antibodies 

  • Prewarm technique 

    • Prewarm serum and cells to 37 degrees Celsius separately and then combine and maintain that temperature throughout the testing 

    • Prevent cold antibodies from reacting 

    • Use with caution!

      • This may result in decreased reactivity of some significant antibodies 

  • Saline replacement 

    • Remove serum and replace with equal volume of saline 

      • Positive if there is true agglutination 

      • Negative when rouleaux id present 

• Enzymes 

  • Complex antibody ID 

  • Modify the red cell surface by removing sialic acid residues and denature or remove glycoproteins 

  • Panel cells treated with enzymes 

    • Modify the surface of the red cell by removing sialic acid or denaturing glycoproteins 

  • Ficin and papain are the most frequently used enzymes 

    • Ficin → figs 

    • Papain → papaya

    • Bromelin → pineapple 

    • Trypsin → pigs 

• Adsorption 

  • Uptake of antibodies onto specific receptors on the red blood cell surface 

  • Methods 

    • Red cells with known antigens are incubated with serum or plasma to remove a suspected antibody from the plasma 

    • Known antibody is incubated with unknown red cells to observe id the cells will uptake the antibody 

    • Autologous

      • Only if they haven't received blood in the past 3 months

      • Patients red cell 

    • Differential or allogenic 

      • Known red cell types 

  • Autoadsorption 

    • Only if DAT is positive for IgG and there is an autoimmune issue

    • The temperature of incubation will depend on the reactivity of the antibodies 

      • Cold antibodies at 4 degrees Celsius 

      • Warm antibodies at 37 degrees Celsius 

  • Adsorbed plasma is then tested to detect any remaining antibodies 

  • Most common use is to remove autoantibodies from the serum or plasma 

Clinical application of adsorption 

• Warm autoantibodies 

  • All cells positive at the same strength in the AHG phase 

  • Positive auto-control 

  • Resolve by removing autoantibody to determine if there is an underlying alloantibody 

• RESt → rabbit erythrocyte stroma 

  • Absorbs cold-reacting autoantibodies 

  • Cannot be used for ABO typing or crossmatching 

• WARM

  • Commercial form of ZZAP 

  • Contain lyophilized preparation of DTT → destroys Kell 

• HPC → Human platelet concentrate 

  • Made from pooled platelets 

  • Removes unwanted HLA antibodies 

  • HLA antigens on platelets are used to absorb Anti-Bg antibodies 

• Cold 

  • Adsorption performed at 4 degrees celsius 

  • Removal of potent cold reactive autoagglutins 

Elution 

• Definition → removal of an antibody from the surface of the red blood cell 

  • Total elution 

  • Partial elution 

• Usually IgG antibodies 

• Methods 

  • Change the thermodynamics 

  • Change the forces 

  • Change the structure 

• Total elution 

  • Red blood cells are destroyed 

  • Not used for phenotyping or autoadsorptino 

  • Methods 

    • 56 degrees Celsius heat where red cells are destroyed 

    • Lui freeze is where red cells are frozen and then rapidly thawed to cause lysis 

    • Acid pH of 3.0 

• Partial elution 

  • Removes the antibody but leaves the red cell membrane intact 

  • Methods 

    • Gentle heat at 45 degrees Celsius that releases the IgG from the surface of the red cell 

    • ZZAP or chloroquine 

    • EGA uses an acidic pH to dissociate the antibody from the cell

• Neutralization 

  • Substance in the body and nature have antigenic properties similar to the red cell antigens 

  • Used to inhibit the reactivity of the corresponding antibody 

  • Lewis substances present in saliva 

  • P1 substances are present in hydatic cyst fluid and pigeon eggs 

  • Sda substances are present in urine 

Calculation 

• Calculation for antigen-negative phenotypes 

• (precentage %)/(percentage) = (# of units) / x

Blood products: