Blood Bank Case Studies Case Studies from the reference laboratory Jackie Ensley, MLS(ASCP)CMSBB
Objectives • Present various case studies and describe the approach to serologic problem solving and antibody identification. • Determine possible causes of pan-reactivity and steps to resolve complex antibody cases. • Briefly review serologic and molecular characteristics of antibodies identified and their respective blood group system, including clinical significance. • Explain the various techniques and methods used in the case studies for antibody identification.
Antibody Identification • Antibody detection and identification is a complex problem-solving process • Many techs may have a “gut-feeling” about the antibody before testing completion and intuitively know what needs to be done for antibody identification • Be prepared to reevaluate your hypothesis if testing results do not fit with initial assessment
Antibody Identification • Use the tools available to help you detect and then identify the antibody: • Gel/solid phase • Tube testing: saline/PeG/LISS/albumin/Room temperature/4˚C • Enzymes such as ficin, papain, trypsin • Chemicals such as 0.2M DTT • Adsorption/elution • Reticulocyte/sickle cell separation • Phenotypically similar cells • Antisera/rare antigen negative cells
Antibody Identification • Know phases of reactivity • Some antibodies react best at room temperature/4˚C (M, N, P1, Lewis, etc) • Some antigens destroyed by enzymes/chemicals (Ficin destroys Fya, Fyb, M, N, etc) • Enzyme treatment of red cells enhances reactivity of some antibodies such as those in the Rh system, Jka, Jkb, Lea, Leb, P1 • Know strength/pattern of reactivity • Some antigens show variable antigen expression and some antibodies show variable reactivity and may show dosage, such as -Jka/-Jkband -M/-N • Note: different strengths may also indicate more than one antibody is present
Antibody Identification • Besides using the blood bank techniques available to detect the antibody, also keep in mind these tips to aid you in the identification process: • Review patient’s records, including medication, age, gender, race, diagnosis and transfusion history • Investigate/repeat any inconsistent or contradictory reactions in the patient’s workup • Phenotype the patient to confirm they are antigen negative for the suspected or identified antibodies
Case Study 1 • Reference Lab testing: • ABO/Rh performed: • DAT Performed:
Serologic Problem Solving Question to ask yourself: So where do we go at this point? • Let’s look at what we know: • Phase of reactivity: AHG • Strength/pattern of reactivity: pan-reactive, about the same strength. • Patient history: recently transfused • DAT/autocontrol: positive/reactive • Other info: eluate is also pan-reactive with same strength
Serologic Problem Solving Question to ask yourself: So where do we go at this point? • Narrowed down possibilities: • Warm autoantibody • Multiple antibodies in plasma (and eluate) • Antibody to a high incidence antigen
Next Step: • Reference tech decides to perform adsorption on plasma only. Why not an adsorption on the eluate? • The patient has been transfused in the last 3 months… Technical Manual States that “newly developed antibodies initially detectable only in the eluateare usually detectable in the serum after about 14 to 21 days”
Blood Bank Technique: Adsorption How is an alloadsorption performed? • Alloadsorption: Patient has been transfused or transfusion is unknown. R1R1 = R2R2 rr Incubate together to adsorb the antibodies onto the donor red cells Patient’s Plasma + Donor RBC’s
How is an alloadsorptionperformed? R1R1 Adsorption Plasma- Test R2R2 Adsorption Cells- discard = rr Incubation allows any antibody to adsorb onto the red cells (alloantibody or autoantibody) Centrifuge the tubes and separate the adsorbed plasma from the red cells for testing
How is an alloadsorptionperformed? rr (D-C-E-c+e+) R1R1 (D+C+E-c-e+) R2R2 (D+C-E+c+e-) Example: anti-E Example: anti-E Run each adsorbed plasma with panel cells to identify any antibodies. Antibodies in adsorbed plasma will depend on the phenotype of the adsorbing cell.
Autoantibody Confirmation Testing • Patient had been transfused in the last 3 months so need to perform reticulocyte separation • Want to be testing patient cells and not donor cells
Blood Bank Technique: Reticulocyte Cell Separation How is a Reticulocyte Cell Separation Performed? • Patient has been transfused so need to separate patient cells from donor red cells Stopper one end of the hematocrit tube with clay. Spin the sample down and fill microhematocrit tubes with the red cells.
Blood Bank Technique: Reticulocyte Cell Separation How is a Reticulocyte Cell Separation Performed? Air Excess saline/plasma Buffy Coat Newer Red Cells Older Red Cells Spin the microhematocrit tubes and then cut the tubes to get the reticulocytes Clay Plug
Autoantibody Confirmation Testing Now that we have the retics: • DAT/IgG had been positive so perform DAT/IgG on retics: Can not proceed with testing to identify warm autoantibody until the DAT is negative
Blood Bank Technique: EGA Treatment EGA Treatment • What is EGA? • EDTA glycine acid dissociates IgG from red blood cells so the treated red cells can be used for further testing or antigen typing using the AHG phase. • Use when direct antiglobulin phase (DAT) is positive • Does not impair red cell surface antigens
Blood Bank Technique: EGA Treatment EGA Treatment • The Process • Wash IgG coated red cells thoroughly • Suspend cells briefly in EGA solution to dissociate bound IgG antibody • Bring mixture to neutral pH • Centrifuge and wash cells with saline • Test treated cells by performing a DAT • Limitation: destroys Kell, Era, Bg antigens
Autoantibody investigation • EGA testing performed and DAT negative retics obtained • To confirm the antibody is warm autoantibody the DAT negative retics are tested against the plasma and eluate: This is what was expected if the antibody was autoantibody! Further testing is not required, the warm autoantibody has been confirmed.
Antibody Confirmation • Lastly need to confirm anti-Jka (JK1) by antigen typing • Use retics so that typing patient cells and not donor cells Patient types Jkanegative
Results • Patient has warm autoantibody and anti-Jka (JK1). • Transfusion recommendations: Transfuse Jka- (JK1), AHG crossmatch least incompatible, red blood cell products.
Kidd Blood Group System • Located Chromosome 18 • Glycoprotein with 10 membrane spanning domains • ·Daniels, G. (2013) Kidd Blood Group System, in Human Blood Groups, 3rd edition, Wiley-Blackwell, Oxford, UK. Kidd antibodies are often difficult to work with and are a common cause of delayed hemolytic reactions
Jka (JK1) Antibody & Antigen Reid, Marion and Christine Lomas-Francis (2012). The Blood Group Antigen FactsBook, 3rd Edition, Elsevier.
Case Study 2 • The 2nd sample was collected on 1/24/2014, 9 days after transfusion. • Sample was sent to the reference laboratory
Case Study 2 • Reference Lab testing: • ABO/Rh performed: • DAT Performed:
Serologic Problem Solving Question to ask yourself: So where do we go at this point? • Let’s look at what we know: • Phase of reactivity: AHG • Strength/pattern of reactivity: pan-reactive, same strength. • Patient history: recently transfused • DAT/autocontrol: positive/reactive • Other info: eluate is also pan-reactive with same strength
Serologic Problem Solving Question to ask yourself: So where do we go at this point? • Narrowed down possibilities: • Warm autoantibody • Multiple antibodies in plasma and eluate • Antibody to a high incidence antigen
Next Step: • Reference tech decides to perform adsorptions on plasma & eluate. Why perform an adsorption? • To adsorb out suspected warm autoantibody and determine if there are any alloantibodies hiding under the pan-reactivity.
Results • Appears to be warm autoantibody • No alloantibodies were detected in the alloadsorbed plasma or eluate Need to confirm warm autoantibody
Autoantibody Confirmation Testing • Patient had been transfused 9 days ago so perform reticulocyte separation. • DAT/IgG had been positive so perform DAT/IgG on retics: Proceed with further testing to identify warm autoantibody
Autoantibody Confirmation Testing • To confirm the antibody is warm autoantibody the retics are tested against the plasma and eluate: This is NOT what was expected if the antibody was autoantibody! Further testing is required and now antibody to a high incidence antigen is suspected