Adv Blood Bank Exam #1

Tests in Pretransfusion & prenatal testing (screen/detection, standards)

• Antibody Screen/antibody detection

• AABB Standards: testing methods demonstrate clinically significant antibodies at AHG phase

Unexpected/Atypical antibodies (what/formed in response)

• antibodies to red cell antigens that are not Anti-A or Anti-B

• Typically formed in response to:

  • Red cell transfusion

  • Exposure to fetal cells in pregnancy or delivery

  • Transplant of organs or hematopoietic stem cells

  • Sharing needles & blood doping

Alloantibodies vs Autoantibodies

• allo = Antibodies produced following exposure to foreign RBC antigens

• auto = Antibodies to self antigens; Can result from a disease process or medication

  • ex: autoimmune diseases & Anti-I

Unexpected Antibody Rates (2)

• 0.5 - 1.5% have detectable unexpected red cell antibodies

• 14 - 50% in chronically transfused populations may demonstrate unexpected red cell alloantibodies

Antibody screen: FDA requires, screen cells type, can miss

• FDA requires screen cells to be antigen typed for the following antigens:

  • D, C/c, E/e, M/N, S/s, P1, Le a/b , K/k, Fy a/b, & Jk a/b

• all screen cells = type O

Antibody screen: What can be missed

• screens can miss low-frequency antigens (most do not have) or ABO antigens

  • rare to find a patient with the corresponding antibody

  • unlikely the patient will receive blood from a donor with this antigen

  • most antibody screen reagents won’t have cell (+) for that low-frequency antigen → screen (-)

False positive reactions: Remember, examples, autocontrol

• remember: not all reactivity is clinically significant

• examples: rouleaux, antibodies to preservatives in reagents, fibrin

• autocontrol falsly (+) due to: antibody to potentiator like LISS [cells weakly (+) & DAT (-)] & rouleux

Autocontrol: AABB, positive result, plasma/red cell, (+/-) results

• some labs perform with antibody panel (not required by AABB)

• autocontrol (+) → some labs perform DAT

• tests patient plasma with their own red cells & includes protentiator used in panel, incubation step, & AHG

• (-) = alloantibodies

• (+) = autoantibodies

Autocontrol: Positive result in what kind of patients

• autocontrol may be (+) in patients with:

  • Autoimmune hemolytic anemia (Warm and/or cold or drug)

  • HTR (acute or delayed)

  • Passively acquired antibodies: Antibodies from donor plasma & Newborns with mom’s antibodies

DAT vs IAT (plasma/red cells)

• DAT: tests patient’s red cells with AHG, no potentiator & incubation step

  • detects antibodies bound to patient cells in vivo

• IAT: tests patient plasma with reagent red cells & AHG

  • detects patient’s antibodies bound to reagent cells in vitro

  • has washing & AHG

Autocontrol vs DAT (what is detected)

• DAT: detects antibodies bound to patient’s red cells in vivo

  • no enhancement reagents & incubation

• Autocontrol: detects antibody bound to patient’s cells in vitro

  • has enhancement reagents & incubation

Patient history (what to look for; 4)

• look for: transfusions/HTR, antibodies ID, antigen typing, pregnancies, transplants

• check for diagnosis: Sickle cell, cancer treatment, pregnancy

• race: anti-U, P(-), Anti-H

• age: female of childbearing potential, elderly (back-typing issues), young people generally don’t have autoantibodies

Antibody panel procedure

• exactly the same as antibody screen for all methods (tube, gel, solid)

• think: panel is more screen cells, screen cells are small panels

Reaction phases interpretations (3)

• Immediate Spin (IS) = IgM, cold antibody (allo or auto)

• 37C = IgM, or an IgG is reactions are not seen at IS

• AHG = IgG, clinically significant

Process of elimination: Rule of 3

• Need 3 homozygous cells to rule out an antibody

  • exception only for C/c & E/e: 1 homo & 2 hetero

  • exception only for K: 3 hetero for rule out

• need 3 (+) cells to rule in an antibody

Homozygous vs. Heterozygous for ABID

• homo = double dose of antigen

• hetero = both antithetical antigens present

• antithetical pairs: C, E, K, Kp, Js, Fy, Jk, Le, M/N, S, Le, Lu

• homo cells used only for rule out

Finding compatible blood

• clinically significant antibody can’t be ruled out/in → treated as if patient has it

• exception: not able to rule out every possible antibodies for low-frequency and high-frequency antigens

Ruling out steps (2)

1. look at cells that have (-) reactions

   1. anything (+) cannot be what the antibody is directed to

2. cross off homo antigens on (-) cells

   1. ex: D is by itself, if antibody has anti-D, these cells would have (+) reaction

3. Fill antibody ID form: document cell # of cross off (helps to rule out)

Selected cells: Define, can’t rule out, panel cells

• reagent red cell chosen from another panel to confirm or eliminate the possibility of an antibody

• can’t rule out → create select cell panel

• use panel cells that haven’t been tested with patient’s plasma

Why choose select cells

• don’t want waste of sample, reagent, time testing that won’t be informative

• don’t run complete cell panels when only need to run selected cells from that panel

Choosing select cells (requirements; 4)

• choose panel cell (from new panel) that is:

  • antigen (-) for suspected antibody & antigen homozygous (+) for the antibody you are trying to rule out

• select cell is (-) = antibody can be ruled out

• selected cell (+) = not ruled out & need to do more testing

• antigen (-) for suspected antibody & antigen homozygous (+) for the antibody trying to rule out

Selected cell not available

• give antigen (-) for all that were not ruled out

• if (+) for antigen then you can rule out that antibody

Proteolytic enzymes (function, treatment, refers to, membranes, antigen vs antibody)

• denature certain proteins

• used to treat RBCs to aid in complex antibody ID

• proteolytic refers to breakdown of proteins into smaller fragments

• enzymes modify RBC membranes by removing (-) charged sailic acid residues → reduce zeta potential

• modify antigens not antibodies

Enhanced vs Destroyed by enzymes

• enhanced: Rh, Kidd, Lewis, I, P1PK

• destroyed: Duffy, MNSs, Xga

• no change: Kell, U, Lutheran

Enzyme panel interpretation (treated): Nonreactive, stronger reaction, no change, some reaction goes away

• treated: nonreactive → Duffy, MNSs, and/or Xga

• treated: stronger reactions → Rh, Kidd, Lewis, I, and/or P1PK

• treated: no change → NOT Duffy, MNSs, or Xga

• treated: some reaction goes away → more than 1 antibody present

Full crossmatch vs Absorption

• full: current or past history of antibody with (-) or (+) AB screen

• absorb: removal of known antibody from serum in order to allow detection of other antibodies present

Interpreting panel results: Single antibody (suspect if & testing)

• suspect if:

  • some cells (+); same strength & phases

  • (-) auto control

  • just some cells (+); different stregnths and/or phases

• testing:

  • Do selected panel to rule out

  • Antigen type patient (except if transfused in past 3 months)

Interpreting panel results: Multiple antibodies (suspect if & testing)

• if:

  • Cells that are (+) have different strengths and/or phases

  • (-) auto control

  • pattern of reactivity does not match exactly (extra cells reacting)

• testing:

  • Do selected cells to rule out

  • enzymes, Lewis or P substances

  • Test patient cells to confirm they lack antigen

Interpreting panel results: Antibody to High incidence antigen (suspect if & testing)

• if: all cells (+); same strength and same phase)

• testing: send to reference lab

Interpreting panel results: Antibody to Low incidence antigen (suspect if & testing)

• if: only 1 cell is (+)

• testing: send to reference lab

• ex: C^w, Kpa, Jpa, Lua

Interpreting panel results: Weakly reactive antibody or antibody showing dosage (suspect if & testing)

• if: weak reactivity with some cells (different reaction strengths)

• testing: May need enhancement techniques (enzymes, increase serum, increase incubation time)

  • Do selected cells to confirm or exclude antibody specificity

  • check expiration date of panel cells

Antibodies that exhibit dosage

Rh, Duffy, MNS, Lu, Kidd

Why enzyme panels are run

• if extra reactions on untreated panel: due to Duffy, MNS, Xga

• When enzyme treated panel is run, Duffy, MNS, Xg will not be present for patient’s antibodies to react

• Allows other antibody reactions to be seen

• note: can only used untreated panel for rule outs

Absorption common uses

• Antibody removed from serum sample by adsorption onto red cells expressing corresponding antigen

• Separating multiple antibodies present in a single serum

• Remove autoantibody activity to permit detection of underlying antibodies

• Confirm presence of specific antigens on RBCs

Absorption

• Need large amounts of red cells so donor units are used for adsorptions

• Once antigen-antibody binding occurs then remaining serum may be tested for other clinically significant antibodies

• testing: Once you have adsorbed the antibody out of the serum → then remaining serum may be tested for other clinically significant antibodies

  • Can do elution on adsorbed red cells to identify antibody absorbed

• Cells used in adsorptions should have corresponding antigen of antibody you are trying to absorb

Elutions

• remove & ID antibody that is bound to RBCs

• Investigation of (+) DAT

• Concentrate antibody if weak

• Remove interfering antibody blocking antigen sites on rbcs to allow for rbcs to be phenotyped

When do to elutions

• (+) DAT (IgG)

• To isolate specific antibodies in multiple antibody patient after absorption

• test for weakly expressed antigens on red cells after incubation with specific antiserums

Elutions

• No single elution method will result in the ID of antibodies

• Thorough washing of red cells before elution ensures antibody in the eluate is only red cell-bound and is not antibody from plasma

• Saline from last wash: used as control, shows all free antibody has been removed by washing, tested in parallel with eluate

Elution types

• acid (for warm reactive autoabs or alloabs

• Glycine-HCl/EDTA

• Heat: preparing IgG free red cells for ABO testing

• Lui Freeze-Thaw (ABO): mainly for anti A, B, or AB

• Methylene Chloride

Acid elution reagents

• acid: lowers pH of medium to cause dissociation of ag-ab bonds

• Buffer: raises pH of eluate so it will not harm RBC reagent cells

• wash solution: washes the antibody coated rbcs free of residual serum proteins & provides correct ionic strength for antibody reactivity

• before elution (antibody on RBCs) → after elution (antibody in eluate supernatant)

Adsorption and Elution Combined

• used to: Separate mixture of antibodies

  • Detect weakly expressed antigens on red cells

  • Help identify weakly reactive antibodies

• Incubate serum with selected rbcs (adsorption)

• Elute antibody from adsorbing red cells

Anti-G

requires both absorption & elution

Inhibition/Neutralization of antibodies

• antibodies can be inhibited or neutralized by soluble antigens

  • anti-P1 & Lewis (soluable antigen)

  • anti-Sda (inhibited by urine)

  • anti-Chido/Rodgers (inhibited by serum/plasma)

• Use: aid in ID & rule-outs

Inhibition of antibodies

• allow to rule-out antibodies to common red cell antigens

  • tested in parallel with saline control

  • saline control should be (+), inhibited plasma (-)

• help ID antibody: if inhibition successful, procides serologic evidence of antibody specificity