Antibody Detection & Identification Flashcards
When to suspect an antibody?
ABO discrepancy pos Ab screen incompatible crossmatch transfusion reaction pos DAT HDFNB
screening cells must have what antigens on them
D,C,c,E,e,M,N,S,s,Lea,Leb,Pi,K,k,Fya, fyb, jka, jkb
3 cell screening kits must include what antigens?
homozygous Duffy & Kidd antigens
R1R1, R2R2 & rr cells must all be included
expiration time frame for all red cell reagents
every 3 weeks
BB labs have standing orders
antibody screen limitations
may miss:
low titered antibodies (Kidd)
Ab to low frequency antigens
Ab that do not react under the experimental conditions (pH, LISS, other ions etc)
BSA
bovine serum albumin in either 22% or 30%
mechanism is unclear
enhances the sensitivity at AHG phase for most antibodies
requires an increased incubation time!
LISS
low ionic strength solution: increases the rate of Ab binding to specific Ag sites on the RBC by decreasing zeta potential
equal volumes of serum & LISS!
limitations of LISS
enhances cold autoantibodies & may miss weak Kell Ab
PEG
polyethylene glycol: removes water molecules to allow greater chance for collision between antigen & Ab
DO NOT CENTRIFUGE until saline has been added
Proteolytic enzymes
papin, ficin, & bromelin
causes breakdown of protein molecules
enhance: Rh, Kidd, Lewis, I, P Ab
denatures: M,N, Fya, Fyb
Enhanced methods
ortho gel or immucor capture systems enhanced sensitivity automated objective cost pulls up non-clinicially-relevant Ab
laboratory information systems
built in systems/checks in place to prevent mistyping, missing antibodies, & other clinically relevant information
always need to confirm all the information on a patient’s file
DAT detects:
Ab that have been formed IN VIVO:
autoantibodies
alloantibodies following transfusion
DAT reagent
polyspecific AHG for IgG & C3d
if positive, one can go back and test with separate reagents to determine if antibody mediated or complement mediated
possible reasons why all 3 cells in a DAT would have various reaction strengths
dosage
multiple antibodies
multiple specificity
possible reasons why all 3 cells in a DAT panel are 1+ at IS
IgM antibody to a common antigen
antibody ID - autocontrol
Pt plasma is combined with their own 4% RBC solution
includes potentiator
read at all stages
IgM antibodies
lewis, Lutheran, M, N, I, & PI
IgG antibodies
Rh, Kell, Kidd, Duffy, SsU, Lub
antibodies that agglutinate strongly even with heterozygous cells
Ab to K, D,E,e,c,C
antibodies that react weakly especially with heterozygous cells
Fya, Fyb, Jka, Jkb, S,s
DTT treatment will eliminate which Ab
Kell & Lutheran antibodies
what to do when autocontrol is positive
can be caused by cold autoantibody, warm autoantibody or delayed transfusion reaction etc etc
initial investigation should include a DAT
can use elution & absorption techniques to ensure absence of underlying antibodies
Cold alloantibodies
Pi, M,N, Lea, Leb
usually insignficiant
prewarming techniques ore neutralization
Neutralization of Lewis antibodies
add commercially available lewis antigen to pt plasma & retest - if neg, then Lewis Ab was present & no other Ab are present
‘short cold’ panel
select cells that will help ID the suspected cold autoAb
always include a cord cell: I, Le & P neg
most common cold autoantibodies are I, IH & H
cold autoabsorption technique
goal: remove the cold autoAb from the plasma so the plasma can be tested for clinically relevant IgG alloantibodies
1. remove plasma
2. treat RBC to remove Ab
3. add plasma back to RBCs
4. incubate at RT so Ab in plasma will bind to RBC
5. remove plasma & test
warm autoantibodies
more common than cold autoAb
DAT is pos for IgG
most common warm autoAb is against the e antigen
warm autoadsorption limitation
must not perform if patient has been transfused in the last 3 months bc plasma may contain alloAb to transfused cells
elution techniques general
performed on pt RBC when the DAT is positive w/ an IgG
goal: ID the Ab bound to RBCs such as in recently transfused patients experiencing reactions & newborn from mother with IgG Ab
Elution methods
acid elution: glycine acid is rapid & sensitive. available in kits & used commonly
