abdetection and identification
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Antibody Detection
Dr. Ali Ibrahim
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Antibody Detection
The test used to detect antibodies iscalled an antibody screen
Antibody screens are used for: Patients needing a transfusion
Pregnant women
Cases of transfusion reactions
Blood and plasma donors
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Antibody Screen
Uses patients plasma/serum againstreagent red cells to detectunexpected antibodies
Unexpected antibodies are found inaddition to the expected anti-Aand/or anti-B
Unexpected antibodies are a result
of red cell stimulation (transfusion,HDN)
Unexpected antibodies may be: Clinically significant (IgG)
Not clinically significant (IgM)
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Clinically significant antibodies
Usually IgG
React best at 37and AHG phase
(IAT) Clinically significant antibodies are
associated with hemolytictransfusion reactions (HTR) and
hemolytic disease of the newborn(HDN)
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Performing an antibody screen
Patients plasma/serum is incubatedwith screening cells
After incubation, an IAT isperformed (indirect antiglobulintest) using AHG reagent
This will detect any IgG antibodies
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Screening Cells
Screening cellsare single or pooleddonor group O cells, however, single-donor vials offer increased sensitivity
Why group O? so anti-A and anti-B wontreact
Screening cells come in sets of 2 or 3vials each
Each vial (donor) has been phenotypedfor each antigen
18 antigens are required on at least oneof the vials: D, C, E, c, e, M, N, S, s, P
1
,Lea, Leb, K, k, Jka, Jkb, Fya, Fyb
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Screening cells
Screening cells come with a sheet ofpaper called an antigram
Screening cells are an alreadyprepared 2-5% RBC suspension
An antigram(2 or 3 cells) will listthe antigens present in each vial
A reaction to one or more cellsindicates the presence of anunexpected antibody
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Cell Antigram
Some manufacturerswill use + instead of
1 to indicate thepresence of theantigen
Screening Cells I &II
IS 37
AH
G
CC
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Screening cells
The Examiner should be aware thatsome antigens demonstrate dosage
An attempt should be made to used
screening cells that are homozygousfor the clinically significant antigens(Rh, Duffy, Kidd). Just be awarethat different strengths can occur
Homozygous antigens will reactstronger
Heterozygous antigens will reactweaker
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Examples
Fya Fyb
SCI + + 2+
SCII 0 + 4+
Fya Fyb
SCI + 0 4+
SCII 0 + 0
If patients serum containsanti-Fya, there will be a
strongerreaction becauseSCI is homozygousfor the
Duffy antigen
In this case, the person hasanti-Fyb. The antibodyreacts weaker with SCI(heterozygous) andstronger with SCII
(homozygous)
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Screening Cells
Screening cells may also containlow-incidence antigens like V, Cw,Jsa,Luaand Kpa
The presence of these antigens isnot required for screening cells
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Pretransfusion Screening
Screening for antibodies is normallyperformed prior to blood transfusion todetect antibodies that react at bodytemperature (37)
Colder reacting antibodies (RT and below)
are therefore considered insignificant andjust cause interference when performinglab testing
The only important thing to remember
concerning cold antibodies is that theymay bind complement if a persons bodytemperature becomes low
Open-heart surgery h othermia
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Autocontrol
Tests patient serum with their own redcells
Some labs may or may not perform anautocontrol (AC) with the screendependson the hospital
However, the AC should be run with theantibody panel
AC is incubated with the antibody screen(or antibody panel)
If a lab uses an AC with the screen and itis positive, they may run a DAT (patientcells + AHG) to detect in vivocoating
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Autocontrol
The AC and DATcan help indetermining whether the antibodiesare directed against the patientscells or transfused cells(allo-orautoantibody)
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Potentiators
Used in antibody detection andidentification to enhance antigen-antibody reaction
Saline (may only enhance if incubatedlong time)
Low-ionic strength solution(LISS)common
Bovine serum albumin (BSA) Polyethylene glycol (PEG)
Proteolytic enzymes (can destroy someantigens)
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Potentiators
Albumin Serum/cell mixture should incubate at least20 minutes, 30 minutes preferred; doesnt
enhance warm autoantibodies
LISS Incubation time of 10 minutes; lowersionic strength allowing better reaction;
sensitive and quick!
PEG Enhances warm autoantibodies; does notreact well with insignificant antibodies
(IgM)
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Limitations
Very effective in detectingantibodies
If negative, then the crossmatchshould be compatible
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Patient History
GET THE HISTORY!!
Mixed red cell populations from aprevious transfusion can remain for up
to 3 months
Patient may have come from anotherhospital
Some diseases are associated withantibodies
Some antibodies occur at a higherfrequency in some races
Get diagnosis, age, race, etc
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Example 1
Screening
Cell
IS 37C AHG CC
I 0 0 0
II 0 0 2+ ND
NotDone
IgG antibody
Singlespecificity
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Example 2
Screening
Cell
IS 37C AHG CC
I 0 0 3+
II 0 2+ 3+
IgG antibody
Multiple specificities
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Example 3
Screening
Cell
IS 37C AHG CC
I 1+ 0 0
II 3+ 0 0
IgM antibody
Single specificity showingdosage
Neg AHG, addCC
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Example 2
Screening
Cell
IS 37C AHG CC
I 0 0 2+
II 0 0 2+
IgG antibody
Allo- or autoantibody?(dont know withoutfurther testing)
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Antibody Identification
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Why do we need to identify?
Antibody identification is needed fortransfusion purposes and is animportant component of
compatibility testing It will identify any unexpected
antibodies in the patients serum
If a person with an antibody isexposed to donor cells with thecorresponding antigen, serious sideeffects can occur
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Key Concepts
In blood banking, we test knowns withunknowns
When detecting and/or identifyingantibodies, we test patient serum(unknown) with reagent RBCs (known)
Known: Unknown:Reagent RBCs + patient serum
Reagent antisera + patient RBCs
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Reagent RBCs
Screening Cells and Panel Cells arethe same with minor differences:
Screening cells
Antibody detection
Sets of 2 or 3 vials
Panel cells
Antibody identification At least 10 vials per set
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Antibody Panel vs. Screen
An antibody panel is just anextended version of an antibodyscreen
The screen only uses 2-3 cells:
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Antibody Panel
An antibody panel usually includesat least 10 panel cells:
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Panel
These are Group O red blood cells
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Panel
Each of the panel cells has beenantigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb
, k, Fya
, and Jka
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Panel
An autocontrol should also be runwith ALL panels
Autocontrol
Patient RBCs+
Patient serum
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Panel
The same phases used in anantibody screen are used in a panel
IS
37
AHG
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Antibody ID Testing
A tube is labeled for each of thepanel cells plus one tube for AC:
AC
1 2 3 4 5 6 7 8 9 10 11
1 drop of each panel cell+
2 drops of the patients serum
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IS Phase
Perform immediate spin (IS) andgrade agglutination; inspect forhemolysis
Record the results in theappropriate space as shown:
2
0
0
Last
tube
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(LISS) 37C Phase
2 drops of LISS are added, mixedand incubated for 10-15 minutes
Centrifuge and check foragglutination
Record results
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(LISS) 37C Phase
2
0
0
2
0
0
2
0
0
2
0
0
0
0
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IAT Phase (or AHG)
Indirect Antiglobulin Test (IAT) were testing whether or notpossible antibodies in patients
serum will react with RBCs in vitro To do this we use the Anti-Human
Globulin reagent (AHG) Polyspecific
Anti-IgG
Anti-complement
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AHG Phase
Wash cells 3 times with saline(manual or automated)
Add 2 drops of AHG and gently mix Centrifuge
Read
Record reactions
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AHG Phase
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
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And dont forget.
.add check cells to
any negative AHG !
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IS LISS37
AHG CC
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
All cells arenegative at
AHG, soadd
CheckCells
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You have agglutinationnow what?
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
??
CC
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Interpreting Antibody Panels
There are a few basic steps tofollow when interpreting panels
1. Ruling out means crossing out
antigens that did not react
2. Circle the antigens that are notcrossed out
3. Consider antibodys usual reactivity
4. Look for a matching pattern
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An antibody will only reactwith cells that havethe
corresponding antigen;antibodies will not reactwith
cells that do not havethe
antigen
Always remember:
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Heres an example:
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1. Ruling Out
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
Cross out antigens that show NO REACTION in any phase; doNOTcross out heterozygous antigens that show dosage.
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2. Circle antigens not crossed out
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
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3. Consider antibodys usual reactivity
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
Leais normally a Cold-Reacting antibody (IgM), so it makessense that we see the reaction in the IS phase of testing;The E antigen will usually react at warmer temperatures
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4. Look for a matching pattern
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
Yes, there is a matching pattern!
E doesnt match andits a warmer rx Ab
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Interpretation
anti-Lea
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Guidelines
Again, its important to look at: Autocontrol
Negative - alloantibody
Positive autoantibody or DTR (i.e.,alloantibodies)
Phases
IS cold (IgM)
37- cold (some have higher thermal range) orwarm reacting
AHGwarm (IgG)significant!!
Reaction strength
1 consistent strength one antibody
Different strengths multiple antibodies or dosage
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About reaction strengths
Strength of reaction may be due todosage If panel cells are homozygous, a strong
reaction may be seen If panel cells are heterozygous,
reaction may be weak or even non-reactive
Panel cells that are heterozygousshould not be crossed out becauseantibody may be too weak to react(see first example)
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Guidelines (continued)
Matching the pattern
Single antibodies usually shows apattern that matches one of the
antigens (see previous panel example) Multiple antibodies are more difficult to
match because they often show mixedreaction strengths
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Rule of three
The rule of threemust be met toconfirmthe presence of the antibody
Ap-value 0.05 must be observed
This gives a 95% confidence interval
How is it demonstrated?
Patient serum MUST be:
Positive with 3 cells with the antigen
Negative with 3 cells without the antigen
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Our previous example fulfills the
rule of three
2
0
0
2
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
3 Negativecells
3 Positive
cells
Panel Cells 1, 4, and 7 are positivefor the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negativefor the antigen and did not give a reaction at immediate spin
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What if the rule of three is not fulfilled?
If there are not enough cells in thepanel to fulfill the rule, thenadditional cells from another panel
could be used
Most labs carry different lotnumbers of panel cells
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Phenotyping
In addition to the rule of three,antigen typingthe patient red cellscan also confirm an antibody
How is this done?
Only perform this if the patient has NOTbeen recently transfused (donor cells
could react) If reagent antisera (of the suspected
antibody) is added to the patient RBCs, anegativereaction should resultWhy?
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Remember Landsteiners Rule
Individuals DO NOTmake
allo-antibodies againstantigens they have
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Multiple antibodies
Multiple antibodies may be more ofa challenge than a single antibody
Why?
Reaction strengths can vary
Matching the pattern is difficult
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So what is a tech to do?
Several procedures can beperformed to identify multipleantibodies
Selected Cells
Neutralization
Chemical treatment
Proteolytic enzymes Sulfhydryl reagents
ZZAP
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Selected Cells
Selected cells are chosen from otherpanel or screening cells to confirmor eliminate the antibody
The cells are selected from otherpanels because of theircharacteristics
The number of selected cells
needed depends on how mayantibodies are identified
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Selected Cells
Every cell should be positive foreach of the antibodies and negativefor the remaining antibodies
For example:
Lets say you ran a panel and identified3 different antibodies: anti-S, anti-Jka,
and anti-P1 Selected cells could help
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Selected Cells
Selectedcells
S Jka P1 IS LISS37
AHG
#1 + 0 0 0 0 2+
#5 0 + 0 0 0 3+
#8 0 0 + 0 0 0
These results show that instead of 3 antibodies, thereare actually 2: anti-S and anti-Jka
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Neutralization
Some antibodies may be neutralizedas a way of confirmation
Commercial substances bind tothe antibodies in the patient serum,causing them to show no reactionwhen tested with the corresponding
antigen (in panel)
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Neutralization
Manufacturers directions should befollowed and a dilutional controlshould always be used
The control contains saline and serum(no substance) and should remainpositive
A control shows that a loss of reactivityis due to the neutralization and nottothe dilution of the antibody strengthwhen the substance is added
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Neutralization
Common substances P1substance(sometimes derived from hydatid
cyst fluid)
Leaand Lebsubstance(soluble antigen found
in plasma and saliva) Isubstancecan be found in breast milk
Sdasubstancederived from human or guineapig urine
**you should be aware that many of thesesubstances neutralize COLD antibodies; Coldantibodies can sometimes mask more clinicallysignificant antibodies (IgG), an important reasonto use neutralization techniques
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Enzymes (proteolytic)
Can be used to enhance or destroycertain blood group antigens
Several enzymes exist: Ficin (figs) Bromelin (pineapple)
Papain (papaya)
In addition, enzyme proceduresmay be One-step
Two-step
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Enzymes
Enzymes remove the sialic acidfrom the RBC membrane, thus
destroying it and allowing other
antigens to be enhanced
Antigens destroyed: M, N, S, s,Duffy
Antigens enhanced: Rh, Kidd,Lewis, I, and P
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Enzyme techniques
One-stage
Enzyme is added directly to theserum/cell mixture
Two-stage Panel cells are pre-treated with
enzyme, incubated and washed
Patient serum is added to panel cellsand tested
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Enzyme techniques
If there is no agglutination aftertreatment, then it is assumed theenzymes destroyed the antigen
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Enzymetreament
Anti-K
Perfect match for anti-Fya
Duffy antigens destroyed
Kell antigens not affected
Enzyme treatment
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Sulfhydryl Reagents
Cleave the disulfide bonds of IgMmolecules and help differentiatebetween IgM and IgG antibodies
Good to use when you have bothIgG and IgM antibodies (warm/cold)
Dithiothreitol (DTT)is a thiol and will
denature Kell antigens 2-mercaptoethanol (2-ME)
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ZZAP
A combination of proteolyticenzymes and DTT
Denatures Kell, M, N, S, Duffyandother less frequent blood groupantigens
Does not denature the Kx antigen
Good for adsorption techniques frees autoantibody off patients cell, so that
autoantibody can then be adsorbed onto anotherRBC
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Autoantibodies.
Warm & Cold Reacting
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Autoantibodies
Autoantibodies can be coldorwarmreacting
A positive autocontrol or DAT may
indicate that an auto-antibody ispresent
Sometimes the autocontrol may bepositive, but the antibody screening
may be negative, meaningsomething is coating the RBC
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Getting a positive DAT
The direct antiglobulin test(DAT)tests for the in vivocoatingof RBCs with antibody (in the body)
AHG is added to washed patient redcells to determine this
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What can the DAT tell us?
Although not always performed inroutine pretransfusion testing, apositiveDAT can offer valuable
information If the patient has been transfused, the
patient may have an alloantibodycoating the transfused cells
If the patient has NOT been transfused,the patient may have anautoantibodycoating their own cells
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Identifying autoantibodies
Auto-antibodies can sometimesmask clinically significant allo-antibodies, so its important to
differentiate between auto- andallo-antibodies
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Cold autoantibodies
React at room temperature withmost (if not all) of the panel cellsand give a positive autocontrol
The DAT is usually positive withanti-C3b AHG (detects complement)
Could be due to Mycoplasma
pneumoniae, infectious mono, orcold agglutinin disease
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Cold autoantibodies
Mini-cold panels can be used to helpidentify cold autoantibodies
Since anti-I is a commonautoantibody, cord blood cells (no Iantigen) are usually included
Group Oindividual with
cold autoanti-I
Group Aindividual with
cold autoanti-IH
Anti-IH is reacting weakly with the cordcells (some H antigen present)
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Avoiding reactivity
Cold autoantibodies can be anuisance at times. Here are a fewways to avoid a reaction:
Use anti-IgG AHG instead ofpolyspecific. Most cold antibodies reactwith polyspecific AHG and anti-C AHGbecause they fix complement
Skipping the IS phase avoids theattachment of cold autoantibodies tothe red cells
Use 22% BSA instead of LISS
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Other techniques
If the antibodies remain, thenprewarmed techniquescan beperformed:
Red cells, serum, and saline areincubated at 37before being combined
Autoadsorptionis anothertechnique in which the autoantibody
is removed from the patients serumusing their own red cells The serum can be used to identify any
underlying alloantibodies
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Warm autoantibodies
Morecommon that coldautoantibodies
Positive DAT due to IgG antibodiescoating the red cell
Again, the majorityof panel orscreening cells will be positive
The Rh system (e antigen) seems tobe the main target although othersoccur
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Warm autoantibodies
Cause warm autoimmune hemolyticanemia (WAIHA)H&H
How do you get a warm autoantibody?
Idiopathic Known disorder (SLE, RA, leukemias, UC,
pregnancy, infectious diseases, etc)
Medications
Several techniques are used whenwarm autoantibodies are suspected
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Elution (whenever DAT is positive)
Elution techniques freeantibodies from the sensitizedred cells so that the antibodies
can be identified
Y
Y
Y
YSensitized
RBC
Positive DAT
Elution
Y
Frees antibody Antibody ID
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Elution
The eluateis a term used for theremoved antibodies
Testing the eluate is useful ininvestigations of positive DATs
HDN Transfusion reactions Autoimmune disease
The red cells can also be used afterelution for RBC phenotyping if needed
When tested with panel cells, the eluateusually remains reactive with all cells if awarm autoantibody is present
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Elution Methods
Acid elutions (glycine acid)
Most common
Lowers pH, causing antibody to
dissociate
Organic solvents (ether, chloroform)
Dissolve bilipid layer of RBC
Heat (conformational change) Freeze-Thaw (lyses cells)
ABOantibodies
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Adsorption
Adsorption procedures can be usedto investigate underlyingalloantibodies
ZZAPor chloroquinediphosphate can be used todissociate IgG antibodies from theRBC (may take several repeats)
After the patient RBCs areincubated, the adsorbed serum istested with panel cells to ID thealloantibody (if present)
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Adsorption
Two types:
Autoadsorption
No recent transfusion
Autoantibodies are removed using patientRBCs, so alloantibodies can be identified
Allogenic (Differential) adsorption
If recently transfused
Uses other cells with the patients serum
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Wash x3 afterincubation
Centrifuge afterincubating; and
transfer serum to 2ndtube of treated cells;
incubate and
centrifuge again
2tubes
Removeserum and
test foralloantibody
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More reagents.
Many of elution tests can damagethe antigens on the RBC
Choroquine diphosphate(CDP)
and glycine acid EDTA reagents candissociate IgG from the RBC withoutdamaging the antigens
Very useful if the RBC needs to beantigen typed
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Chloroquine diphosphate
Quinilone derivative often used asan antimalarial
May not remove autoantibody
completely from DAT positive cells
Partial removal may be enough toantigen type the cells or to be used
for autoadsorption of warmautoantibodies
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THE END!!
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