abdetection and identification

7/26/2019 Abdetection and Identification

1/94

Antibody Detection

Dr. Ali Ibrahim


2/94

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


3/94

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)


4/94

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)


5/94

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


6/94

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


7/94

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


8/94

Cell Antigram

Some manufacturerswill use + instead of

1 to indicate thepresence of theantigen

Screening Cells I &II

IS 37

AH

G

CC


9/94

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


10/94

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)


11/94

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


12/94

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


13/94

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


14/94

Autocontrol

The AC and DATcan help indetermining whether the antibodiesare directed against the patientscells or transfused cells(allo-orautoantibody)


15/94

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)


16/94

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)


17/94

Limitations

Very effective in detectingantibodies

If negative, then the crossmatchshould be compatible


18/94

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


19/94

Example 1

Screening

Cell

IS 37C AHG CC

I 0 0 0

II 0 0 2+ ND

NotDone

IgG antibody

Singlespecificity


20/94

Example 2

Screening

Cell

IS 37C AHG CC

I 0 0 3+

II 0 2+ 3+

IgG antibody

Multiple specificities


21/94

Example 3

Screening

Cell

IS 37C AHG CC

I 1+ 0 0

II 3+ 0 0

IgM antibody

Single specificity showingdosage

Neg AHG, addCC


22/94

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)


23/94

Antibody Identification


24/94

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


25/94

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


26/94

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


27/94

Antibody Panel vs. Screen

An antibody panel is just anextended version of an antibodyscreen

The screen only uses 2-3 cells:


28/94

Antibody Panel

An antibody panel usually includesat least 10 panel cells:


29/94

Panel

These are Group O red blood cells


30/94

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


31/94

Panel

An autocontrol should also be runwith ALL panels

Autocontrol

Patient RBCs+

Patient serum


32/94

Panel

The same phases used in anantibody screen are used in a panel

IS

37

AHG


33/94

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


34/94

IS Phase

Perform immediate spin (IS) andgrade agglutination; inspect forhemolysis

Record the results in theappropriate space as shown:

2

0

0

Last

tube


35/94

(LISS) 37C Phase

2 drops of LISS are added, mixedand incubated for 10-15 minutes

Centrifuge and check foragglutination

Record results


36/94


37/94

(LISS) 37C Phase

2

0

0

2

0

0

2

0

0

2

0

0

0

0


38/94

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


39/94

AHG Phase

Wash cells 3 times with saline(manual or automated)

Add 2 drops of AHG and gently mix Centrifuge

Read

Record reactions


40/94

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


41/94

And dont forget.

.add check cells to

any negative AHG !


42/94

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


43/94

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


44/94

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


45/94

An antibody will only reactwith cells that havethe

corresponding antigen;antibodies will not reactwith

cells that do not havethe

antigen

Always remember:


46/94

Heres an example:


47/94

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.


48/94

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


49/94

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


50/94

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


51/94

Interpretation

anti-Lea


52/94

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


53/94

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)


54/94

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


55/94

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


56/94

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


57/94

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


58/94

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?


59/94

Remember Landsteiners Rule

Individuals DO NOTmake

allo-antibodies againstantigens they have


60/94

Multiple antibodies

Multiple antibodies may be more ofa challenge than a single antibody

Why?

Reaction strengths can vary

Matching the pattern is difficult


61/94

So what is a tech to do?

Several procedures can beperformed to identify multipleantibodies

Selected Cells

Neutralization

Chemical treatment

Proteolytic enzymes Sulfhydryl reagents

ZZAP


62/94

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


63/94

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


64/94

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


65/94

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)


66/94

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


67/94

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


68/94

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


69/94

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


70/94

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


71/94

Enzyme techniques

If there is no agglutination aftertreatment, then it is assumed theenzymes destroyed the antigen


72/94

Enzymetreament

Anti-K

Perfect match for anti-Fya

Duffy antigens destroyed

Kell antigens not affected

Enzyme treatment


73/94

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)


74/94

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


75/94

Autoantibodies.

Warm & Cold Reacting


76/94

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


77/94

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


78/94

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


79/94

Identifying autoantibodies

Auto-antibodies can sometimesmask clinically significant allo-antibodies, so its important to

differentiate between auto- andallo-antibodies


80/94

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


81/94

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)


82/94

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


83/94

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


84/94

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


85/94

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


86/94

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


87/94

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


88/94

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


89/94

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)


90/94

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


91/94

Wash x3 afterincubation

Centrifuge afterincubating; and

transfer serum to 2ndtube of treated cells;

incubate and

centrifuge again

2tubes

Removeserum and

test foralloantibody


92/94

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


93/94

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


94/94

THE END!!

abdetection and identification

Documents