Unit 10 Identification of

Unexpected Antibodies

Terry Kotrla, MS, MT(ASCP)BB

Introduction Unexpected alloantibodies are antibodies

other than anti-A or –B. Found in 0.3-2% of population. Immunization to RBC antigens may result

from: Pregnancy Transfusion Deliberate injection of immunogenic material Event unknown

Introduction Once antibody detected must determine

specificity and clinical significance. Clinically significant antibody will

Shorten survival of antigen positive RBCs. Cause HDFN if fetus inherits antigen.

Clinical significance varies RBC destruction may occur within minutes. RBC survival may decrease by only a few days. Use documented experience with other

examples of antibodies with same specificity.

Introduction May be no data for some antibodies Consider that it is not clinically significant

if no reactivity at 37C or IAT Clinical significance important in donor

blood selection and prenatal testing. Must select antigen negative blood if clinically

significant. During pregnancy may predict likelihood of

HDFN.

General Procedures Need adequate quantity of serum/plasma

and RBCs to resolve. EDTA RBCs preferred for study of RBCs. Medical History CRUCIAL and include:

Number of pregnancies Transfusion history, especially if recent Drug therapy

General Procedures Test serum/plasma at all phases of activity

initially detected. Additional antibodies may be detected at

different phases. Reactivity may be increased by:

Extending incubation time Lowering temperature if cold antibody suspected Increase serum:cell ratio Use of enzyme treated cells.

General Procedures - Enzymes

Enhance reactivity of Rh, P, I, Le and Jk antibodies.

Denatures M, N, S and Fy antigens, if multiple antibodies present and one has MNS or Fy

specificity, aids in identification. Reactivity of antibodies to M, N, S or Fy will go away

or weaken tremendously. Allows underlying antibody activity to be detected.

General Procedures Serum/plasma tested against panel of 8 or more

Group O RBCs of KNOWN phenotype. Must be able to identify WITH CONFIDENCE most

frequently encountered, clinically significant alloantibodies.

Distinct pattern should be apparent for single alloantibodies.

Must be sufficient RBCs that lack and carry antigens to which antibodies most frequently made- usually three of each.

Autocontrol MUST be run

Considerations in Interpreting Serological Results

Alloantibodies of some blood group specificities FREQUENTLY display consistent serologic characteristics: Evaluate effects of temperature, suspending

medium or enzymes with sample. Variation in strength of reactivity? Hemolysis present? Is autocontrol positive or negative?

Single Alloantibody Usually easy to recognize, clear cut pattern of

reactions. Test phase suggestive of specificity BUT

exceptional samples will be encountered IS/RT think anti-P1, Le, M, N, I or Lua

37C or AHG think anti-Rh, Jk, K, Fy or Lub

Varying strength of reactivity within 1 phase may be due to: Antibody showing dosage Multiple antibodies present

ALWAYS look for additional antibodies

Special Considerations Rh Antibodies If anti-E identified look for anti-c

Determine Rh phenotype of patient. If patient is E and c negative (R1R1) transfuse

with R1R1 even if anti-c NOT detectable Anti-c common cause of delayed HTR in

R1R1 patients with anti-E Reverse not a problem, almost all c

negative donors will be E negative.

Phenotype Autologous Cells After identification of alloantibody type

patient for corresponding antigen. If interpretation correct patient will be

NEGATIVE. Provides additional confirmation of specificity If patient is positive indicates misinterpretation

of results. CANNOT antigen type recently transfused

patient, use pretransfusion sample or perform cell separation.

Probability of Identification Three RBCs antigen positive that react

with antibody. Three RBCs antigen negative that DO

NOT react with antibody.

Multiple Antibodies May be very challenging. Clues

Pattern of reactive and non-reactive RBCs do not fit single antibody specificity.

Variable strength of reactions. Different RBCs react at different test phases. Antigen negative donor incompatible with

patient sample.

Antibody to High Frequency Antigen Very difficult to work up. Most, if not all, RBCs on panel will be

antigen positive. Must find additional negative RBCs to rule

out “hidden” or “masked” antibodies. Selected Cell Panel

There is only one e negative RBC on panel Must find additional e negative to rule out

antibodies not ruled on with negative RBC. Technique also used for multiple antibodies.

Antibodies to High Incidence Antigen Suspected when ALL reagent RBCs are

positive BUT autocontrol is negative. Rule out multiple antibodies. Will most likely have to send to reference

laboratory which possess rare cells. Patient’s siblings most likely source of

antigen negative blood.

Antibodies to Low Incidence Antigen Serum/plasma has negative screen but

ONE donor incompatible. Check for ABO incompatibility if reaction

at IS only. Perform DAT on donor if reaction at AHG

If positive, not the problem. If negative most likely alloantibody.

Perform a panel, be sure to test cells positive for low frequency antigens.

Inappropriate to delay transfusion.

Anomalous Serological Reactions Antibodies to drugs or additives may be

present. If drug induced autocontrol will be positive. If additive suspected try different additive.

Some times nothing can be done to increase strength of reactivity.

Interpretation, “Unable to determine antibody specificity”.

Transfuse with serologically compatible blood.

Selected Serological Procedures When pattern fails to indicate specificity other

procedures may be performed: Enzyme Techniques Temperature reduction Increase serum to cell ratio Increase incubation time Decrease pH Change enhancement media Use of thiol reagents Prewarmed technique Inhibition tests Titration Adsorption Elution

Enzyme Techniques Treatment of RBCs with proteolytic

enzymes ENHANCES reactivity of Rh, P, I, Lewis and complement binding alloantibodies such as anti-Jka.

Antigens of M, N, S and Fy are depressed or destroyed, antibody CANNOT react.

Enzyme techniques used whenever weakly reactive antibody may be Rh antibody OR when a patient has multiple antibodies and one of them is a Fy.

Temperature Reduction Alloantibodies (e.g., anti-M, -P1) that react better

at cold temperatures. Specificity may become apparent at or below 22

C. Auto-control especially important for tests at cold

temperatures, because many sera contain cold reactive auto-antibodies.

Anti-I specificity confirmed by testing patient serum with adult (I+) and cord (i+) cells at 4 C. Positives with adult cells and negative with cord cells

confirms anti-I specificity.

Increase Serum/Plasma to Cell Ratio Increases amount of antibody in test

system which may increase the strength of reactivity of antibodies present in low concentrations.

Increase to 4 or more drops. May have to add additional washes. NOTE: LISS requires EQUAL volume of

serum and RBCs

Increase Incubation Time Use 30-60 minutes to allow more time for

more antibody to attach to RBCs. NOTE: DO NOT exceed incubation time

recommended by enhancement media manufacturer.

Decrease pH Decrease pH to 6.5 by addition of 0.2 N

HCl. Weak anti-M GREATLY enhanced.

Change Enhancement Media Some blood group antibodies react

preferentially in test systems utilizing Low Ionic Strength Salt (LISS) solutions.

A variety of LISS procedures have been described.

PEG

Thiol Reagents Strong IgM class antibodies may have high

thermal amplitude and may mask other antibodies.

Two reagents - dithiothreitol (DTT) and 2-mercaptoethanol (2-ME)

Cleave inter-subunit disulfide bonds of IgM molecules.

The IgG molecules relatively resistant to such cleavage, treatment results in destruction of IgM but leaves IgG intact able to react in-vitro.

Applications of DTT and 2-ME Determining the immunoglobulin class of

an antibody, especially if the antibody has potential to cause HFDN.

Dissociating RBC agglutinates caused by IgM antibodies (e.g., spontaneous agglutination of RBCs caused by potent cold reactive auto-antibodies).

Identifying specificities in a mixture of IgM and IgG antibodies, particularly when an agglutinating IgM antibody masks the presence of IgG antibodies.

Prewarmed Technique Cold auto-agglutinins may demonstrate

high thermal amplitude resulting in false positive reactions at 37C and AHG.

Can Confirm that only a cold auto-agglutinin is

present or Detect the presence of clinically significant

underlying alloantibodies.

Prewarmed Technique Place RBCs to be tested in appropriate test tubes

and place in 37C heat block. Prewarm tube of serum to 37C. Add 3 to 4 drops of prewarmed serum to

prewarmed cells and incubate 1 hour. Without removing tubes from heat block add

saline that has been prewarmed to 37C. Immediately spin and wash 2 additional times

with the warm saline. Perform the AHG procedure.

Inhibition Tests Some blood group antigens exist in soluble form

in such body fluids as saliva, urine or plasma. These substances useful in antibody identification

studies, either to confirm antibody specificity by inhibition or to neutralize antibodies that mask the presence of concomitant non-neutralizable antibodies.

The following soluble blood group substances can be used in antibody identification tests. Lewis P1

Sda

Lewis Inhibition Tests Lea and Leb substances present in saliva of

persons with appropriate Lewis phenotype Lewis substance can be prepared from saliva. Most blood banks use commercially prepared

Lewis substance. Lewis substance will neutralize Lewis

antibodies in a patient specimen, allowing the detection of underlying, clinically significant alloantibodies.

P1 Inhibition Tests Soluble P1 substance is present in hydatid

cyst fluid as well as pigeon eggs. Anti-P1 may mask the presence of

underlying alloantibodies. Addition of P1 substance to the patient's

serum causes neutralization of the anti-P1, allowing the detection of underlying alloantibodies

Sda Inhibition Sda blood group substance is present in

soluble form in various body fluids, with the most abundant source being urine.

Urine from Sda positive individual added to patient serum, causing neutralization of the anti-Sda.

Aids in the detection of underlying alloantibodies.

Titration Titer of an antibody usually determined by

testing serial two-fold dilutions of the serum against selected RBC samples.

Results expressed as reciprocal of highest serum dilution causing macroscopic agglutination.

Three applications: Prenatal Studies Antibody identification HTLA antibodies

Titration – Prenatal Studies If antibody is of a specificity known to

cause HDFN OR when the clinical significance of the antibody is unknown, results of titration studies and outcome of previous pregnancies are used to assess the need for amniocentesis.

Rising titers are indicative of active immunization of the mother.

Titration – Antibody Identification Some antibodies cause agglutination of

virtually all reagent RBC samples, but specificity is indicated by differences in the strength of reactivity with each sample in titration studies.

Titration procedure is not used very commonly for this purpose.

Titration - HTLA HTLA antibodies react very weakly in

undiluted state but, unlike most weakly reactive antibodies (e.g., anti-D with a titer of 4), react at a high dilutions (e.g., 1 in 2000).

Such antibodies include anti-Ch, -Rg, -Cs, -Yk, -Kna, -McCa and -JMH.

When weak reactions are observed in the IAT, titration studies may be used to establish whether or not the reactions are due to the presence of an HTLA antibody.

Adsorption Antibody can be removed from a serum by

adsorption to RBCs carrying the corresponding antigen. Incubate serum with antigen positive cell. Antibody forms complex with RBC membrane-

bound antigens. When the serum and RBCs are separated, the

antibody remains attached to the RBCs. Subsequent elution of the bound antibody can

often give additional useful information.

Use of Adsorption Techniques Removing auto-antibody activity to permit

detection of coexisting alloantibodies. Removing unwanted antibody from serum that

contains an antibody suitable for reagent use. Confirming the presence of antigens on RBCs

through their ability to remove a specific serum antibody.

Confirming the specificity of an antibody by showing that it can be absorbed only to RBCs of a particular blood group phenotype.

Separating multiple antibodies present in a single serum sample.

Elution Elution techniques free antibody

molecules from sensitized RBCs so the recovered antibody can be tested.

A variety of methods are employed with the primary objective being breaking the bond between the antigen and the antibody.

Use of Elution Techniques Identification of an antibody coating a baby's

RBCs in the case of HDFN. Identification of an antibody causing an acute or

delayed hemolytic transfusion reaction. Investigation of a positive DAT. Concentration and purification of antibodies, the

detection of weakly expressed antigens and the identification of multiple antibodies.

Preparation of antibody-free intact RBCs for use in phenotyping or autoabsorption.

Elution Technique – Technical Factors Incorrect technique. Incomplete washing

If cells are incompletely washed contaminating serum antibody will cause false positive reactions.

An aliquot of saline from the last wash is saved and tested in parallel. Positive reactions with the last wash invalidates the test.

Binding of proteins to glass surfaces. Dissociation of antibody before elution. Instability of eluates.

Summary If an unexpected antibody is detected in a

patient’s serum or plasma it must be identified.

Once identified the clinical significance must be determined.

Summary If the antibody is clinically significant

antigen negative donors must be found and crossmatched for the patient, a Coomb’s crossmatch must be done.

If the antibody is not clinically significant it is not necessary to provide antigen negative blood, but the donors must be compatible by the Coomb’s crossmatch.

ReferencesAABB Technical Manual, 16th

edition, 2008Basic and Applied Concepts of

Immunohematology, 2nd edition, 2008