Discuss/compare current testing methods/technologies used for antibody screening and identification. Provide an overview of modes of reactivity of cold reacting antibodies. Discuss techniques associated with cold autoantibody workups.

Current common methods:

Tube Testing: "Traditional method".

Tube testing Gel testing


Quick, less expensive. Most familiar method. Sensitivity of test system can be varied by choice of potentiators. Requires washing phase and precise agglutination reading techniques. More subjective vs. other methods, especially concerning microscopic readings. More difficult for new techs to master.


Solid Phase testing

Choice of antiglobulin reagent: Choice of antiglobulin reagent:

Polyspecific AHG: IgG + C3 component. May detect complement dependent antibodies (i.e. some Kidd system antibodies). Tends to enhance the reactivity of autoantibodies and clinically insignificant antibodies.

Monospecific IgG reagent: IgG only.

Usually preferred to limit detection of clinically insignificant antibodies. Issitt: monospecific AHG used in conjunction with a strong potentiator (LISS or PeG) can detect antibodies previously classified as complement dependent only. Gamma IgG lacks IgG 4 subclass component.


Choice of potentiator: Choice of potentiator:

No enhancement: perform incubation for at least 30 minutes. Albumin: Due to high protein content, more clinically insignificant antibodies may be detected.

LISS (Low Ionic Strength Solution)

Reduces incubation time to as short as 10 minutes. Increases sensitivity by reducing the zeta potential. May decrease sensitivity in the detection of Kell system antibodies. Cannot have any dilutional factor associated with LISS testing. Must retain proper serum to cell ratio as described in the package insert.

Choice of potentiator:

Choice of potentiator:

PeG (Polyethylene Glycol)

Enzymes (ficin, papain, bromelain, etc.)

Removes water molecules between cells, allowing for enhanced agglutination. Strongest of common potentiators. Not recommended when patient has a high protein count, may result in false negative results. 98% sensitivity, however specificity is lower. Enhances the detectability of autoantibodies and clinically insignificant antibodies. (Use of monospecific AHG reagent is preferred).

Removes sialic acid, thus reducing the net negative charge on RBCs. May enhance the detection of some antibodies. Rh, Kidd, ABO, autoantibodies Destroys some antigens: M,N,S, Duffy, Xga. Effect on s antigen may be variable.

Gel testing (ID-MTS, Ortho)


Principle: For aby screening and ID, patient's serum and 0.8% test cells are added to gel cards that have microtubes containing IgG. Cards are allowed to incubate at 37C to allow for sensitization to occur and then centrifuged. During centrifugation, if antibody coated cells are present they will react with the IgG and cause clumping in the microtubes.

Less subjective than tube testing. Cards can be covered and stored upright for another technologist to interpret if necessary. System can be automated. Easy to determine mixed cell populations when performing DAT testing. Easier interpretation, especially for generalists who rotate in blood bank.


Gel- advantages, cont.

Gel-advantages, cont.

Smaller sample volume required and test system volumes are measured and consistent. No cell washing phase. Easy to learn. Decreased detection of nuisance antibodies- weak cold autos and cold reacting allos.

If antibodies are fully IgM, antibody would not be expected to react using the gel technique (IgG card). Gel in conjunction with enzyme treated red cells is an excellent technique to detect weak Rh antibodies that may not be readily detectable using other methods.

Gel- Disadvantages


May not be able to resolve samples with strong cold autoantibodies. Cost (may be relative), consider staff productivity. May get false positives due to strong rouleaux. Highly recommended to use plasma vs. serum to help avoid false positives. May even need to centrifuge plasma to remove particulate matter. Selected cells: need to manually prepare 0.8% RBC suspensions. May be cumbersome.

Incubation and centrifugation times longer than performing PeG or LISS tube testing. May still have to use tube testing as a backup since strong rouleaux and moderate to strong cold autoantibodies cause interference. If you still have to retain tube testing as a backup, consider cost of retaining both systems or cost of sending more samples to a reference laboratory.

Solid Phase Testing (Immucor)

Solid Phase- Advantages

Principle: Microwells are coated with test RBC's (antigens). Incubate patient's serum and potentiator at 37C, followed by addition of indicator RBC's and then centrifugation. Positive results: Adherence of red cells to the microwell. Negative results: Red cells pellet to the bottom of the microwell.

Plates can easily be covered and stored and interpreted by another staff member. System can be automated. Highly sensitive. Requires small sample volume. Platelet testing kits available for crossmatching and/or platelet antibody screening.


Solid Phase-Disadvantages

Which method to use?

Should use plasma vs. serum. Serum is known to cause false positive results. Manual interpretation of red cell adherence in microwells is more subjective than gel testing. More costly than tube testing. Due to high sensitivity, may detect more clinically insignificant antibodies.

All 3 methods are good.

Compare variables within your own laboratory and choose the system that offers the most benefit.

Antibody ID Tips: Cold reacting antibodies

Antibody ID Tips: Alloantibodies typically cold reacting.

ABO system:

Anti-A1: Usually not clinically significant. Give crossmatch compatible or prewarm compatible units. Anti-H and Anti-IH: Usually are benign cold autoantibodies that react at colder temperatures. Anti-H is clinically significant in the rare Bombay phenotype and reacts at broad temperature ranges.

Lewis: Antibodies common in pregnancy. May see hemolysis at 37C when Anti-Lea is present. React more commonly at lower temperatures. When a Lewis antibody is present, it is not necessary to give antigen negative blood, give crossmatch compatible units.

Antibody ID Tips: Alloantibodies typically cold reacting.

Antibody ID Tips: Blood Group System Reactivity

Lewis, cont.

P blood group system:

Weak Lewis system antibodies may not be clear-cut due to variations in Lewis antigen expression on reagent red blood cells. Lewis neutralization procedures may be helpful, especially when suspected Lewis antibody is reacting variably.

Most common antibody is Anti-P1, reacts more commonly in lower temperatures. Antibody specificity may not be clear-cut since there is a wide variance concerning the P1 antigenic expression on panel cells. Use "reverse rule-out" technique. See if all positive reactions are with P1 positive cells, even though there may be negative reactions obtained with some P1+ cells. If this is the case, do not rule out Anti-P1 and investigate further.


Antibody ID Tips: Blood Group System Reactivity.

Antibody ID Tips: Blood Group System Reactivity

If Anti-P1 is suspected, P1 neutralization may be helpful. If Anti-P1 is present, give crossmatch compatible or prewarm compatible units. (P1 negative units not indicated). Ro cells from African-Americans tend to have the strongest P1 expression.

MNSs system Anti-M and Anti-N: Usually clinically insignificant. May be IgM, or less commonly IgG. Giving M or N negative units is not necessary. Give crossmatch compatible or prewarm compatible units. Antibodies commonly react at lower temperatures and commonly show dosage. Anti-N: Specificity may be seen with dialysis patients. Overall Anti-N is rare.

Antibody ID Tips: Blood Group System Reactivity

Antibody ID Tips

MNSs system Anti-M: when is clinical significance crucial?


Need to determine whether antibody is IgG or IgM. IgG antibodies can cross the placenta, IgM antibodies cannot.

If IgG vs. IgM differentiation is necessary for a pregnant patient, can differentiate by the use of the prewarm technique and treatment of serum/plasma with a thiol reagent (DTT or 2ME). (Non-reactive with prewarm, likely IgM). If Anti-M is not reactive using IgG gel card with cells with a homozygous expression of the M antigen, additional testing for differentiation would not be necessary; this would be consistent with an antibody of IgM composition only.

Antibody ID: Cold reactive antibody, no pattern...

What is good supporting evidence of a cold autoantibody?

Autoantibody Perform an autocontrol and/or DAT. If positive, consider a cold or warm autoantibody. Cold autoantibody: Cold screen shows reactions at 4C, IS, or RT with the screening cells and/or autocontrol. DAT may be positive due to complement. Interfering reactions usually abolished by the prewarm technique. May have to use RESt technique or cold auto/allo adsorptions for very strong cold autos.

Red Cross criteria for reporting of a cold auto: For reactivity reported as a cold autoantibody, confirmation will include an autocontrol reactive in at least one of the following phases: IS, RT, or 4C. And... At least one of the following must be met:

Serum reactivity is seen in at least one of the following phases: IS, RT, or 4C. DAT is reactive with Anti-C3. Autoadsorptions with autogeneic red blood cells at 4C will remove all autoantibody reactivity.


Prewarm technique

Prewarm technique

If there is sufficient evidence that a cold autoantibody is present and the antibody is interfering with the 37C/AGT phases of testing, it would be appropriate to proceed to the prewarm technique. The prewarm technique has been controversial (primarily due to misuse/inappropriate use).

If prewarm technique is attempted, it is recommended that you follow the procedure as outlined in the AABB Technical Manual. Other literature has suggested the use of potentiators (LISS, PeG) in conjunction with the prewarm technique. However, there have been reports of the loss of clinically significant alloantibodies when the prewarm is used in conjunction with a potentiator. Therefore, this aspect of the prewarm technique remains controversial and current evidence shows that use of potentiators in conjunction with the prewarm are not effective in enhancing the reactivity of underlying alloantibodies.


Serologic testing

Will not detect direct agglutinins or hemolysins. Newly formed IgM antibodies (whether in a clinically significant or clinically insignificant blood group) may not be detected. May not detect complement binding antibodies.

When performing serologic testing in the presence of cold-reactive autoantibodies, it is strongly recommended to use Anti-IgG rather than polyspecific antiglobulin serum.

Strong cold auto, prewarm does not abolish reactivity:

RESt Technique

If the patient has not been transfused within the past 3 months, the cold autoadsorption technique is the preferred method.

Patient's cells are treated with a proteolytic enzyme solution as to free up antibody binding sites and then subsequently used for adsorbing of the patient's serum/plasma at 4C. Cannot use this technique if patient has been recently transfused. Transfused cells may adsorb clinically significant alloantibodies.

May be used when strong cold autoantibodies are present and laboratory does not perform autoadsorptions.

RESt advantages: Can use when the patient has been recently transfused. RESt advantages: Kit is commercially available and easy to use. RESt adsorbs AntiI, Anti-IH, and Anti-H.


RESt Technique, cont.

Cold alloadsorption

RESt disadvantages

Do not use RESt adsorbed serum for crossmatching or ABO reverse testing, rabbit stroma can adsorb Anti-B. Per the package insert, performing RESt adsorptions can reduce the detectability of certain antibodies if the suspending diluent is not completely aspirated after centrifugation. Careful technique is very important.

Per AABB Technical Manual, it has been known that some examples of Anti-D and Anti-E have been removed by this method.

This is the most complex technique and usually is performed in reference laboratories. Can be used as an alternative to RESt when a patient has a potent cold autoantibody and has been recently transfused. Selection of allogeneic cells for adsorptions is critical; there must be multiple cell lines (usually R1, R2, and rr) in which the three cell lines (in combination with each other) are negative for K, Jka, Jkb, Fya, Fyb, S, and s so that autoantibody can be adsorbed and potential clinically significant alloantibodies can be identified in the adsorbed serum. If patient's full phenotype is known, a phenotypically similar allogeneic cell line may be used.

Use of the Autocontrol

Autocontrol, cont.

When evaluating cold autoantibodies, the results of the autocontrol are important. If an antibody is reacting at colder temperatures in conjunction with a negative autocontrol, proceed conservatively.

If SCs are positive at colder temperatures and AC is negative, consider the patient's blood type and with regards to H antigen expression.

If screening cells are positive and AC is negative, consider the possibility of a clinically significant alloantibody such as Anti-Vel, Anti-PP1Pk, or Anti-H in a Bombay individual. Be sure to evaluate for hemolysis at IS and/or 37C.

O>A2>B>A2B>A1>A1B = H antigen expression. Occasionally group A1, A1B, or (less commonly) type B persons may have so little converted H antigen on their red cells that they produce Anti-H. This Anti-H is not considered clinically significant, but this scenario would support the finding of a cold autoantibody where the antibody reacts with type O screening cells but not with the patient's autologous cells.

Autocontrol, cont.

Rarer cold autoantibody specificities

H expression:

If the patient has a blood type with higher H antigen expression, (type O, A2) and has a cold reacting antibody with a negative autocontrol, serious consideration should be given to the possibility of an antibody with potential clinical significance. (Most notably Anti-Vel and Anti-PP1Pk.)

If an antibody is reacting at colder temperatures, but not with enzyme treated cells: Possible specificity is Anti-Pr. Donath-Landsteiner antibody: IgG antibody (usually with anti-P specificity) that reacts with red cells in colder areas of the body and causes complement to bind to RBCs at cold temperatures. Complement dissociates from the red cells at warm temperatures and hemolysis occurs. (Antibody is referred to as a biphasic hemolysin and is associated with PCH).


PCH, cont.

Prewarm controversy, revisited

Serological picture in PCH:

DAT positive due to complement. Eluates are non-reactive. Autoantibody may agglutinate normal red cells at 4C, but rarely reacts above 4C. (Usually does not interfere with pretransfusion testing). As mentioned, the antibody of PCH is usually of P specificity, reacting with all red cells (including the patient's own red cells) except those of the very rare p or Pk phenotypes.

While studies have shown that there may be a decrease in antibody activity when the prewarm technique is used, it is still acknowledged by most that the prewarm technique is necessary when appropriate.

Inappropriate approach:

Appropriate Approach:

Blood bank technologist observes weak reactivity at the Coombs phase. The prewarm technique is utilized to see if the reactions "go away" without any evidence that a cold reacting antibody is present.

Weak reactivity is noted of undetermined specificity. Prior to proceeding to the prewarm technique, the technologist ensures that reactivity is noted at either 4C, IS, and/or RT with the screening cells and autocontrol. If this reactivity is proven, there can be confidence that usage of the prewarm technique is appropriate.

Known cold antibody, tips:

Cold autoantibody:tips

Ensure that your patient sample and reagents are at room temperature prior to testing. (Including RBC suspensions of donor units if crossmatches are to be performed.) If DAT is positive due to Anti-C3 and a cold autoantibody is present, use plasma vs. serum in conjunction with monospecific IgG antiglobulin reagent. These simple measures may allow for decreased interference and possible avoidance of the need for the prewarm technique vs. standard techniques.

Use of enhancement: the use of PeG with the prewarm technique is the most controversial due to reports of loss of clinically significant alloantibody activity. The use of LISS is less controversial. While the use of LISS can allow for a shortened incubation time, there is little evidence that the use of LISS will enhance the detectability of possible underlying alloantibodies.


Cold autoantibody tips: ABO typing

Cold antibody-reverse ABO typing

Forward typing discrepancy:

Cold alloantibody

Perform several washes with warm saline and repeat forward typing. For very strong interference, warm washes may not be effective. Can treat cells with a thiol reagent (0.01M DTT) to remove the bound IgM and repeat forward typing.

Use antigen negative A1 and B cells. If antigen negative A1 and B cells are not available, the prewarm technique could be utilized.

Do not perform reverse typing using prewarm if testing reverse cells at 37C is prohibited by manufacturer's insert.

Cold autoantibody-reverse ABO typing

ABO discrepancy:

Can perform prewarm if not prohibited by manufacturer's insert. If antibody has I or IH specificity, type A1 and B cord cells may be useful. Cold autoadsorbed or cold alloadsorbed serum.



Do not use RESt technique.



Regarding test methods: If moderate to strong cold autoantibodies are present, it is likely that gel methodology will not resolve interference. Tube testing is generally used as a back-up when interference due to cold autoantibodies is still evident using the gel prewarm technique.

Even though the prewarm technique is criticized, when used appropriately, it is helpful. Simply ensure that you have supporting evidence that it is likely that a cold autoantibody is the source of carryover to the 37C and/or Coombs phase of testing.


If you are unsure??

Consult/send to your facility's reference lab. We are happy to help!!




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