Immunoheme Chapter 9

what are the limitations of the antibody screen?

- Antibody titer may be below the level of sensitivity - Low-prevalence antigens and antibodies may be present - Lack of homozygous expression of the target antigen on screening cells

What are the clinical applications of antibody titration?

- Hemolytic disease of the newborn (HDN) - To differentiate immune anti-D from passively acquired anti-D to RhIG adminstration (for Rh-neg mother pregnant with Rh-pos baby) - To confirm the presence of antibodies previously known as HTLA (high titer, low avidity)

What factors influence sensitivity of an antibody screen?

- Serum to cell ratio - Temperature and phase of reactivity - Length of incubation and pH

How could you avoid cold autoantibodies?

- Use monospecific anti-IgG - Skip IS phase on the antibody screen *after a cold antibody has been identified you can then - use 22% albumin instead of LISS (remember that LISS will enhance cold autoantibodies) - Perform prewarm technique - Perform adsorption techniques

What are antibody screens performed? (4)

1. Crossmatch recipients 2. Prenatal workup 3. Blood donors 4. Transfusion reaction workup

A technologist has decided to test an enzyme-treated panel of RBCs against a patient's serum. Which of the following antibody pairs could be separated using this technique? A. Anti-Jk^a and anti-Jk^b B. Anti-S and anti-Fy^a C. Anti-D and anti-C D. Anti-Jk^a and anti-Fy^a

D. Anti-Jk^a and anti-Fy^a *Duffy (Fy^a), MNS are INACTIVATED by enzymes. While, Kidd (Jk^a and Jk^b) and Rh are ENHANCED. Therefore, using Jk^a (to be enhanced) and Fy^a (to be inactivated) will allow for separation of the two.

What antibodies are IgG that will react at AHG?

Duffy, Kidd, and Kell antibodies

What are the 3 different type of elution techniques?

- Acid elution - Lui freeze thaw elution - Heat elution

When is a antibody titer considered significant?

When there is a fourfold or greater increase, or if there is an increase in score of 10 or more

How can you calculate the probability of finding compatible blood? What is the equation?

# of units needed/Incidence of phenotype = # of units to type and screen

The crossmatch has many limitations. A compatible crossmatch will NOT: (What 6 things?)

1. Guarantee normal survival of transfused RBCs; 2. Prevent immunization of the recipient; 3. Detect all unexpected red blood cell antibodies in the recipient serum; 4. Prevent delayed hemolysis due to an anamnestic antibody response to antigens against which the patient has previous but undetectable immunization; 5. Detect all ABO grouping errors either in donor or recipient; 6. Detect most D grouping errors in the donor or recipient.

List methods that may be used when working with a multiple- or high-frequency antibody or antibodies. (4 things)

1. Test additional cells from a different panel. The cells selected for testing should have minimal overlap in the antigens they possess 2. Enzyme treatment: enzymes modify the RBC surface by removing sialic acid residues and by denaturing or removing glycoproteins. the effect is to destroy certain antigens, thereby enhancing the expression of others. A sensitive method is to treat the panel RBCs first and then perform the antibody identification panel. 3. Neutralization: neutralization of antibodies in serum allows for separation of antibodies or confirmation that a particular antibody is present. Patient's serum is incubated with neutralizing substance, allowing the soluble antigens in that substance to bind with the antibody and prevent it from reacting when introduced to the panel RBCs during antibody identification. 4. Adsorption: Antibodies are removed from serum by adding the target antigen and allowing the antibody to bind to the antigen. The antigen-antibody complex is then removed by centrifugation. Used for the detection of unabsorbed alloantibodies.

How could you enhance weak IgG antibodies?

1. Use a different enhancement media (PEG) 2. Increase serum to cell ratio (normally 2:1) 3. Increase incubation time

Panel cells come in a set of ____ or ____

10 or 20

What kind of panel can be run to destroy MNS and Duffy blood group antigens, and enhance Rh, Kidd, and Lewis antigens?

A ficin panel *This is an enzyme that will result in those occurrences.

What is an antigram?

A profile of antigen typings of each donor in the manufacture of commercially supplied screening and panel cells. An antigram lists the antigens present in the reagent red cell suspension.

What is Elution?

A technique used to remove sensitized IgG from red blood cells. The antibody is recovered in a solution for further testing (re-tested at AHG using panel cells)

an antibody demonstrates weak reactivity at the AHG phase when using a tube method.with no enhancement reagent and monospecific anti-IgG AHG reagent. when repeating the test, which of the following actions may increase the strength of the positive reactions? A. Adding an enhancement reagent, such as LISS or PEG B. Decreasing the incubation time form 30 minutes to 10 minutes C. Employing the prewarm technique D. Decreasing the incubation temperature to 18C

A. Adding an enhancement reagent, such as LISS or PEG

Antibodies are excluded using RBCs that are homozygous for the corresponding antigen because: A. Antibodies may show dosage B. Multiple antibodies may be present C. It results in a P value of .05 for proper identification of the antibody D. All of the above

A. Antibodies may show dosage

What is the difference between alloantibodies and autoantibodies definition-wise?

Alloantibodies are antibodies formed in response to transfusion or pregancy. Also called atypical/unexpected antibodies. Autoantibodies are antibodies made to one's own red blood cells

How can you differentiate an alloantibody from an autoantibody? Give me an example screen and DAT

Alloantibodies will be positive for the antibody screen, but negative for the DAT. This is because a DAT detects antibodies that are attached to the patient's OWN RBCs. Alloantibodies are formed in response to pregnancy, transfusion, or transplantation targeted against a blood group antigen that is not present on the person's red blood cells.

What is an alloantibody?

An antibody formed in response to pregnancy, transfusion, or transplantation targeted against a blood group antigen that is not present on the person's red blood cells.

What is an unexpected/atypical antibody?

An antibody other than anti-A, anti-B, or anti-A,B. All other antibodies directed against RBC antigens are considered unexpected and must be detected and identified before blood can be safely transfused, no matter their reaction strength of profile. Alloantibodies are considered to be atypical/unexpected antibodies

What is a high-frequency antigen?

An antigen present in >98% of the population

What antibodies are Igm antibodies that are likely to be reactive at IS? (5)

Anti-Le, anti-M, anti-N, anti-I, and anti-P (Think P-Le-MIN)

What are some examples of HTLA antibodies (antibodies against high frequency antigens)?

Anti-Yt^a Anti-Ch^a Anti-Rg^a

What are HTLA antibodies?

Antibodies to high frequency antigens (such as k, kp^b, Js^b, and Lu^b) that react weakly (1+) with panel cells but have a high titer (>64)

What is a low frequency antigen?

Antigens that have a frequency UNDER 10% of peoples

What is the purpose of an autocontrol?

Autocontrols can be used when a technologist wants to omit immediate spin and RT phases to limit detection of insignificant cold antibodies. Autocontrol should be included when resolving reverse typing discrepancies that may be caused by autoantibodies.

The physician has requested 2 units of RBCs for patient DB, who has two antibodies, anti-L and anti-Q. The frequency of antigen L is 45%, and the frequency of antigen Q is 70% in the donor population. Approximately how many units will need to be antigen-typed for L and Q to fill the request? A. 8 B. 12 C. 2 D. 7

B. 12

Which of the following methods may be employed to remove IgG antibodies that are coating a patient's red blood cells? A. Adsorption B. Elution C. Neutralization D. Titration

B. Elution *Elution is good for dissociating antibodies from the RBC surface to allow for identification (specifically those autoantibodies that are IgG identified via positive monocloncal AHG DAT). *Adsorption is good for removing antibodies in the serum *Neutralization is good for neutralizing antibodies in serum *Titration is good for quantification of the identified antibody

Why would you not want to perform IS if you are using LISS as an enhancer?

Because LISS enhances cold autoantibodies

Why is washing important when performing elution?

Because washing removes unbound antibodies. If those antibodies are allowed to remain in the test system, they would contaminate the final eluate and yield false-positive results. As a control, wash supernatant should be tested in parallel with the eluate to detect the presence of unbound antibody. The last wash should be nonreactive, or the eluate results will be invalid

A request for 8 units of packed RBCs was received for patient LF. The patient has a negative antibody screen, but one of the 8 units was 3+ incompatible at the AHG phase. Which of the following antibodies may be the cause? A. Anti-K B. Anti-Le^a C. Anti-Kp^a D. Anti-Fy^b

C. Anti-Kp^a

Anti-Sd^a has been identified in patient ALF. What substance would neutralize this antibody and allow detection of other alloantibodies? A. Saliva B. Hydatid cyst fluid C. Urine D. Human breast milk

C. Urine

Patient JM appears to have a warm autoantibody. She was transfused 2 weeks ago. what would be the next step performed to identify any alloantibodies that might be in her serum? A. Acid elution B. Warm autoadsorption using autologous cells C. Warm differential adsorption D. RESt^TM adsorption

C. Warm differential adsorption *When phenotyping the patient is difficult because of a positive DAT or recent transfusion, differential absorption may be performed. For this method, the patient's serum sample is divided into a minimum of 3 aliquots. Each aliquot is adsorbed using a different cell. One must be negative for K, another neg for Jk^a, and the third negative for Jk^b. The cells are treated with enzyme to render them negative for antigens of the Duffy and MNS systems. Following adsorption, antibody identification panels are performed separately on each aliquot, and the reactivities are compared to reveal underlying alloantibodies. *Autoadsorption would not work because those work on autoantibodies *Elution is used for positive IgG autoantibodies (found via DAT) *RESt^TM adsorption is good for cold autoantibodies.

Give me 2 examples of low frequency antigens

C^w and Lu^a

How are unexpected antibodies found?

Clinically significant antibodies are typically IgG antibodies that react at 37C or that react in the antihuman globulin (AHG) phase of the indirect antiglobulin test. Autoantibodies can be detected via autocontrol. IgM/cold antibodies can typically be detected during the IS phase

What can be performed if a anti-I or anti-i are suspected and you want to be sure?

Either perform autoadsorption or a mino cold panel. Mini cold panel is a panel of selected cells to help identify a suspected cold autoantibody. Adult group O cells (containing I antigen), and cord O cells (containing i antigen) are tested with patient plasma. if patient has anti-I, he will react against adult O cells, if has anti-i, he will react against cord cells, and if he has anti-IH, he will react against all cells.

T/F Autoadsorption technqiue should be the first to use to get rid of interfering cold autoantibodies

F If the cold autoantibody still persists after implementing the other techniques (prewarming, skip IS, using 22% albumin instead of LISS, and using monospecific anti-IgG), then you can perform an autoadsorption technique

How can you differentiate an alloantibody from an autoantibody?

If the antibody is an alloantibody, it will show positive reaction in antibody screen but negative reaction for DAT. If it is an autoantibody, it will show positive reaction for both the screen and DAT

How would you know if a person has autoantibodies?

If their autocontrol is positive *Remember that autoantibodies can be either warm or cold

How can you differentiate an IgM and IgG antibody for both alloantibodies and autoantibodies?

IgM alloantibody: IS has positive reaction, but no other phases do. DAT is negative. IgM autoantibody: IS has positive reaction, and so does Poly and C3. IgG alloantibody: positive reactions for AHG and maybe 37C. DAT is negative. IgG autoantibody: AHG is positive, DAT is positive for Poly and IgG.

What is the purpose of antibody titration and how is it performed?

It quantifies the amount of antibody present. Determine the titer level via twofold serial dilutions of serum tested against a suspension of RBCs possessing the target antigen.

What blood group system antibodies are enhanced by enzymes?

Lewis, Kidd, and Rh

What blood group system antibodies are destroyed by enzymes?

MNS and Duffy

How could anti-P1, anti-Le^a, and anti-Le^b be neutralized?

P1 subtance can be added to neutralize anti-P1, while Lewis substance can be added to neutralize anti-Le^a and anti-Le^b

What potentiator is used to enhance weak antibodies?

PEG

If the AC is positive, what is the next step?

Perform DAT to determine if antibodies are auto or allo

If the screen does not meet the rule of 3 (wherein 3 antigen positive cells show agglutination, 3 antigen negative cells show no agglutination, and there is therefore a 95% confidence level), what do you do next?

Perform a selected cell panel

How would you get rid of warm autoantibody interference?

Perform a warm autoadsorption using patient RBCs to remove warn autoantibodies that may be masking clinically significant alloantibodies

Once the antibody has been identified, what is done next?

Phenotyping of the patient for the corresponding antigen using manufactured antisera. *This keeps in mind Landsteiner's rule. The reaction should ALWAYS be negative. (Because the person should not be producing antibodies against an antigen that they have)

Albumin enhances what type of antibodies?

Rh-Antibodies

Explain why patient info regarding history, age, race, and diagnosis helps in the process of antibody identification.

Some diseases are associated with the development of certain antibodies. For ex, a patient with systemic lupus erythematosus or carcinoma is frequently associated with warm autoantibody, whereas pneumoniae may result in a cold autoimmune process. Patients with sickle cell disease can have both multiple alloantibodies and autoantibodies. Generally, patients younger than 50 years old or healthy blood donors do not possess autoantibodies. Some antibodies are associated only with certain races because of the frequency of antigens in certain populations. For example, anti-U is more frequently associated with persons of African descent because most U-negative individuals are found in this population.

T/F Cold alloantibodies are IgM antibodies that react best at IS. They are not clinically significant if they are non-reactive at 37C

T

T/F During massive transfusion, if the patient is known to have clinically significant unexpected antibody, all infused units should be tested for and lack the corresponding antigen, if time permits. The antibody in the patient's serum may not be demonstrable because of dilution with large volumes of plasma and other fluids. however, a rapid rise in antibody titer level and subsequent destruction of donor RBCs may occur if antigen-positive units are infused.

T

T/F Even if the person has the antibody in their system, you should provide the person with blood that adheres to that antibody - NOT to the presence of the antigen on the patient RBCs. ex: warm autoantibody anti-e is in patient serum and patient has e antigen on RBCs. You would transfuse this patient with blood from e-negative person.

T

T/F Selected cells are often used to complete the Rule of 3 and are selected to run an IAT without doing another entire panel. These are cells chosen from another panel to confirm or eliminate the possibility of an antibody

T

T/F Adsorption technique cannot be performed if the patient has been transfused within the last 3 months

T *In this case, you would perform allogenic adsorption or use stroma for adsorption

T/F You would want to rule out antigens found on homozygous cells that had a negative reaction

T *Some antigens may not have had a strong enough strength due to their being expressed on heterozygous cells.

T/F Antibodies to low frequency antigens may not be detected during routine antibody screen but may be suspected if the crossmatch results are incompatible

T *To find compatible blood, select another random unit for crossmatching.

T/F Alloantibodies and autoantibodies can be either IgM or IgG

T Any antibodies can be IgM or IgG - its where the reaction occurs (IS, 37C, AHG) that can lead you to detect whether it is IgM and IgG. Alloantibodies will be positive for antibody screen but negative for DAT. Autoantibodies will be positive for both

Describe the reagent red blood cell panel

The antibody screen involves incubating the patient's serum or plasma with screening cells at 37C and performing an indirect antigloblin test (IAT) for the detection of IgG antibodies. Antibody screening cells are grouped "O" reagent red cells. These cells are tested with the patient's serum to determine whether an unexpected antibody exists. A reaction to one or both of the screen cells demonstrates the presence of an atypical antibody.

he current AABB standards state that tests to detect ABO incompatibility are sufficient if... (what 2 things?)

The current AABB standards state that tests to detect ABO incompatibility are sufficient if no clinically significant antibodies were detected in the antibody screening process and if no historical record exits of clinically significant unexpected antibodies being detected.

What is reaction strength indicative of?

The number of antibodies present. Single antibody reacts in a similar strength EXCEPT when demonstrating dosage effect. Multiple antibodies may be suspected if varying reaction strengths are observed.

If the autocontrol is positive, what does this indicate?

The presence of autoantibodies

How does reaction strength contribute to antibody resolution?

The strength of the reaction does not indicate the significance of the antibody, only the amount of antibody available to participate in the reaction. A stronger reaction may be due to dosage (cells with homozygous antigen expression). Different reaction strengths could also indicate the presence of more than one antibody. A cell that possesses more than one of the target antigens may react more strongly than a cell possessing only one of the target antigens. A third possibility is an antigen with variable expression. I, P1, Le^a, Le^b, Vel, Ch/Rg, and Sd^a antigens are expressed more strongly on some RBCs than on others; antibodies to these antigens may react more strongly with one panel cell than another.

How could you identify HTLA antibodies (antibodies to high frequency antigens)?

They would have weak reactions at AHG phase (1+) They are NOT enhanced by potentiators They are NOT clinically significant

When would you use an O cell control?

When you are resolving reverse typing discrepancies that may be caused by alloantibodies. Antibody screening cells are group O, so performing an antibody screen is the same thing as completing an O control. O control = patient plasma + reagent O cells

What is the purpose of adsorption?

To absorb the autoantibody in order to identify any clinically significant alloantibodies

What is the purpose of the antibody screen?

To detect clinically significant antibodies in both the blood donor and the intended recipient as part of pretransfusion compatibility testing. The screen will not detect antibodies when the antibody titer has dropped below the level of sensitivity for the screening method employed.

What are potentiators used for?

To increase the speed/sensitivity of the antibody-antigen reaction

What is the rule of 3?

To make a scientific conclusion, these reactions must be statistically greater than the reactions in a random event. The p value, or probability value, must be 0.05 or less for identification to be considered valid. To obtain this probability, at least three antigen-positive red cells that react and three antigen-negative red cells that do not react should be observed. In this example, three antigen-positive cells and seven antigen-negative cells were observed. Therefore the "rule of three" has been met. if there were not enough cells in this panel to determine sufficient probability, additional cells from another panel would be selected for testing.

Explain why patient info regarding transfusion or pregnancy helps in the process of antibody identification.

Unexpected antibodies can be made in response to a transfusion of red cells or exposure to fetal cells during pregnancy. Transfusions within the last 3 (book says 3 - Myra says 4) months present the possibility of a mixed red cell population and recent antibody stimulation. Antigen typing results must be interpreted carefully when the patient has recently received a transfusion because positive reactions may be caused by the presence of donor RBCs remaining in the patients circulation. Positive reactions caused by donor RBCs usually show mixed-field agglutination, but this depends on how recently the transfusion was given and how much blood was transfused. Also, within the first 3 months after transfusion, a positive DAT may indicate a delayed hemolytic transfusion reaction.

How could you get rid of the interference of autoantibodies if you suspect the presence of an alloantibody?

Use autoadsorption technique

When is an autocontrol performed? During antibody screen or during antibody identification?

Usually performed during antibody identification rather than during and antibody screen

What is the difference between warm autoadsorption and differential (allogenic) adsorption?

Warm autoadsorption uses patient RBCs to adsorb the autoantibody Differential (allogenic) adsorption uses allogenic RBCs to adsorb the autoantibody

DAT is used to detect in-vivo sensitization of RBCs. If a DAT is (IgG) positive, what is the next step?

When IgG antibodies are detected, the next step is to dissociate the antibodies from the RBC surface to allow for identification. This can be done through elution (wherein we want to take those antibodies and test them separately)

What antibodies are IgG that will react at 37C? (4)

anti-C, anti-E, anti-c, and anti-e

What are some examples of cold autoantibodies?

anti-I, anti-i, and anti-P

Antibodies that react more strongly with cells having homozygous antigen expression are said to show ______

dosage

What antigens are considered to be high-frequency antigens?

k, (Kp^b), (Js^b), and (Lu^b)