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Transfusion Science > Chapter 8 Antibody Detection and Identification > Flashcards

Chapter 8 Antibody Detection and Identification Flashcards

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1
Q

What are antibody screens used for?

A
  1. Patients needing a transfusion.
  2. Pregnant individuals
  3. Blood and plasma donors.
  4. Patients who have had transfusion reactions.
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2
Q

What reading does the tube test in an antibody screen need to be for it to be positive?

A

Any agglutination with screening cells (1+ or even weaker) means it is detecting an antibody to one of the antigens present on the screening cells.

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3
Q

What are symptoms for hemolytic transfusion reactions (HTRs)?

A
  1. Fever and chills
  2. Hemoglobinuria
  3. Less common: pain, hypotension, nausea/vomiting, dyspnea, renal failure, DIC.
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4
Q

Screening cells are single or pooled donor group O cells, but what is the advantage of single donor vials?

A

They offer increased sensitivity.

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5
Q

What type of cells are screening cells and why?

A

Group O cells are used so that anti-A and anti-B antibodies will not react.

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6
Q

What documentation do screening cells come with?

A

A sheet of paper called an antigram that lists all the antigens present in each vial.

18 antigens are required on at least one of the vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb

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7
Q

What is an autocontrol?

A

Tests a patient’s serum with his or her own RBCs.

The autocontrol is incubated with the antibody screen (or antibody panel)

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8
Q

What is the next step if the autocontrol is positive?

A

A DAT may be ran (patient cells with AHG) to detect in vivo coating.

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9
Q

What is the purpose of the autocontrol (AC) and the direct antiglobulin test (DAT)?

A

The AC and DAT can help to determine if the antibodies are directed against the patient’s cells or transfused cells (allo vs autoantibody).

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10
Q

Why are potentiators (like PEG) used in antibody detection and identification?

A

To enhance ag:ab reactions.

See Table 8.1 in textbook.

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11
Q

When is PEG not recommended to be used?

A

If a patient has increased proteins (e.g. in case of multiple myeloma in can cause false agglutination).

See Table 8.1 in textbook.

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12
Q

What technique is the analyzer at CBS in Manitoba?

A

Solid phase (SPRCA) technique.

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13
Q

What is the first step in the investigation once you get a positive antibody screen?

A

Get the patient’s medical history, these facts may be relevant:
1. Transfusion history.
2. Come from another hospital.
3. Some diseases are associated with antibodies (esp. IgM autoantibodies).
4. Diagnosis, age, and race of patient.
5. Pregnancies.

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14
Q

How long can mixed RBC populations from a previous transfusion last?

A

Can remain in a person’s body for up to 3 months.

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15
Q

What is an antibody panel?

A
  1. Extended version of an antibody screen (10 to 20 cells)
  2. Group O red cells.
  3. Each panel cell is antigen typed (phenotyped)

The antigram lists the phenotypes of each panel cell and is used to record and interpret results.

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16
Q

What three different phases are ran during an antibody
identification?

A
  1. IS / RT
  2. IgG (checking immediately after water bath before washing and adding AHG).
  3. With AHG after incubation at 37C
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17
Q

What does a positive autocontrol or DAT indicate?

A

Indicates that auto antibody or alloantibody is present to recently transfused cells.

A positive AC may indicate a delayed hemolytic transfusion reaction.

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18
Q

What does a negative autocontrol result indicate with a positive screen?

A

Alloantibody

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19
Q

What does a positive autocontrol plus a negative DAT indicate?

A

False positive.

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20
Q

What can reaction strength indicate in the antibody panel testing?

A
  1. Strength is affected by dosage (small variance - e.g. 2+ to 3+ range).
  2. Varying strengths may indicate multiple antibodies (wider variance) (1+ to 3+ or 2+ to 4+).
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21
Q

What is the first simple way to interpret an antibody screen?

A

Match positive reactions of one of the antigen columns.

This may mean a single antibody is present if you get a match. Multiple antibodies show varying patterns.

Think simple first - complicated next.

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22
Q

How do you rule out antibodies in an antibody screen?

A

Panel cells that are negative in all phases can be used to rule out antibodies.

Begin with the first panel cell that is negative in all phases and cross out any antigens present, except those that are heterozygous - as that Ab may be too weak to react.

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23
Q

What is the rule of three for antibody panel interpretation guidelines?

A

3 antigen positive cells must react and 3 antigen negative cell must not react with the patient’s serum or plasma. If this does not occur, additional panel cells are selected to be used.

If multiple antibodies are present you must prove rule of three for each antibody and the cells shall be negative for the other antigens that could react with other potential antibodies not ruled out yet.

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24
Q

How does phenotyping the patient help confirm what antibodies are present in their plasma?

A

Phenotype the patient. Individuals do not make alloantibodies toward antigens on their own RBCs.

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25
Q

How can phenotyping be done to ID Abs if the patient has recently been transfused?

A

RBC separation technique should be used before phenotyping is done.

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26
Q

What are some techniques that can be used to id multiple antibodies in a panel?

A
  1. Selected cells - out of other panocell sets.
  2. Proteolytic enzymes - destroys Duffy, M, N, S, s antigens on panel cells.
  3. Other chemicals (e.g. dithiothreitol used to denature Kell antigens).
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27
Q

How are enzymes used in antibody identification?

A

Enzymes are used to eliminate or enhance antibody activity.

Duffy and MNS antigens are destroyed.

Rh, Kidd, Lewis, I, and P1PK antigens are enhanced.

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28
Q

What is the one stage test with proteolytic enzymes?

A

Enzymes, RBCs, and serum are simultaneously incubated.

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29
Q

What is the two stage test with proteolytic enzymes?

A

Panel cells are pretreated with enzymes, washed, and then used as usual.

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30
Q

What do you suspect if most panel cells are positive?

A

Suspect an alloantibody to a high-frequency antigen if most panel cells are positive. High frequency antigens occur in the population at a frequency of 98% or higher.

Additional testing may be needed to confirm the Ab such as using enzymes or dithiothreitol to destroy Kell antigens.

31
Q

What are high-titer, low-avidity antibodies and their main characteristics?

A

1.Antibodies to high-frequency antigens that react weakly.
2. React at AHG phase.
3. Not implicated in transfusion reactions or HDFN.

32
Q

When are antibodies to low-frequency antigens suspected?

A

When they occur alone OR screen is negative and crossmatching is positive.

Only one reactive cell suggests this type of antibody.

33
Q

What are common antibodies that are seen as response to low frequency antigens?

A

Anti-C2,
anti-Wr_a,
anti-V,
anti-Co_b,
anti-Bg_a,
anti-Kp_a, and
anti-Lu_a

34
Q

What are strategies for enhancing weak IgG antibodies?

A
  1. Incubate longer
  2. Phenotype if not recently transfused
  3. Increase serum to cell ratio, within limits of reagent.
  4. Repeat with a different enhancement such as enzymes or PEG
  5. Check antigen dosage on weak or missing reactions.
  6. Select different cells from a new panel.
35
Q

How do cold alloantibodies act during testing?

A

IgM antibodies that react during immediate spin, crossmatching and sometimes at 37C.

Not clinically significant.

36
Q

What cold alloantibodies have variable reactions?

A

Anti-P1,
Anti-M
Anti-N
Have variable reactions.

37
Q

What can be done to enhance cold alloantibodies variable reactions?

A

To enhance reactions, incubate below room temperature.

38
Q

What can be done to avoid cold alloantibodies reactions?

A

To avoid reactions, neutralization can be performed so that clinically significant antibodies may be detected.

39
Q

What is autoimmune hemolytic anemia?

A
  1. Immune destruction of “self” red cells (auto).
  2. Immune system is suppose to destroy non-self but in AIHA the immune system is malfunctioning and destroys self.
40
Q

What does sensitization mean for the patient?

A

Spleen will remove the sensitized red cells (extravascular removal) which will result in red cell destruction and therefore anemia. Antibodies coating red cells cause them to be less pliable and unable to get through the spleen.

41
Q

What do we see in the lab with autoimmune hemolytic anemia?

A
  1. Positive DAT and/or autocontrol from IgG or C3 from the patient attached to the patient’s own red cells in vivo.
  2. Autoantibodies usually react with all reagents and self and donor RBCs, regardless of the antigens present.
42
Q

What technique is done to remove autoantibodies when testing? Why?

A

Adsorption techniques usually performed to remove autoantibodies.

It is important to identify underlying alloantibodies when an autoantibody is encountered.

43
Q

What are the three different types of autoimmune hemolytic anemia (AIHA)?

A
  1. Cold AIHA (18%)
  2. Warm AIHA (70%)
  3. Drug-induced AIHA (12%)
44
Q

When do we see agglutination for typical cold autoantibodies? What should their autocontrol and DAT results be?

A

During immediate spin (crossmatching) and have a positive autocontrol and DAT.

45
Q

What antibodies specifically are most cold autoantibodies?

A

Autoanti-I
Autoanti-H
Autoanti-IH

46
Q

What type of panels can be used to help identify cold autoantibodies? Describe it.

A

“Cold panels”
- Screen and panel cells are always adult cells and type O, but adult cells have I on their cells (reacts with Autoanti-I & IH) and O cells have lots of H antigens (reacts with Autoanti-H & IH). Therefore typical panel cells will all react & agglutinate.

Cold panels have cord blood cells with i Ag and no I Ag and A1 cells with far less H plus an O type (with I and H).

47
Q

In what individuals may Cold Agglutinin Disease show up in?

A
  1. May occur in people with a history of mild anemia, Mycoplasma pneumoniae infection, or infectious mononucleosis.
  2. Chronic forms could be seen in elderly patients with lymphoma, chronic lymphocytic leukemia, or Waldenstrom macroglobulinemia.
48
Q

At what temperature do cold autoantibodies react strongest at?

A

Typically stronger reactions at the cold phase and lesser or no reactions at the warmer phases (strongest at 4 deg C).

Strongest at 4degC
Strong at RT
Positive at 37degC
Note: IAT may still show reaction but weaker 1+ or w+)

Serologically they have a higher titre of antibody present.

See slide 42 for example tests results.

49
Q

What is the clinical result for the patient with cold agglutinin disease?

A

Causes hemolytic anemias that can be mild to life-threatening.
There is no cure - can try to live in a warmer climate.

50
Q

What cold autoantibody causes Paroxysmal Cold Hemoglobinuria (PCH)?

A

Autoanti-P

51
Q

What is another term of Autoanti-P and its unique characteristics?

A

Also known as Donath-Landsteiner antibody.

IgG
Complement binds to P+ red cells at the extremities (lower temp) and causes red cells to lyse when they are warmed at the core. Called Biphasic hemolysin (or biphasic antibody).

52
Q

What individuals are more prone to get Paroxysmal Cold Hemoglobinuria (PCH)?

A

Transient in kids with viral infection, chicken pox, H influenza, adults with tertiary syphilis.

53
Q

List 5 ways you can you avoid cold autoantibody reactions in the lab?

A
  1. Use a monospecific anti-IgG rather than a polyspecific.
  2. Skip immediate spin on crossmatching and testing at 37C.
  3. Use 22% bovine serum albumin instead of LISS or PEG.
  4. Pre-warm all tubes to 37C will avoid reactivity.
  5. Use adsorption techniques if all else fails.
54
Q

What percentage of autoimmune hemolytic anemias are the warm type? What does it mean to be a warm type?

A

70% - more problematic!

IgG autoantibodies that attach to RBCs at 37C.

Extravascular hemolysis (sensitized cells removed by spleen).

Causes either 1. Idiopathic or 2. Secondary (from Lymphoma, systemic lupus erythematosus, carcinoma or drug therapy).

55
Q

What makes it hard to detect a warm autoantibody?

A
  1. ABO results usually show no discrepancies, but sometimes the spontaneously agglutinate (see slide 46).
  2. DAT isn’t always positive 2-7% have a negative DAT result. 67% are positive for IgG and C3d. 20% are + for IgG only, and 13% are + for C3d only.
56
Q

What is the key for suspecting a warm autoantibody?

A
  1. All cells with IAT method are positive. May not show specificity - e.g. cases of autoanti-e.
  2. Positive autocontrol.
57
Q

What is important to find out if you suspect a warm autoantibody?

A

If it is hiding an alloantibody!

58
Q

How can warm autoimmune hemolytic anemia affect the patient in terms of onset?

A

Onset can be gradual with minimal symptoms or life threatening rapid hemolysis.

Depends on amount of autoab present, ability of autoab to attach to RBC, or its ability to bind complement. If complement activation is not sufficient the result is spherocytes.

59
Q

What is the treatment for warm AIHA?

A
  1. Treat primary cause, administer steroids, or other immunosuppressive therapies.
  2. Plasmapheresis - blood is filtered to remove autoantibodies, and then replaced.
  3. Splenectomy - spleen enlarges because cells can not properly pass through. Last resort.
60
Q

What happens to the Hgb and Hct in warm AIHA?

A

Low due to red cell destruction.

61
Q

What morphology of red cells are seen in warm AIHA?

A
  1. Spherocytes or fragmented RBCs.
  2. Bone marrow compensates - w/ shift to the left.
  3. See retics, polychromasia and NRBCs.
  4. High MCV because of immature cells.
62
Q

Why may you perform an autoadsorption? What else can you do?

A
  1. Perform autoadsorption because autoanti-e can interfere with antibody testing and can mask alloantibodies.
  2. Use panel cells that are e-negative but 98% of population is e-positive.
  3. Perform elution.
63
Q

What is drug induced AIHA?

A

Removal of red cells by extravascular or intravascular hemolysis due to antibody formation against drugs. Look similar to a warm autoantibody.

Problem stops with stopping the drug.

See table 8.12 for more details.

64
Q

What makes you suspicious of a drug induced autoimmune hemolytic anemia?

A
  1. ABO results usually show no discrepancies - so not that.
  2. DAT is positive with poly specific (IgG and C3d), IgG, and C3d –> clue!
  3. No interferences with antibody screen or antibody panel. So not that.
65
Q

What is an elution?

A

Elution is when you recover an antibody in solution called an eluate. The eluate is used in an antibody panel to identify the antibody.

66
Q

When may one use the elution technique?

A

When DAT is positive, the IgG must be detached by using the elution technique.

Elution is performed in suspected cases of HDFN and may not be reactive in all cases of warm autoantibodies.

67
Q

What cautions are needed regarding the elution technique?

A

Risk of lab error: Chemicals can destroy blood groups (Duffy, MNS, etc.) Incorrect technique or contamination.

68
Q

What is auto-adsorption? And when can you use it?

A

Complete removal of autoantibodies to test for underlying alloantibody.

Use, only if the patient has not been transfused within the last 3 months.

69
Q

What does auto-adsorption do?

A

In vitro attachment of the patients antibodies to the patients own red cells therefore removing the antibody from the plasma.

70
Q

If the patient has been transfused recently how could you do adsorption?

A

You can do an allo-adsorption. Allogenic red cells are used for adsorption.

71
Q

What chemicals or techniques are used for elution technique?

A

Glycine acid to remove Abs off red cells. (Current method).

Historically heat freeze-thaw and Ether Methylene chloride Chloroform had been used but they are either not effective or too hazardous.

72
Q

What are the steps to the autoadsorption method?

A
  1. RBC Pretreatment; remove patient plasma, treat RBCs with DTT, treat with enzymes.
  2. Attachment of antibody. Plasma combined with treated RBCs and incubated for 30-60 mins. Ab attaches to RBCs.
  3. Testing. All interfering autoantibodies are now attached to RBCs. Centrifuge sample and remove plasma to a separate tube. Test plasma for alloantibodies.

See slide 56 & 57 for more details.

73
Q

What are the steps to take if someone with AIHA needs a transfusion?

A
  1. Patient history
  2. Discover if cold, warm or drug induced.
  3. Determine if any alloantibodies.
  4. Issue compatible blood.
    a) Cold AIHA blood should be put through a warmer.
    b) Warm AIHA may need to use blood with least amount of agglutination. Not ideal.

Transfusion Science (17 decks)

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