Chapter 3 Blood Banking Reagents

Question 1

What are four different categories of reagents for blood banking?

Answer 1

4 basic categories of reagents for blood banking:
1. RBCs with known antigens
2. Antisera with known antibodies
3. Antiglobulin reagents: anti-IgG and / or complement
4. Potentiators to enhance antibodies.

Question 2

Who licenses reagents for the blood bank?

Answer 2

Commercial blood banking reagents are licensed by the Center for Biologics Evaluation and Research (Food and Drug Administration [FDA]).

Question 3

What are the FDA’s criteria for blood banking reagents?

Answer 3

The FDA’s criteria can be found in the Code of Federal Regulations.

Reagents must meet min standards before being licensed:
1. Specificity -recognition of the antigenic determinant and its corresponding antibody.
2. Potency - strength of the reaction (e.g. anti-A and anti-B are made to agglutinate to 3+ and 4+).

Question 4

Name 3 main components of a QC program for reagent quality.

Answer 4

QC Program (Blood bank)
1. Statement of the criteria for acceptable reagent performance.
2. Documentation of reagent use
3. Corrective actions for lack of performance.

Question 5

What are Polyclonal antibodies?

Answer 5

Polyclonal antibodies:
1. Made from several different clones of B cells that secrete antibodies of different specificities
2. Produced by immunizing rabbits with purified human IgG molecules
3. Recognize multiple epitopes

Example: antihuman globulin (AHG)

Question 6

What are Monoclonal antibodies?

Answer 6

Monoclonal antibodies:
1. Made from single clones of B cells that secrete antibodies of the same specificity
2. Use hybridoma technology
3. Recognize a single epitope

Examples: anti-A, anti-c, and anti-IgG antibodies

Question 7

What antigen is Anti-A antisera directed against?

Answer 7

Antisera are directed toward specific antigens on the patient’s RBCs
Anti-A: directed toward A antigen
Anti-B: directed toward B antigen

Question 8

Your have one antisera is that is blue and the other yellow, what type are they?

Answer 8

Antisera is Colour Coded as follows:
Anti-A contains blue dye
Anti-B contains yellow dye

Question 9

What does it mean to do an immediate spin?

Answer 9

Testing is performed in the immediate-spin (IS) phase since A and B antibodies are IgM. IgM antibodies prefer binding at room temperature or lower.

Immediate spin means spinning at 3400rpm for 15 seconds after adding the antisera to the sample. We just want the cells to get close to each other to allow lattice formation if the antibodies in the reagent bind to antigens on the cell surface.

Question 10

What does it mean to do forward typing? reverse typing?

Answer 10

Forward typing is when you add antisera to the patient’s red cells to see if it agglutinates or not.

Reverse typing is when you add commercial red cells to patient’s plasma containing their antibodies to confirm the forward typing.

Question 11

What antibodies should have Type A blood person have in their plasma?

Answer 11

A type A person should have B antibodies in their plasma.

Question 12

The Rh group blood system contains several antigens, why is the D antigen the most important?

Answer 12

The Rh blood group system contains several antigens, but the D antigen is the most important because of increased immunogenicity.

Question 13

When do you determine if the person is type D or not?

Answer 13

The Standards for Blood Banks and Transfusion Services requires that all blood samples be typed for the D antigen (recipients, donors, and expectant females).

Question 14

Why is a negative reagent control perform during typing for D in the Rh blood group?

Answer 14

When Commercial anti-D is combined with the patient and donor RBCs any agglutination is significant.

A negative reagent control ensures that a false-positive result has not occurred.

Question 15

What is the Rh control for anti-D?

Answer 15

The Rh control includes all the other ingredients that are found in the D antisera minus the actual antibodies. Occasionally a person will have something that causes agglutination without having the antigen present. We expect a negative result in the control vial (patient cell and control). If there is agglutination in the control we cannot interpret the D results.

Question 16

What is the difference between low protein and high protein antisera? Which one is used and why?

Answer 16

High-Protein Reagents:
1. Contain polyclonal antibodies,
2. Approximately 20% bovine albumin
Low-Protein Reagents;
1. Contain monoclonal antibodies (IgM) or monoclonal and polyclonal blends (to detect Weak D)
2. Approximately 6% bovine albumin

High protein reagents tend to promote false-positive agglutination so therefore low protein reagents are used. The low protein reagents do not necessarily always need a control to be run for each patient sample.

Question 17

What are the ingredients in the Rh negative control?

Answer 17

Contains everything found in anti-D reagent except the antibody:
- Buffered saline solution
- Bovine serum albumin
- EDTA
- 0.1% sodium azide (preservative)

Question 18

What causes a false positives to anti-D (or other antisera)?

Answer 18

False-positive agglutination can result from
Strong cold autoantibodies
Protein abnormalities

Question 19

When is a separate control used for Anti-D forward typing?

Answer 19

When is a separate control used?
1. A separate control is used if RBCs agglutinate with all ABO antisera.
2. A separate control is not needed if there is a negative result using any of the ABO low-protein reagents.

Question 20

What are reagent RBCs?

Answer 20

Commercial reagent RBCs contain known antigens to confirm the presence of antibodies in patient serum or plasma

Question 21

What are the characteristics of A1 and B commercial red cells to have?

Answer 21

Can be from a single donor or a pool of donors
Washed and re-suspended to a 2% to 5% concentration
Negative for Rh antigens (D, C, and E)
Should be a 2+ to 4+ grading

Question 22

Name some reasons give for why commercial red cells may lyze?

Answer 22

Commercial Red cells lyze over time due to:
- Age, short life span
- Frozen in transport
- Mixed too harshly

Question 23

How can you tell if a vial of commercial red cells have lyzed and how can that affect the results of reverse typing?

Answer 23

The supernatant in the vial will start looking reddish.

If too many cells lyze you will get a weakened reaction and may miss agglutination, resulting in a false negative result.

Question 24

What are screening cells?

Answer 24

Screening cells are used in antibody screen (detection) tests for clinically significant, unexpected antibodies. They are group O red cells to prevent any interference from naturally occurring antibodies.

Question 25

What comes with each screening cell reagent?

Answer 25

• Each lot of screening cells comes with an antigram that shows the antigen profile
• The cells must express antigens associated with the most clinically significant antibodies
• Screening cells are regulated by the FDA for: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and Jkb
• 7 different blood group systems
• Will detect presence of all clinically significant antibodies

Question 26

What are panel cells?

Answer 26

Used when the antibody screen is positive to identify the specific antibody(ies) present –> Antibody panel

Question 27

Describe a lot of panel cells - what comes with it and group type are they made from?

Answer 27

Each lot of panel cells will have an antigram that shows the antigenic profile of each vial.
Are group O red cells with varying antigenic expression of other blood group systems

Question 28

What is an antiglobulin test?

Answer 28

Commercial antibody with a specificity toward human globulins is used to agglutinate antibody-coated (sensitized) RBCs.

Question 29

What do antiglobulin test reagents contain? How is it created?

Answer 29

Antiglobulin Test Reagent contains antibodies toward:
• IgG (anti-IgG) and/or
• Complement (anti-C3d and anti-C3b)
• Created by injecting human IgG and/or complement (C3d and C3b) into rabbits

Question 30

Give four reasons for a positive DAT (Direct Antiglobulin Test)?

Answer 30

A positive DAT could result from the following scenarios;
1. Transfusion reaction - donor cells coated w/ IgG
2. Hemolytic disease of the fetus and newborn - fetal RBC coated with IgG
3. Autoimmune hemolytic anemia - IgG or C3 on patients RBCs
4. Drug-related mechanism - IgG drug complex attached to RBCs.

Question 31

How times are the RBC’s washed for the DAT and why?

Answer 31

Washed x 4 with saline. Decanting the saline each time. This would get rid of any residual plasma, unattached antibodies, extra proteins, etc. Now we have the patient’s red cells that may have IgG and/or complement binding to them.

Question 32

What is the indirect Antiglobulin test (IAT)?

Answer 32

INDIRECT Antiglobulin Tests
• Detects IgG bound to RBCs in vitro (outside the body)
o This test uses the patient’s plasma.

Question 33

What are applications for the IAT?

Answer 33

IAT is used for:
1. Antibody screening
2. Antibody identification
3. Cross-match
4. Antigen typing.

Question 34

What is polyspecific AHG?

Answer 34

Polyspecific AHG:
• Contains both anti-IgG and anti-C3d antibodies
• Derived from polyclonal or monoclonal sources

Question 35

What is monospecific AHG?

Answer 35

Monospecific AHG:
Contains either anti-IgG or anti-C3b/C3d, but not both
C3d is usually the only complement protein that will remain attached to RBC

Question 36

When is a differential DAT performed?

Answer 36

Differential DAT is an immunohematologic test that uses monospecific anti-IgG and monospecific anti-C3d/anti-C3b reagents to determine the cause of a positive DAT with polyspecific antiglobulin reagents.

Question 37

When are IgG sensitized check cells used?

Answer 37

IgG sensitized check cells used:
1. Required as a control system when AHG results are negative
2. When added to a negative AHG test, reagent should cause agglutination as they will bind with free AHG. Should be 2+

Question 38

What are some causes for a false negative DAT or IAT?

Answer 38

False-negative results are caused by
1. Failure to add the AHG reagent
2. Failure of the AHG reagent to react
3. Failure to wash the RBCs adequately (neutralization)

Question 39

What is neutralization?

Answer 39

Neutralization can occur if we did not wash well enough and causes false negatives. If there is lots of left-over antibodies present in the tube the anti-human globulin will bind with them.

If there is a great deal of unbound antibodies it will tie up all the AHG and we won’t get lattice formation or see agglutination.

Question 40

What are potentiators?

Answer 40

Potentiators: Reagents that enhance the detection of IgG antibodies by enhancing uptake and/or promoting direct agglutination.

Question 41

List the various potentiators that can be used and the main reason it is used?

Answer 41

1. Low Ionic Saline Solution (LISS) - has clusters of free Na+ and Cl- ions around RBCs that maintains RBC integrity while helping with antibody uptake (sensitization)
2. Polyethylene Glycol (PEG) - Water soluble polymer that removes water from the test mixture, therefore increasing the concentration of the antibody for better antibody detection
3. Proteolytic Enzymes - Modifies RBC membrane by removing negatively charged molecules, reducing the zeta potential, does not work in all cases to help agglutination.
4. Bovine Serum Albumin - Allows antibody-sensitized cells to come closer together than is possible with saline and hence helps with lattice formation.

Question 42

What are lectins?

Answer 42

Lectins are not potentiators
• Seed extracts that have specificity toward certain RBC antigens (mimic antibodies)
• Bind to carbohydrate determinants of RBC antigens
They mimic antibodies.

Question 43

Describe gel technology for agglutination testing?

Answer 43

• Uses dextran acrylamide gel particles to trap agglutinated cells
• Gel particles are combined with reagents and pre-dispensed in microtubes of plastic cards
• RBCs or plasma/serum are added to the microtube and allowed to incubate before centrifugation
• Large agglutinates are trapped at the top of the microtubes (4+)
• Nonagglutinated cells travel unimpeded to the bottom of the microtube (0)
• Eliminates variation in tube shaking between technologists

Question 44

Describe the microplate method of agglutination testing?

Answer 44

• A 96-well microtiter plate replaces test tubes
• Applies same principle as test tube method
• A concentrated button indicates a reaction
• Dispersed cells indicate no reaction

Question 45

Describe the solid phase method of agglutination testing?

Answer 45

Solid Phase:
1. Uses microplate wells with immobilized reagent
2. Cells that adhere to the sides and bottom of the wells are POSITIVE
3. Cells that settle to the bottom of the wells are NEGATIVE