Unit 2 - Anti-Human Globulin (AHG) Testing & Quality Control

Commonly used in both antibody screening and ID procedures to INCREASE SPEED and SENSITIVITY of the antibody attachment to the RBC antigen

(antibody) potentiators or enhancement media

Polyspecific

- Combine a bunch of different animal's blood (combining poly clonal anti-IgG and polyclonal anti-C3) -->Has both Anti-IgG and Anti-C3 so detect either attached...but can't distinguish which one. Dye GREEN

LISS (Low ionic strength solution (0.2% NaCl))

-"Increases rate of antibody uptake" (sensitization) -Reduces zeta potential -Allows positively charged antibody to react more effeciently with antigen on RBC membrane -Allows for decrease incubation time...only need 5-15 minutes -Normal physiologic saline ions cluster around the antigen and antibody molecules...hindering the Ab-Ag complex formation. When the ionic strength is reduced the Ag-Ab molecules combine at a faster rate. -Great for antibody screening...fast incubation!!!

Define quality control (QC)

-Analytical Phase of the laboratory. It monitors the test equipment and reagents, to ensure accuracy of results. -Testing to determine the accuracy and precision of the equipment, reagents, and procedures

Monospecific

-Contains only one specificity...EITHER IgG or C3 -Clear -preferred at many blood banks due to clinically significant findings involving IgG

Cite the common sources of error that affect AHG testing

-For both DAT and IAT-----CELL WASHING -For IAT only ---Incubation temp and time, antibody-antigen ratio

Check Cell

-Group O RBCs sensitized with human IgG (so they would react with anti-IgG) -Used to verify QC of reagent AHG... Make sure it was added to test and that it was working properly

Monoclonal reagents (In blood bank we use mostly monoclonal)

-Hybridoma technology (hybrid cell used for production of antibodies in large amounts fused with myeloma cells) -Mostly use mice -The spleen cells that contain the antibody producing lymphocytes are fused with myeloma cells -Use myeloma cells because they can grow permanently (indefinitely) in cell cultures...so easy supply -Disadvantage is that you don't have a variety produced. Only one per clone

Define root-cause analysis and state its purpose

-Investigation to identify factors contributing to errors -Components: Identify root cause, Corrective strategy, Implement, Review

Explain the appropriate use of polyspecific and monospecific AHG

-Most clinically significant findings are IgG...so Monospecific is preferred at many blood banks. Polyspecific reagents have unwanted false positive reactions that are not clinically significant. Complement only is not significant

Describe resources used in determining appropriate quality control protocols and acceptability.

-Package inserts -Equipment manuals -Regulatory agencies specifications

Explain the IAT procedure

-Patient sample (EDTA purple tube) is spun and plasma is extracted -Reagent RBCs added that would react with the ANTIBODY we are trying to detect " Incubate at 37C " Wash 3 times " 1 drop AHG " Spin, read " If negative....check cell

Explain the DAT procedure

-Patient sample (EDTA purple tube) is spun and plasma is extracted. -->Because EDTA negates the in vitro activation of complement pathway, the test detects only complement proteins that have been bound to the RBC IN VIVO (sensitized red blood cells). -1 drop of 2-5% suspension -Wash 3 times -1 drop AHG -Spin, read, incubate, read -If positive...can use monospecific to determine if IgG or C3 sensitized -If negative...check cell

Differentiate between quality assurance (QA) and quality control (QC)

-QA is the big picture, all the stages and steps in the laboratory and facility -QC is analytical phase and monitors test equipment and reagents

Differentiate between monoclonal and polyclonal reagents

-Reagent used to detect immunoglobulins (antibodies) or complement attached to RBCs are AHG (Anti-human globulins) -They may be monoclonal or polyclonal

List the regulatory agencies involved in blood bank quality and safety

-Regulatory - Federal/State/Local Laws...MUST FOLLOW 1. FDA...Blood is a drug 2. CMC (Center for Medicare & Medicaid Services) -CLIA, HIPAA 3. OSHA -State Agencies (WI Dept. of Health Services, DNR)

In Vitro

-Sensitized in a test tube

In Vivo

-Sensitized inside the body (immune system)

IAT (Indirect Antiglobulin Test)

-Test used to detect antibody bound to red cells in vitro -Two stage test

Why do Anti-Human Globulin test? (DAT)

-To detect IgG or complement bound to patient cells -Detect HCFN (hemolytic disease of the fetus and newborn) -AIHA (Autoimmune hemolytic anemia) Antibodies produced against "self" -A positive DAT is an important indicator of potential immune mediated red cell destruction in the body and often leads to anemia

Why do Anti-Human Globulin test? (IAT)

-To see if the patient /donor has any unexpected antibodies (they would come from pregnancy or transfusion)...Antibody screen -Crossmatch or compatibility testing of donor to patient -Antibody identification -Determine RBC phenotype

Polyclonal reagents

-Use a pool of different people's IgG/C3 to give a broader spectrum of reactivity -Use sheep, rabbit, goats -Animal immunized with purified IgG OR Complement (C3) from a pool of normal human sera. Use the animals blood. Limited supply...only last until they die. -Advantage is that you can use many different donors and produce many different Anti-IgG

Discuss the role of potentiators in routine blood bank testing.

-Zeta potential---influences the agglutination reaction and with IgG visible agglutination might not occur compared to a large IgM...so....Enhancement media (Antibody Potentiators) to the rescue -Potentiators - reagents that enhance the detection of IgG antibodies by increasing their reactivity. They can reduce the zeta potential by adjusting the environment -Used in IAT tests ONLY****** -Commonly used in both antibody screening and ID procedures to INCREASE SPEED and SENSITIVITY of the antibody attachment to the RBC antigen

Neutralization can cause...

-a false negative in an antiglobulin test (DAT or IAT)...that is why we use a check cell

Monospecific AHG

-can be either anti-IgG or anti-C3d

Enzymes (papain, ficin, bromelin)

-come from plants (ficin from figs, bromelin from pineapple, papain from papaya) -May enhance, decrease or destroy antigen-antibody complex formation (depending on the antibody present) -remove sialic acid from the RBC membrane -decreases neg. charge and zeta potential -enhances uptake of antibody to SOME antigens (those not destroyed by the enzyme)

Polyspecific AHG

-detects both IgG and complement attached to RBCs

Polyspecific reagents have unwanted...

-false positive reactions that are not clinically signficant -complement only is not significant

Zeta potential

-force of repulsion between red cells in saline -influences the agglutination reaction and with IgG visible agglutination might not occur compared to a large IgM -So....Enhancement media (Antibody Potentiators) to the rescue

Clonal

-manufactured

Neutralization can...

-mask a positive Antiglobulin test (False negative). -->So for a negative result, a check cell (IgG sensitized cells) is added to the tubes that will give a positive reaction

Lectins

-plant extracts useful as blood banking reagents -they bind to carbohydrate portions of certain red cell antigens and agglutinate the red cells -can be useful in identifying antigen present on the patient or donor red cells

Albumin

-promotes direct agglutination (second stage of agglutination) but not antibody uptake (first stage of agglutination) -Not widely used -Reduces Zeta potential allowing RBCs to come closer together, but does not shorten incubation time -Bovine serum albumin (BSA) is commonly used

Antibody Potentiators or enhancement media

-reagents that enhance the detection of IgG antibodies by increasing their reactivity -they can reduce the zeta potential by adjusting the environment

PEG (polyethylene glycol)

-removes water molecules...thus increasing antibody conc. and increasing probable collision between antigen and antibody. -combine with LISS..."Increases rate of antibody uptake" (sensitization) -best for warm antibodies like IgG -must be used along with monospecific AHG

Specific

-specificity (what it will react with)

DAT (Direct Antiglobulin Test)

-test used to detect antibody bound to red cells in vivo -One stage test

Indirect Antiglobulin Technique (IAT)

-used to demonstrate in-vitro sensitization of RBCs

Three types of reagent red cells for routine testing include:

1) A1 and B cells in ABO serum testing 2) Screening cells to detect red cell antibodies 3)) Panel cells to identify red cell antibodies

Describe calibration, preventive maintenance and QC requirements in the blood bank; discuss the importance of each in accurate patient results

1) Calibration - Maintain equipment accuracy examples: Instruments in Blood Bank: Pipettes, Thermometers, Centrifuges, .. 2) Preventive maintenance - decreases downtime and avoids costly repairs 3) Reagent QC - performed daily...ensure reagents are reacting with appropriate strength and specificity

IAT is used in these situations for immunohematology in these situations

1) Detection of unexpected antibodies (Antibody screen) (IgG) 2) Identification of unexpected antibodies (Antibody Identification Panel) (IgG) 3) Compatibility testing (sometimes referred to as crossmatch) 4) Detecting a specific RBC antigen(s) (phenotyping) 5) Special studies such as identifying WBC or platelet antibodies

DAT is used in these situations

1) Diagnosis of HDFN 2) Diagnosis of autoimmune hemolytic anemia 3) Investigation of transfusion reactions and RBC sensitization to drugs

Coombs Test principle

1) Human IgG or complement is purified and injected into an animal (rabbit or goat). 2) The animal then makes an antibody against the foreign (human) protein-- antihuman globulin or AHG. 3) The AGH will react specifically with the protein that stimulated its production. 4) If that protein is attached to a RBC, it will cause agglutination.

Three reasons for a negative reaction with check cells

1) No AHG was added to the tubes 2) the cells were inadequately washed 3) the AHG reagent was contaminated or no longer reactive

Recognize the basic components of a blood bank QA program

1) Records and Documentation -Management plan, Audits, Storage, Audit trail, Trace, Logs, ... 2) Personnel Qualifications -Training, Job descriptions, Competency, ... 3) Supplier Qualification 4) Error Management -Root Cause Analysis 5) Validation -Prove process and systems are reliable 6) Proficiency Testing 7) QC -Monitors test systems -Calibration (accuracy of readings) Process of standardizing an instrument against a known value for accuracy. -Preventive Maintenance -Reagent QC (preformed daily)

Factors affecting antiglobulin tests (IAT only)

1) Temperature of incubation (IgG and complement both react best at 37 deg.) 2) Suspending/reaction/enhancement medium (LISS and PEG are most common) 3) Antibody: antigen ratio (zone of equivalence) - increasing the proportion of antibody to antigen usually increases the sensitivity...2 drops serum to 1 drop of RBCs) 4) Incubation time (Albumin = 30-60 min; LISS or PEG = 10-15 min)

Explain the principle of Anti-Human Globulin (AHG) testing

1) The test uses a reagent that has been prepared by injecting animals with human antibodies (IgG) and complement proteins. 2) The animals then produce and antibody against that human antibody& complement....thus producing Anti-IgG and Anti-C3 (complement). (This reagent is called AHG (Anti-human Globulin)) 3) This eagent will now react with human IgG antibody and complement...whether unbound in the serum or bound to antigens on the RBCs.

Common sources of false positive errors in antiglobulin testing

1. dirty glassware --> particles/contaminants 2. red cells already agglutinated --> cold reactive antibody of patient origin 3. improper centrifugation (overcentrifugation) --> red cell button packed so tightly that nonspecific clumping cannot be dispersed

Check cells are required by

AABB as a control system to be used when AHG tests are negative

5. Why are group O red cells used as a source for commercial screening cells?

ABO antibodies do not react with group O cells

List the accrediting agencies involved in blood bank quality and safety

Accrediting agencies - FOLLOW IS VOLUNTARY example: AABB (American Assoc. of Blood Banks) Scientific Societies - VOLUNTARY example: ISBT (International Society of Blood Transfusion

AHG

Anti-Human Globulin

What reaction do check cells cause?

At least 1+

Validation

Challenge to a process or system to prove it is reliable before it is implemented

9. In which source are the regulations regarding the manufacturing of blood banking reagents published?

Code of Federal Regulations

Distinguish between the direct antiglobulin test (DAT) and the indirect antiglobulin test (IAT)

DAT = In vivo sensitization of RBCs IAT = In vitro sensitization of RBCs

7. What regulatory agency provides licensure for blood banking reagents?

FDA

Hemolytic Disease of the Fetus and Newborn

HDFN

In 1945, Coombs, Mourant, and Race described a test that detected "incomplete" ____ antibodies in the serum of a woman that had lost a baby to ________

Rh, HDFN

Why do red blood cells need to be washed?

To remove the reactive unbound antibodies and complement from messing with the results. (these unbound antibodies can inhibit and neutralized the AHG from reacting fully with the bound antibodies we are trying to detect)

Check cells

aka Coombs control cells -added to all negative AHG reactions -cause a 1+ reaction

Direct Antiglobulin Test (DAT)

aka Direct Coombs Test -used for in-vivo RBC sensitization

8. What antibodies are present in polyspecific AHG reagent?

anti-IgG and anti-C3d

If an antibody screen test is positive, an __________________ is performed

antibody identification panel

4. Part of the daily quality control in the blood bank laboratory is the testing of reagent antisera with corresponding antigen-positive and antigen negative red cells. What does this procedure ensure?

antibody specificity

First stage of agglutination

antibody uptake (sensitization)

Neutralization

blocking antibody sites, causing a negative reaction -is a source of error -can mask a positive antiglobulin test

Quality assurance

combined activities of an organization to ensure the quality of products and services offered; a comprehensive documentation practices program monitoring the entire testing process

13. What source of antibody is selected to determine the specificity of a red cell antigen in a patient sample?

commercial antisera

14. What source of antigen is selected to determine the presence of a red cell antibody in a patient sample?

commercial reagent red cells

2. What characteristic is associated with monospecific AHG reagents?

contain either anti-IgG or anti-C3d antibody specificities

Polyspecific AHG reagent

contains both anti-IgG and anti-C3d antibodies and detects both IgG and C3d molecules on red cells

Reagent QC is performed _________ in the transfusion service.

daily

Proteolytic enzymes

enzymes that denture certain proteins

12. Select the method that uses the principle of sieving to separate larger agglutinates from smaller agglutinates in Ag-Ab reactions.

gel technology

Check cells are commercially made...

group O positive human red cells coated with IgG antibodies

Autoimmune hemolytic anemia

immune destruction of autologous (self) red cells

Sensitized

immunoglobulin or complement attached to the cells from the immune system (in vivo) or from a test procedure (in vitro) -stage 1 of agglutination (stage 2 is lattice formation

The DAT test is used to detect antibody bound to red cells ___________________; the IAT is used to detect antibody bound to red cells _____________________

in vivo; in vitro

If the Coombs control cells do not make the reaction turn positive, the test is _____________________

invalid

15. What reagents are derived from plant extracts?

lectins

Quality control

monitors test system components such as reagents and instrumentation; assures accuracy in the analytical phase of testing

Agglutination via check cells does not prove a test was truly _____________________

negative

no agglutination at the completion of the antiglobulin test is interpreted as a __________________ and indicates that no IgG or complement proteins were attached to the red cells

negative antiglobulin test

To detect potential _________________________, IgG-sensitized cells are added to tubes with negative reactions

neutralization

Reaction phase

observation of agglutination at certain temperatures, after incubation or after addition of AHG

10. After the addition of anti-D reagent to a patient's red cell suspension, agglutination was observed. The result with anti-A reagent was negative. What is the interpretation of this patient's D typing?

patient is D-positive

A __________ reaction with check cells proves only that AGH was present and working

positive

the formation of agglutinated red cells after the addition of AHG shows that IgG or complement proteins were attached to the red cells; this is interpreted as a __________________- (DAT)

positive antiglobulin test

LISS and PEG are commonly used as...

potentiators in routine blood bank testing

6. Where can you locate information regarding reagent limitations?

product inserts

Second stage of agglutination

promote direct agglutination (lattice formation)

Monospecific AHG reagents

reagents prepared by separating the specificities of the polyspecific AHG reagents into individual sources of anti-IgG and anti-C3d/anti-C3b

11. Select the reagent to use for detection of unexpected red cell antibodies in a patient's serum sample.

screening cells

After IgG-sensitized cells are added to a tube with an initial reaction of negative, a positive reaction should be observed to confirm that _________________ was adequate

washing

Differential DAT

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

Indirect antiglobulin test

test used to detect antibody bound to red cells in vitro

Direct antiglobulin test

test used to detect antibody bound to red cells in vivo

3. You have added IgG-sensitized red cells to a negative indirect antiglobulin test. You observe agglutination in the tube. What situation was not controlled for in testing by adding these control cells?

the addition of patient serum (AHG does NOT ensure that the AHG reaction is negative)

1. What is the purpose of including a reagent control when interpreting group AB, D-positive red cells after testing with a low protein anti-D reagent?

to detect false positive agglutination reactions