05 AHG

IgG antibody(ies) and/or complement (C') bound to RBCs.

Due to its size, IgG cannot cause immediate visible hemagglutination when attached to RBCs.

Bridging

An antibody solution made from immunized animals (e.g., rabbit) that makes human IgG or C' antibodies bind to human immunoglobulins found on RBCs.

Direct Antiglobulin Test (DAT) or Direct Coombs' Test (DCT)

Indirect Antiglobulin Test (IAT) or Indirect Coombs' Test (ICT)

In-vivo; inside

It detects in-vivo sensitization of RBCs, where sensitization occurs inside the body.

In-vitro; outside

It is used in the Antibody Screen (ABS) Test and the Antibody Identification (Ab ID) Test.

Detection of in-vivo sensitization of RBCs.

Detects both IgG and/or C'.

vivo; RBCs

IgG and/or C'

Hemolytic Disease of the Fetus/Newborn - maternal IgG crosses the placenta and binds to infant RBCs (up to a 4+ reaction).

Hemolytic Transfusion Reaction - transfused recipient antibodies bind to donor RBCs.

Autoimmune Hemolytic Anemia - a condition or drug-induced phenomenon that creates allo-antibodies or auto-antibodies toward RBCs.

Cold AIHA: Mediated by IgM.

Warm AIHA: Mediated by IgG

Class/Type II

IgG

IgM

Wash unknown (patient) RBCs at least 3x with saline to remove unbound globulins.

Polyspecific AHG reagent.

Mix the sample and centrifuge (accelerates agglutination) and then read the results.

The reaction is graded on a scale from 0 to 4+ and recorded.

Coombs' Check Cells (CCCs) / Check Cells (CCs).

IgG antibodies.

Mix, spin, read, and record the reaction.

The test is valid.

The test is invalid and must be repeated.

Repeat the DAT/DCT using monospecific AHG reagents (individual IgG reagent and individual C' reagent).

Evidence of in-vivo hemolysis or sequestration of RBCs (intra- or extra-vascular).

Recently transfused, unexpected IgG allo-Ab.

Medical condition, IgG auto-Ab, or C'.

Medication (IgG or C').

Positive DAT/DCT results with no clinical significance may be detected in up to _____ of the population.

Antibody (Ab) titer, primary cause for antibody production, binding of C', etc.

It removes unbound globulins and ensures accurate test results.

The proper amount must be added, and inadvertent omission can lead to invalid results.

To ensure accurate detection of IgG and/or C' binding to RBCs.

Optimal

Allo/auto antibody + complement (C').

Allo/auto antibody only.

Complement (C') present, which may suggest:

Cold Hemagglutinin Disease (CHD).

Paroxysmal Cold Hemoglobinuria (PCH).

Detection of in-vitro sensitization of RBCs.

Unexpected IgG allo- or auto-antibodies in recipient sera that may be incompatible with donor RBC antigens.

_% to _% of the general patient population have irregular RBC antibodies.

____% to _____% of multiply-transfused populations have irregular RBC antibodies.

Identification of unexpected allo-antibodies in unknown sera and titration of IgG antibodies.

Transfusion of incompatible RBCs.

Decreased survival of donor RBCs.

Clinical effects of Hemolytic Transfusion Reactions (causing morbidity).

The IAT/ICT helps avoid the __________ of incompatible RBCs.

The IAT/ICT helps avoid decreased _________ of donor RBCs.

Clinical effects of __________ are avoided by performing the IAT/ICT.

Safe and effective RBC transfusion.

RBC longevity to maintain oxygen-carrying capacity to vital organs.

Normal body functions without compensations (e.g., tachycardia, hyperventilation).

The IAT/ICT promotes ______ and effective RBC transfusion.

The IAT/ICT ensures ______ to maintain oxygen-carrying capacity to vital organs.

Safe and effective RBC transfusion

Rapid

Heartbeat

Elevated

Breathing

Allows time for antibodies to attach to RBC antigens.

Removes free globulin molecules.

Forms RBC agglutinates for visibility.

Accelerates agglutination by bringing RBCs together.

Interprets the test as "positive" or "negative."

Determines the strength of the reaction.

Verifies the validity of the test.

Refers to the ratio of serum to red blood cells, which influences the sensitivity of the test.

The medium in which the test is conducted, which can affect the interaction between antibodies and antigens.

Ensures optimal conditions for the reaction, typically 37°C for IAT/ICT tests.

Allows sufficient time for antibodies to bind to antigens.

Removes unbound globulins to avoid false-positive results.

Ensures the proper amount of antiglobulin reagent is added to detect bound antibodies.

Optimizes agglutination by facilitating interaction between red blood cells and antibodies.

type of Coombs' reagent that is clear and contains antibodies to human IgG and human C3d.

It may detect C'-binding antibodies on RBCs (e.g., Jkᵃ).

It may detect C' on cells that may not be clinically significant (nuisance reactions).

A Coombs' reagent that targets either IgG or C3d specifically.

IgG monospecific AHG is green in color and contains antibodies to human IgG only.

C3d monospecific AHG is clear, comes in a small vial, and contains antibodies to human C3d only.

It produces fewer nuisance positives and is very specific in targeting either IgG or C3d.

Made by hyper-immunizing animals with human globulins and bleeding them for antisera to obtain high titer and high avidity specificity to human globulin.

onoclonal reagents are produced using hybridoma cells from mice, collecting culture or ascites fluid to yield antisera.

The potency of reagents is regulated by the FDA.

Polyclonal reagents are derived from animal sera with multiple antibody types, while monoclonal reagents are derived from hybridoma cells producing a single antibody type.

Animal hyper-immunized with human globulins, bled for antisera to obtain high titer, high avidity specificity to human globulin.

Use hybridoma cells from mice; collection of culture or ascites fluid yields antisera.

Product is regulated (by ____ ) for its potency.

Polyclonal reagents are derived from animals hyper-immunized with human globulins to produce antisera with high titer and high avidity specificity to human globulin.

Hybridoma cells from mice, with culture or ascites fluid collection.

Reagent RBCs coated with IgG that are added to all negative AHG test results

To check for neutralization of AHG by free globulin molecules or inadvertent omission of Coombs' reagent.

Other acronyms or symbols for Check Cells include:

1._____
2._____
3. √ = Check Cells

To all negative AHG test results.

They verify that AHG was not neutralized or omitted during testing.

In blood banking, "knowns" are tested against "unknowns."

Reagent antisera
Reagent RBCs

Patient RBCs
Patient serum

"Knowns" against "unknowns."

The DAT test checks if antibodies (Ab[s]) are coating the patient's RBCs.

To determine if antibodies (Ab[s]) are coating the patient's RBCs.

Reagent AHG antisera.

Patient RBCs

Antibodies (Ab[s]).

The IAT test checks the patient's serum/plasma to see if the patient has any antibodies (Ab[s]).

It checks the patient's serum/plasma to determine if there are any antibodies (Ab[s]).

Reagent Screening Cells. After incubation and washing of screening cells, Reagent AHG antisera (known) is added.

Patient serum.

For the IAT test, we test the patient's __________ to see if the patient has any Ab(s).

What is added after incubation and washing in the IAT test?

Detects IgG and/or C' already affixed to patient RBCs.

Detects antibodies (Ab) in patient sera, allowing IgG and/or C' to bind to reagent RBCs.

Within the patient's body (in-vivo).

As a result of performing a test or in-vitro.

One-stage procedure.

Two-stage procedure.

No, the patient's RBCs are tested with AHG without incubation.

Yes, it requires incubation before addition of AHG.

The DAT test is used to detect IgG and/or C' __________ affixed to patient RBCs.

The IAT test detects Ab in patient sera, allowing __________ to bind to reagent RBCs.

What clinical conditions are tested with the DAT test?

In antibody (Ab) tests in blood banking.

Detects IgG and/or C' already affixed to patient RBCs.

Detects antibodies (Ab) in patient sera, allowing IgG and/or C' to bind to reagent RBCs.

Occurs within the patient's body (in-vivo).

Occurs as a result of performing a test (in-vitro).

One-stage procedure.

Two-stage procedure.

Does not require incubation before adding AHG.

Requires incubation before addition of AHG.

Hemolytic Disease of the Newborn (HDN) and Hemolytic Transfusion Reaction (HTR).

Antibody (Ab) tests in blood banking.

Refrigerated or clotted samples lead to increased C' attachment.

Glass bits inside tubes, often caused by broken tubes in a box, can cause false positives.

Cells that self-agglutinate can give a false positive AGT result.

Bacterial contamination of cells or saline can lead to a false positive AGT.

Using cells with a positive DAT for IAT (auto control) may result in a false positive.

Excessive centrifugation or over-reading results can falsely indicate agglutination.

Cells with polyagglutination properties can produce a false positive AGT.

Antibodies dependent on preservatives in LISS reagents may lead to false positives.

Contaminants in the AHG reagent can lead to false positive reactions.

Saline containing heavy metals or colloidal silica can cause false positive results in A

Fails to remove unbound globulins, leading to false negatives.

Skipping the addition of AHG reagent results in no agglutination, giving a false negative.

Occurs due to inadequate washing, allowing free globulin molecules to neutralize AHG.

Aged or improperly stored AHG reagent loses potency, leading to false results.

Caused by the deterioration of antibodies (Ab) or complement (C'), resulting in a false negative reaction.

Poor incubation temperature or time prevents proper sensitization of RBCs.

Suspension that is either too weak or too heavy can skew test results.

Suboptimal centrifugation affects cell pellet formation and reading accuracy.

CLS shaking the tube too hard can disrupt agglutination, resulting in a false negative.

Misinterpreting the reaction leads to inaccurate results.

Missing serum in the test prevents antibody reactions, resulting in a false negative.