Coomb's Test and Compatibility Testing Flashcards
used primarily to detect globulins immunologically bound to red cells.
ANTIGLOBULIN TEST or COOMB’S TEST
basic type of reagent used in routine blood banking procedures, such as compatibility testing and antibody screening
Polyspecific or multivalent or broad-spectrum reagent
Polyspecific or multivalent or broad-spectrum reagent contains
anti—lgG and anti—C3d (anti-complement component)
________ reagent may contain antibodies of other specificities, such as anti—lgM, anti—lgA anti—c3b or anti—C4
Polyspecific or multivalent or broad-spectrum reagent
Example of this is the anti—lgG and the anticomplement C3b + C3d (previously called anti—non—gamma)
Monospecific or monovalent reagent
can detect most clinically significant antibodies
Anti—lgG
Monospecific reagent is useful in
differentiating agglutination produced by IgG antibodies
It isassociated with immune hemolysis; therefore, it is important that this component be included in anti—complement reagents to facilitate investigation of immune hemolytic anemias
C3d component
Monospecific sera containing _____ or _____ are not routinely used
anti— IgM or anti—lgA
used for the detection of in vivo red blood cell sensitization
DIRECT ANTIGLOBULIN TEST
Washed red blood cells from the patient are directly tested with antiglobulin serum
DIRECT ANTIGLOBULIN TEST
2 drops of _________ are to be tested in direct antiglobulin test
Patients red cells to be tested
used for the detection of antibodies that may cause red blood cell sensitization in vitro-
INDIRECT ANTIGLOBULIN TEST
antibody—containing serum is incubated with specified red blood cell
Indirect Antiglobulin Test
______ is to be tested in indirect antiglobulin test
Patien’ts serum
Method that uses ANTIGLOBULIN REAGENT and CENTRI 1 minute
DIRECT ANTIGLOBULIN TEST
This test uses 2 drops of cell suspension, incubation in water bath for 37 deg C for 15-30 mins
Indirect antiglobulin test
Extreme reticulocytosis
Lead poisoning
Drug—induced hemolysis
Viral diseases
Results in: False ______
False positive DAT is seen in:
Factors affecting the antiglobulin test in vitro:
Sensitization:
a)Temperature
b)Medium (saline, albumin, serum or enzyme)
c)Time of incubation
d)Proportion of serum to cells
Washing phase in antiglobulin should be ———– to minimize loss of cell bound antibody by elution
rapid and uninterrupted
Factors affecting the antiglobulin test
- Sensitization phase (in vitro only)
a)Temperature
b)Medium (saline, albumin, serum or enzyme)
c)Time of incubation
d)Proportion of serum to cells - Washing phase — it should be rapid and uninterrupted to minimize loss of cell-bound antibody by elution
- Effects of centrifugation
- Methods of reading result
- Controls used
Inadequate washing of red cells result in neutralization of the antiglobulin serum by trace amounts of residual globulin will result ______
False negative
Contamination with human serum will neutralize the reagent resulting in
False negative result
Elution of antibody from the red cells may take place if the test is interrupted or delayed, particularly during the washing phase
result:
False negative
The optimum temperature for reactivity of the antibody must be maintained during incubation to achieve maximal coating of the cells resulting in _______
False negative results
Preferred percentage in cell suspension for Coomb’s test
2% to 5% suspension of RBC
A cell suspension that is too heavy will not permit optimum coating with the antibody; if too weak, reading agglutination may be difficult. A 2% to 5% suspension of RBC is preferred.
False negative result
Test cells, test serum and antiglobulin serum lose reactivity if improperly stored resulting in
False negative result
Some antibodies may be detected only in the presence of active complement Anticoagulants such as EDTA will chelate calcium, preventing activation of complement Thus, the use of plasma rather than serum may lead to a ________ reaction
False negative
Old serum will also have impaired complement activity resulting in
False negative
Undercentrifugation or overcentrifugation causes
False negative reaction
Failure to check negative reactions, microscopically
False negative reaction
Cells have a positive direct antiglobulin test cannot be used with reagent antiserum that require an antiglobulin phase, because all such cells have agglutinated the antiglobulin serum
False positive results
Bacterial contamination of test cells or septicemia causes
False positive
Extreme reticulocytosis because of transfusion bound to reticulocytes reacting with antitransferrin in the globulin reagent
False positive reaction
Saline stored in glass bottle may contain colloidal silica leached from the container
False positive reaction
Saline stored in metal containers or used in equipment with metal parts, may contain metallic ions which may bring about nonspecific protein—coating of the red cells.
False positive reaction
Improperly prepared antiglobulin serum may contain traces of species specific antibodies.
When all the antiglobulin tests are weakly positive, the cause may be improperly cleaned glasswares or other forms of contamination.
False positive reaction
Overcentrfugation
False positive reaction
Patients or donor’s serum contain a naturally occurring cold autoantibody that can sensitize their own or other complement
False positive reaction
Red blood cells may be autoagglutinated before they are washed, and this agglutination may persist through veshing leading to a ______ reaction when antiglobulin serum is added
False positive
test between a patient or recipient of a blood transfusion and his prospective or donors.
crossmatch or compatibility test
Purpose of cross matching
To prevent any possible transfusion reaction due to antibody.
To ensure maximum benefit to the recipient.
crossmatch that uses patient’s serum and donor’s red cells (PS—DR)
prevent a transfusion reaction by detecting antibodies in the patient’s serum which would reduce the survival of the donors cells and to ensure maximum benefit to the recipient.
Major
uses patients red cells and donors serum (DS—PR).
detects antibodies in the donor which may be capable of affecting the recipients’ red cells
Minor
crossmatch WILL DETECT the following
Most recipient antibodies directed against antigens on the donor red blood cells.
Major errors in ABO grouping, labeling and identification of donors and recipients
1.Guarantee normal donor cell survival
2.Prevent recipient immunization
3.Detect errors of Rh and ABO typing
4.Detect all antibodies unless antibody is specific to the donor antigen being crossmatched
5.Detect antibodies to white blood cells or platelets
Detect errors of labeling and collection of sample
Crossmatch will not
Specimen for crossmatch
Fresh, not inactivated serum, less than 48 hours old must be used for the crossmatch
Hemolyzed patient samples should be avoided, because they may ask for hemolysis of donor red blood cells
washed red blood cell suspension may be prepared
are designed to detect IgM antibodies react optimally at room temperature (22 deg C) or lower.
Agglutination or hemolysis in this procedure at 22 deg C will detect
ABO incompatibility, often due to incorrect typing.
Cold agglutinin or autoagglutinins.
acting antibodies such as anti—M, anti—N, anti—S, anti—Lu, anti—F and anti— Le.
Saline Technique
The saline crossmatch at 37 deg C detects
Saline acting anti—Rh antibodies.
Some anti—M, anti—N and anti—S antibodies.
Some anti—K and anti—Le antibodies
act to increase the dielectric constant of the medium, thus allowing IgG antibodies to be demonstrated, Most IgG antibodies are detected
Bovine albumin
albumin crossmatch at 37 deg C detects
a.Most anti—Rh antibodies
b.Some anti—M, anti—N and anti—S antibodies
c.Some anti—Le antibodies
most important and most widely used serologic procedure in modem blood banking. Compatibility testing should be considered incomplete unless the technique is included as part of the test
Antiglobulin technique
serve as an irnportant “back up” test for the indirect antiglobulin test and detect many clinically significant IgG and saline—inactive IgM antibodies
Enzyme technique
enzyme used in enzyme technique
Trypsin
Papain
Ficin
Bromelin
developed to be an all-inclusive test, one which would detect clinically significant antibodies without sacrificing speed — so essential in an emergency, or simplicity so important in prevailing errors
Broad spectrum Compatibility Testing
Three phases of broad spectrum compatibility test
a.Protein or room phase
b.Thermo or incubation period
c.Antiglobulin phase
Cells appear to be agglutinated, yet they have the appearance of a “stocks of coins’ The formation can be dispersed or diminished by the addition of a drop of saline to the slide (diluting the serum-cell mixture with saline).
Roleaux formation
often encountered in certain disease states — notably myelomatosis and macroglobulinemia. It is also caused by a raised serum giobulin concentration and is often seen in patients with serum protein abnormalities. Certain synthetic plasma expanders, such as dextran can also cause —— formation.
rouleaux
simply the spontaneous clumping of all cells against a given serum. There are two main types of this reaction
Panagglutination
due to a bacterial contaminant, which exposes a latent receptor known as T. All human red cells contain this antigenic receptor but are usually non—reactive with ant—T. Since all human adult sera contain anti—T in varying amounts, agglutination would occur with all such cells.
Bacteriogenic panagglutination / Huebner Thomsen—Friedenrich phenomenon
may be used by acquired hemolytic anemia or by rare specific antibodies. The reaction takes place at 37 deg C and occasionally in the cold. The direct antiglobulin test is often positive
Nonbacteriogenic panagglutjnation
reaction of red cells with varying proportions of fresh normal allogeneic or intert AB sera. The reaction takes place at 20deg C and becomes weak or inactive at 37 deg C.
Polyagglutination
are relatively common problems in the laboratory, They are simple antibodies reacting at temperatures between 4 deg C and 20 deg C and they may be specific or nonspecific
Cold grouping
Usually present in cord samples that have been collected by cutting the umbilical cord and allowing the blood to drain into a collection tube
Wharton’s jelly
phenomenon occasionally occurs in the antiglobulin technique and is thought to be caused by partial neutralization of the antiglobulin reagent (i — the antibody is loosely bound or does not remain on the cells).
Prozones
undergo a diminishing of reactivity on storage. The deterioration of antibodies is dependent on the specificity of the antibody and the length and method Of storage.
Antigen or antibody deterioration
