IMMUNOHEMATOLOGY Flashcards
Gel test was invented in ______ by ________.
1985, Dr. Yves Lapierre
Two antigen/antibody reactions
agglutination and hemolysis
In gel testing, ________ is detected however hemolytic reaction is not detected
hemeagglutination
PARALLEL TESTING
gel test + tube test
immortalized antibody producing cell
hybridoma cell
the antibody producing capacity of hybridoma cell is due to
plasma cell
what is elution
detachment/dissociation of antigen or antibody
IMPORTANCE OF WASHING IN DAT
(1) presence of other unbound antibodies
(2) washing phase
(3) addition of AHG
(4) IgG bridge/ lattice formation
(5) agglutination
Steps in Direct AHG
- Washed patient’s red cell (3x), sensitized in vivo
- Addition of AHG reagent (Coomb’s sera)
- Visual red cell agglutination
Steps in Indirect AHG for human red cells
- Human red cells + Human (patient serum) IgG antibody
- Red cell (in vitro) sensitization
Steps in Indirect AHG for sensitized red cells
- sensitized red cells + Coomb’s sera AHG reagent
- Visual red cell agglutination
Gel test principle
size exclusion chromatography
what gel is used in gel test
dextran polyacrilamide gel
steps/procedure in gel test
- addition of cells
- addition of plasma/serum
- incubation for IgG - body temp
- centrifugation - 5 mins
- results - more than 10 mins
the bigger the size, the ______ the position of cells in the medium
higher
reagent is already pre-dispensed, only add the sample to microtube
a. specific
b. low ionic antiglobulin
c. neutral/plain
a
reagent is not added to the gel, it is added along with the sample in microtube
a. specific
b. low ionic antiglobulin
c. neutral/plain
c
specific for sensitization test. IgG antibodies are detected. AHG reagent is pre-dispensed in gel matrix
a. specific
b. low ionic antiglobulin
c. neutral/plain
b
advantages of using gel testing
- standardization
- stable and well defined agglutination reaction
- decreased sample volume <1mL
- enhanced specificity and sensitivity, no need for cell washing
ABO forward/direct cell typing is and example of
a. specific
b. low ionic antiglobulin
c. neutral/plain
a
INDIRECT/reverse/backward typing is an example of
a. specific
b. low ionic antiglobulin
c. neutral/plain
c
In gel testing, stability is up to _______
3 days
In conventional tube method, stability is up to ____________
an hour
In slide method, stability is up to _______
1 min
If RBC are unagglutinated, it will pass thru the gel and will form __________ at the bottom
cell button
one solid clump, 100 percent antigen reacted with antibodies
a. 3+
b. 4+
c. 1+
d. +/-
b
several large sized clumps, 75 percent antigen reacted with antibodies
a. 3+
b. 4+
c. 1+
d. +/-
a
several medium sized clumps, 50 percent antigen reacted with antibodies
a. 3+
b. 4+
c. 2+
d. +/-
c
several numerous small sized clumps, <25 percent antigen reacted with antibodies
a. 3+
b. 4+
c. 1+
d. +/-
c
weak reaction, very small sized clumps, <25 percent reacted with antibodies
a. 3+
b. 4+
c. 2+
d. +/-
d
RBC on top most part of the cell
a. 3+
b. 4+
c. 2+
d. +/-
b
RBC on upper part of gel matrix
a. 3+
b. 4+
c. 2+
d. +/-
a
RBC is disseminated on gel matrix with most RBC found at the center
a. 3+
b. 4+
c. 2+
d. +/-
c
RBC are found on lower part of the gel
a. 3+
b. 1+
c. 2+
d. +/-
b
RBC found near at the bottom of the tube
a. 3+
b. 4+
c. 2+
d. +/-
d
What antibody cannot agglutinate on its own and is the smallest so it can cross the placenta
IgG
A technique for detecting cell-bound immunoglobulin and is used to detect incomplete antibodies (IgG).
AHG Coomb’s test
acts as a bridge/link so IgG can form lattice sequence
AHG
color of AHG/Coomb’s test
GREEN
Complement binding via classical pathway from MOST - LEAST potent
a. IgG subclass 1, 2, 3
b. IgG subclass 2, 1, 3
c. IgG subclass 3, 1, 2
d. IgG subclass 1, 3, 2
c
natural, big pentamere
a. IgG
b. IgM
b
complete best agglutinating antibody
a. IgG
b. IgM
b
cold-reacting, 1-6 deg
a. IgG
b. IgM
b
saline reactive
a. IgG
b. IgM
b
ABO Antibodies
a. IgG
b. IgM
b
complement binding and is more potent
a. IgG
b. IgM
b
Immune
a. IgG
b. IgM
a
incomplete, coating/sensitizing
a. IgG
b. IgM
a
warm-reacting, 37 deg and is clinically significant
a. IgG
b. IgM
a
albumin/AHG reagent
a. IgG
b. IgM
a
Rh antibodies
a. IgG
b. IgM
a
consists of a pool of rabbit anti-human IgG and mouse monoclonal anti C3b and anti C3d
a. monospecific AHG reagent
b. polyspecific AHG reagent
b
Also referred to as broad spectrum coombs reagent
a. monospecific AHG reagent
b. polyspecific AHG reagent
b
method of preparation in polyspecific AHG reagents
conventional/classic method (hyper immunization of rabbit)
contains only one antibody specificity
a. monospecific AHG reagent
b. polyspecific AHG reagent
a
can either be Anti-IgG, Anti-C3b or C3d
a. monospecific AHG reagent
b. polyspecific AHG reagent
a
METHOD of preparation in monospecific AHG reagents
hybridoma method (KOHLER and MILSTEIN technique)
hybridoma cell is made up of
mouse plasma cell AND myeloma cell
first stage in Ag-Ab reaction
sensitization
it occurs when antibodies react with antigens on the cells and coat the cells
sensitization
antigen binding fragment
Fab
determinant, reacts with antibody
epitope
second stage in Ag-Ab interaction
agglutination
it occurs when abs on coated cells form cross-linkages between cells resulting in visible clumping
agglutination
cross linking of Abs adjacent to antigen
lattice formation
single most important serologic test to diagnose HDFN
DAT
detects in vivo sensitization of red cells with IgG and/or complement
DAT
Investigation of transfusion reactions
a. IAT
b. DAT
b
diagnosis of autoimmune and drug-induced hemolytic anemias
a. IAT
b. DAT
b
cells used for DAT should be collected into either ———— to minimize the possibility of ——- attachment of complement components
EDTA or citrate containing anticoagulant
IN VITRO
the ———— is needed to demonstrate antibodies in the event of in vivo erythrocyte sensitization
DAT
a two-step procedure that determines in vitro sensitization of red cells
a. IAT
b. DAT
a
in IAT, what are the two procedures involved
sensitization (incubation/thermophase)
agglutination (AHG step)
incubation/thermophase
a. agglutination
b. sensitization
b
AHG step
a. agglutination
b. sensitization
a
detection of incomplete antibodies in compatibility testing or to screening cells in antibody
a. DAT
b. IAT
b
identification of antigen specificity, using a panel of red cells
a. DAT
b. IAT
b
determination of red cell phenotype using known antisera (Du testing)
a. DAT
b. IAT
b
titration of incomplete antibodies
a. DAT
b. IAT
b
used for confirmatory testing for weak D testing
IAT
In IAT, we use this as confirmatory testing for weak D specifically what type?
low grade type
report for low grade weak D is
Rh positive
If anti-D is negative, report immediately
TRUE or FALSE
false
If IAT is positive it is considered as —-
Rh pos because IAT is used to confirm for the presence of low grade Du antigen
AHG is also referred to as
anti-antibody
Minimum ratio of serum to cells
40:1, 2 drops serum and 1 drop of 5 percent v/v cell suspension
optimal temperature for AHG testing
37 degC
incubation time for AHG testing in saline suspension
30-120 mins
incubation time for AHG testing in LISS suspension
10-15 mins
PROZONE
a. excessive Ag
b. excessive Ab
b
POSTZONE
a. excessive Ab
b. excessive Ag
b
in AHG reaction medium, ___ minute saline test = ___ minute albumin test
a. 30, 20
b. 15, 30
c. 30, 60
d. 60, 30
d
____ is said to reduce the zeta potential between RBCs thus increasing the rate of Ab uptake on the cell
22 % albumin
____ also increases the sensitivity and shortens incubation times
LISS
LISS is an example of _____ which enhances ag/ab interaction
potentiator
in AHG test, saline for washing should be fresh and buffered to a pH of ______
7.2-7.4
Addition of AHG reagents should be added to washed cells immediately after washing
TRUE or FALSE
True
In AHG test, centrifugation is done for
1000 rcf for 15-20 seconds
factors affecting AHG test
- ratio of serum to cells
- temp
- incubation time
- reaction medium
- washing of cells
- saline for washing
- immediate addition of AHG reagent
- centrifugation
Incomplete washing
a. neg
b. pos
a
extreme pH (cause protein denaturation)
a. neg
b. pos
a
too fast, too long
a. false pos
b. false neg
a
too slow, too short
a. false pos
b. false neg
b
spontaneous elution
a. false pos
b. false neg
b
autoagglutinable cells
a. false pos
b. false neg
a
bacterial contamination or other contamination in cells or saline
a. false pos
b. false neg
a
cells with a (+) DAT used for IAT
a. false pos
b. false neg
a
overcentrifugation or overreading
a. false pos
b. false neg
a
polyagglutinable cells
a. false pos
b. false neg
a
dirty glasswares
a. false pos
b. false neg
a
saline contaminated with silica or heavy metals
a. false pos
b. false neg
a
specificity is DECREASED
a. false pos
b. false neg
A
sensitivity is DECREASED
a. false pos
b. false neg
b
inadequate or improper washing of cells
a. false pos
b. false neg
b
most common cause of false neg in AHG technique
inadequate or improper washing of cells
AHG reagent nonreactive owing to deterioration or neutralization
a. false pos
b. false neg
b
AHG reagent not added
a. false pos
b. false neg
b
serum not added in the indirect test
a. false pos
b. false neg
b
serum non reactive owing to deterioration of complement
a. false pos
b. false neg
b
inadequate incubation conditions
a. false pos
b. false neg
b
postzone and prozone (cell suspension either twoo weak or too heavy)
a. false pos
b. false neg
b
undercentrifugation
a. false pos
b. false neg
b
poor reading technique
a. false pos
b. false neg
b
to allow sensitization reaction, contains agent to promote interaction of red blood cell
a. LIP- low ionic polybrene technique
b. ELAT- enzyme linked antiglobulin test
c. Solid phase method - direct/indirect
a
similar to ELISA, uses RBC (contains IgG). Its primary antibody uses AHG.
a. LIP- low ionic polybrene technique
b. ELAT- enzyme linked antiglobulin test
c. Solid phase method - direct/indirect
b
occurs in microwells, Antigen are bound to bottom of well and patient’s plasma/serum is incubated in the well
a. LIP- low ionic polybrene technique
b. ELAT- enzyme linked antiglobulin test
c. Solid phase method - direct/indirect
c
substrate changes in color when there is AHG sensitized red cell binding.
a. LIP- low ionic polybrene technique
b. ELAT- enzyme linked antiglobulin test
c. Solid phase method - direct/indirect
b
in ELAT, color of substrate is _________ to quantity of sensitized Antibodies present in sample
directly proportional
uses colorimeter as principle
a. LIP- low ionic polybrene technique
b. ELAT- enzyme linked antiglobulin test
c. Solid phase method - direct/indirect
b
used to detect as many clinically significant antibodies outside ABO system
antibody screening procedure
in antibody screening procedure, ________ is used to differentiate different types of antibodies
enzyme procedure
enzyme reagents
FICIN, BROMELIN, PAPAIN, TRYPSIN
for IgG alloabs (qualitative)
patient serum + screening cells
for autoantibodies
patient serum + red cells
______________ now replaces the minor cross-matching procedure in routine pre-transfusion
antibody screening procedure
what blood type is used to as screening cell to prevent ABO abs from producing reactions
O
if the antibody screen is reactive, this must be determined
antibody specificity
panel used for antibody identification
11 reagent panel
use of ____, _____ and ______ will inactivate some antigens specially ________.
AET, DTT and ZZAP
KELL
in the prewarm procedure, this may be removed
clinically insignificant cold antibodies
in the prewarm procedure, patient serum, reagent red cells and enhancement medium can be warmed separately at
37 degC for 5-10 mins prior to mixing
these reagents denatures IgM antibodies by breaking disulfide bonds
sulfhydryl or thiol reagents *DTT and 2-ME
use of _____________ techniques to remove unwanted antibodies such as cold or warm autoantibodies, or to help resolve multiple antibodies
adsorption and elution
used to remove unwanted antibodies from Serum
a. elution
b. adsoprtion
b
used to dissociate IgG abs from sensitized cells
a. elution
b. adsoprtion
a
this may also be used as adsorbents for anti-I since they are rich in I antigen.
rabbit cells
these autoantibodies can be adsorbed onto the patient’s enzyme pretreated cells at 4 degC
I, H, or IH
the recovered antibody, eluate can be tested like ____ to determine antibody’s _____
serum, specificity
contains detached antibodies
eluate
example of elution techniques
heat, freeze-thaw, use of organic solvent, acid eluates, using ZZAP or chloroquine diphosphate
mixture of DTT and papain that is used to remove Ab from sensitized red cells and to enzyme treat them at the same time
a. AET
b. DTT
c. ZZAP
d. chloroquine disphosphate
c
reagent used to remove IgG Abs from the surface of sensitized cells; inactivates Bg antigens
a. AET
b. DTT
c. ZZAP
d. chloroquine disphosphate
d
landsteiner-miller heat
a. first generation
b. second generation
c. third generation
a
lui-freeze thaw
a. first generation
b. second generation
c. third generation
a
uses organic solvents
a. first generation
b. second generation
c. third generation
b
uses non-hazardous chemical agents: acid elution
a. first generation
b. second generation
c. third generation
c
another technique for facilitating antibody identification is
NEUTRALIZATION
Hydatid cyst fluid
a. anti P-1
b. anti I
c. anti ABH
d. anti Lea and anti Leb
a
Plasma or serum with Le subs
a. anti P-1
b. anti I
c. anti ABH
d. anti Lea and anti Leb
d
Pooled serum or plasma
a. anti P-1
b. anti I
c. anti chido anti rogers
d. anti sda, tamms horsefall protein
c
urine
a. anti P-1
b. anti I
c. anti chido anti rogers
d. anti sda, tamms horsefall protein
d
saliva of ‘secretors’
a. anti P-1
b. anti ABH
c. anti chido anti rogers
d. anti sda, tamms horsefall protein
b
human milk
a. anti P-1
b. anti ABH
c. anti I
d. anti sda, tamms horsefall protein
c
in heat elution, it will produce
dissociated Abs and destroyed erythrocytes
in ether elution, name the parts in the tube
upper part- ether
middle- destroyed erythrocytes
lower part- dissociated abs
pre-transfusion procedure, composed of series of tests to ensure the safety of the recipient during blood transfusion, to select the appropriate blood unit for transfusion
compatibility/crossmatch
in compatibility/crossmatch, what is the preferred specimen
serum
in compatibility/crossmatch, why is serum preferred than plasma?
- may cause small fibrin clots, difficult to distinguish from true agglutination
- may inactivate complement, antibodies will not be detected
in compatibility/crossmatch, age of specimen must be
as fresh as possible
in compatibility/crossmatch, age of specimen if the patient has been transfused or pregnant within the past 3 months must be
less than 3 days old
in compatibility/crossmatch, the sample storage required by AABB must be stored between
1-6 degC for at least 7 days after transfusion
in compatibility/crossmatch, the sample storage required by AABB must be stored between
1-6 degC for at least 7 days after transfusion
most critical pretransfusion serologic test
ABO grouping
if the patient’s ABO group cannot be satisfactorily determined and immediate transfusion is essential, what blood type should be utilized
blood type O
if Rh type of the recipient cannot be determined and transfusion is essential, what Rh blood type should be given
O -
routinely done on crossmatching
major or minor
major
Major x-match
a. Donor’s serum + recipient’s cells
b. Donor’s cells + recipient’s serum
b
Minor x-match
a. Donor’s serum + recipient’s cells
b. Donor’s cells + recipient’s serum
a
Patient’s serum + donor’s red cells are tested in saline medium. IgM antibodies are detected
a. AHG technique
b. Broadspectrum technique
c. Saline technique
d. High protein/albumin technique
c
PS+DR tested in high protein media. IgG abs are detected (Rh Abs)
a. AHG technique
b. Broadspectrum technique
c. Saline technique
d. High protein/albumin technique
d
PS+DR tested in AHG medium. Non-agglutinating IgG are detected
a. AHG technique
b. Broadspectrum technique
c. Saline technique
d. High protein/albumin technique
a
composed of 3 stages; (1) IS (2) Thermophase/Incubation (3) AHG
a. AHG technique
b. Broadspectrum technique
c. Saline technique
d. High protein/albumin technique
b
immediate spin in saline detects
IgM
thermophase/37degC incubation detects
IgG
Check cells/Coombs control cells (IgG sensitized cells) should be added to tubes that demonstrate no agglutination. For results to be considered valid, _____ must occur.
agglutination
invalid check cell is due to
failure to wash cell
unbound Abs neutralized AHG is due to
reagent already deteriorating, not reacting
What is the possible problem in this crossmatch
Ab screen: (-)
AC: (-)
Major Xmatch: (+)
ABO/Rh typing error
Donor unit with (+) DAT
Patient with low incidence Ab
What is the possible problem in this crossmatch
Ab screen: (+)
AC: (-)
Major Xmatch: (+)
patient alloantibody
What is the possible problem in this crossmatch
Ab screen: (+)
AC: (+)
Major Xmatch: (+)
patient autoantibody
rouleaux
substances that are able to carry oxygen in the absence of intact red cells
blood substitutes
common antibody encountered in BB
anti big K
______ is tested when AHG is negative
check cell
advantages of SFHS
longer shelf life
very stable
not immunogenic
no req for blood typing procedures
disadvantages of SFHS
short intravascular halflife
possible toxicity
high O2 affinity
high oncotic effect
advantages of PFCs
biologic inertness
not immunogenic
easily synthesized
disadvantages of PFCs
adverse clinical effect
high O2 affinity
retention in tissues
