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BB LAB FINALS > midterms > Flashcards

midterms Flashcards

(53 cards)

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1
Q

Provides optimum antigen
concentration for cell typing

A

● 3-5% Concentration
RCS

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2
Q

TUBE FOR RCS

A

EDTA

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3
Q

RCS PROCEDURE

A

Procedure
○ Compute for the required “VOLUME
OF CELLS” and “VOLUME OF NSS”
to be added
○ Wash 2 mL of EDTA-blood 3x using
NSS
○ After the last wash, decant the
supernatant
○ Transfer volume of cells to another
test tube

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4
Q

NSS Preparation (0.85%)

A

● Add 85g of NaCl to 1L of warm
distilled water
● Shake until all salt crystal dissolves
● Autoclave for 15 min at 121°C

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5
Q

IMMUNOHEMATOLOGIC REACTION
SENSITIZATION

A

● Antigen-antibody ratio
● pH
● Temperature
● Incubation time
● Ionic Strength

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6
Q

LATTICE FORMATION

A

● Zeta potential
● Zone of equivalence
● Centrifugation

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7
Q

AB BINDING WITHOUT CLUMPIN

A

SENSITIZATION

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8
Q

ANTIBODY CROSSLINKING LEADING TO VISBLE AGGLKUT

A

LATTICE FORMATION

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9
Q

WHICH IS IGG SENSITIZATION OR LATTICE

A

SENSITIZATION

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10
Q

Process of freeing antibody molecules from
sensitized red cells

A

ELUTION

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11
Q

Performed when Coombs Test is POSITIVE

A

ELUTION

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12
Q

Disrupting the linkage between
antigen and antibody

A

Elution is done by:

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13
Q

Used for eluting warm-reacting auto- or allo-
antibodies

A

ACID ELUTION
● Glycine-HCl
● Most common

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14
Q

HOW DOES ACID ELUTION WORK

A

● Lowers pH – causing antibody to dissociate

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15
Q

CLINICAL SIGNIFICANCE
OF ACID ELUTION

A

● Investigation of HTR
● Diagnosis of AIHA
● Diagnosis of ABO HDFN
● Identification of specificity when multiple
antibodies exist in a patient’s serum
● Phenotyping red cells in patient with a
positive DAT

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16
Q

ORGANIC SOLVENTS
ELUTIKON

A

ORGANIC SOLVENTS
● Ether, chloroform
● Dissolved lipid bilayer of erythrocytes

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17
Q

HEAT ELUTION
PROCEUDRE

A
  1. Centrifuge the sample with positive DAT
  2. Separate the RBC properly in a labeled test
    tube
  3. Prepare 5% RCS from the sample
  4. Wash 6 to 8 times
  5. Add 20 drops of 6% albumin to the washed
    RBC
  6. Place the tubes at 56 degree Celsius for 10
    minutes. Agitate periodically
  7. Centrifuge the solution at 3400 rpm for 5
    minutes
  8. Immediately transfer the supernatant in a
    clean test tube
  9. Test the eluate against panel of cells
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18
Q

HEAT ELUTION whatreagent when to incubate and what centri

A

Add 20 drops of 6% albumin to the washed
RBC

Place the tubes at 56 degree Celsius for 10
minutes. Agitate periodically

Centrifuge the solution at 3400 rpm for 5
minutes

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19
Q

● Cells are lysed in the process -18 °C

A

LUI FREEZE THAW

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20
Q

process of removing antibodies present in the
serum of the patient or donor using rcs

A

autoADSORPTION

pag using rbc with known phenotype alloadsorption

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21
Q

Performed for patients that has not
been transfused or pregnant in the
last 13 weeks

A

AUTOADSORPTION

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22
Q

AUTOADSORPTION process

Study These Flashcards
A
  1. To 0.5 ml of test serum, add an equal amount
    of the corresponding adsorbing cells to be
    used.
  2. Shake gently and spin down.
  3. Remove supernatant and then add an equal
    amount of the corresponding adsorbing cells
    again.
  4. Shake gently and spin down.
  5. Repeat the addition and removal of
    supernatant until all antibodies present in
    the serum have been removed.
  6. Final solution should show no agglutination or
    hemolysis.
  7. Record number of washings performed.
23
Q

DIAGNOSTIC SIGNIFICANCE
of autoadosption

Study These Flashcards
A

● Removing autoantibody permits detection
and identification of coexisting alloantibodies.
● Reagent preparation
● Separating multiple antibodies present in
plasma or eluate to aid in identification
● Confirmation of the presence of a weak
antigen on red cells (ABO discrepancies)
● Confirmation of antibody specificity

24
Q

Uses RBC with known phenotypes to
selectively remove alloantibodies from
patient’s serum
● Used for patient with recent transfusion

Study These Flashcards
A

ALLOADSORPTION

25
Determination of the level of antibodies present in a patient’s blood
ANTIBODY TITRATION
26
Examples where antibody titration is useful
● Estimating antibody in allo-immunized pregnant women ● Elucidating autoantibody specificity ● Characterizing HTLA
27
ANTIBODY TITRATION
Process 1. Prepare 5% RCS of reagent cells. 2. Serially dilute serum in 12 tubes. (1:1 to 1:2048) 3. Add appropriate cells into each tube 4. Centrifuge for 1 minute at 3400 rpm. 5. Agitate tubes and evaluate for agglutination 6. Set aside the positive tubes 7. Add 22% BSA to the negative tubes then incubate for an hour 8. Wash the incubated tubes for 3 times. Decant the last wash 9. Add 2 drops of AHG then resuspend 10. Centrifuge for 1 minute at 3400 rpm. Then check for agglutination
28
DIAGNOSTIC SIGNIFICANCE of antibody titration
DIAGNOSTIC SIGNIFICANCE ● Monitoring the risk of HDFN in alloimmunized mothers ● Isohemagglutinin titration (in donor or patient) ● HTLA reactivity
29
monitored to determine eligibility to receive a non-ABO identical organ
ISOHEMAGGLUTININ TITER
30
HTLA REACTIVITY is usually directed against
Usually directed against Knops, Ch/Rg, Cost, JMH
31
Used to detect red cell sensitization with IgG alloantibodies, IgG autoantibodies complement proteins
ANTIGLOBULIN TEST
32
used to detect these sensitized cells by binding to bound globulins increasing the grade of agglutination
AHG serum
33
Anti Human globulins are produced from
immunized animals.
34
POLYCLONAL AHG is from
animals hyperimmunized with human globulins
35
High tittered and High Avidity
● POLYCLONAL AHG
36
Advantage: May detect complement dependent antibodies (Kidd, Kell, Duffy)
● POLYCLONAL AHG
37
MONOCLONAL AHG is from
○ Hybridoma cells from mice
38
AHG Reaction
When Coombs reagent is added, the Fab portion of AHG will attach to the Fc portion of the TWO ADJACENT IgG molecules Thereby, bridging the gap between the two cells and causes AGGLUTINATION
39
● Detects circulating antibodies against RBC
INDIRECT COOMBS TEST (IAT)
40
Sensitization happens IN VITRO; unbound antibodies
INDIRECT COOMBS TEST (IAT)
41
IAT PROCEDURE
PROCEDURE ● Place 2-4 drops of patient’s serum in a test tube ● Add 1 drop of 2-5% Reagent RCS ● Mix and incubate for 60 minutes ● Add 1-2 drops of AHG serum ● Centrifuge at 3400 for 1 minute ● Gently agitate to dislodge and examine for agglutination ● Add check cells if negative
42
– may cause clinically significant antibodies to not react in IAT
Albumin
43
○ – 30-60 minutes incubation time
LISS
44
may cause aggregation of RBC; thereby reading after 37 deg Celsius is omitted in IAT
PEG
45
IAT CLINICAL SIGNIFICACE
Clinical Significance ● Presence of Maternal Antibodies against fetus ● Detection of antibodies not detected by other techniques (Le, Fy, Jka) Compatibility/Incompatibility of patient and blood unit ● Presence of unexpected antibodies
46
DIRECT COOMBS TEST (DAT) PROCEDURE
● Prepare 3-5% RCS of the patient’s blood ● Transfer 2-3 drops RCS in another test tube ● Add 2-3 drops of AHG ● Incubation for 15-30 minutes at 37 deg Celsius ● Centrifuge under 3400 rpm 1 minute ● Gently agitate to dislodge the cells ● Check for agglutination ● Add Coombs check cells to negative tubes
47
To assess a true negative reaction,____are used
To assess a true negative reaction, Coombs check cells are used
48
QUALITY CONTROL IN AHG TESTING
For IgG coated CC: Use RH antibodies ○ For C3b coated CC: Incubate cells in LISS or anti-Lea or anti-I ○ For C3d coated CC: Incubate C3b sensitized cells with fresh serum or Trypsin
49
Preparation of Check Cells
1) Prepare 5% suspension of Blood Type O RhD positive sample 2) Mix equal amounts of Anti-D and the freshly prepared RCC. Incubate for 15 minutes at 37 deg Celsius 3) Wash 3x with isotonic saline 3-5 times 4) Decant the last wash 5) Resuspend using NSS to make 5% suspension of Coombs Control Cells
50
CHECK CELL INTERPRETATION
INTERPRETATION ● (+) AGGLUTINATION = TRUE NEGATIVE REACTION ➢ AHG reagent was not neutralized during the process ● (-) AGGLUTINATION = FALSE NEGATIVE REACTION ➢ AHG reagent was neutralized during the process
51
● Carbohydrate-binding proteins
LECTINS
52
Preparation of Lectin g
1) Grind the seeds until “Coarse sand” texture using mortar and pestle 2) Place the ground seen in a test tube or a small beaker and add NSS 3) Incubate at room temperature for 4 to 12 hours with occasional stirrinjjjjjjjjjjjj 4) After centrifugation, filter the supernatant fluid 5) Determine the activity and adjust the strength of the lectin
53
(Phaseolous vulgaris)
kidney Bean Lectin

BB LAB FINALS (4 decks)

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