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Special Techniques Flashcards

1
Q

What are the sulfhydryl reagents?

A

DTT - dirhiothreitol
2-ME - 2-Mercapto-Ethanol
AET - 2 aminoethylisothiouronium

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2
Q

How do sulfhydryl reagents work?

A

Reduce the double bond of the cysteine residue which breaks the secondary structure of the polypeptide

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3
Q

What is 0.01 M DTT used for?

A

Disperse autoagglutinins from the cell

Differentiate IgM and IgG

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4
Q

What is 0.2 M DTT used for?

A 
Denatures antigens (KEL, LU, DO, ect.)
Used in ZZAP or WARM reagent
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5
Q

What is the procedure to disperse autoagglutinins?

A

RBC’s coated with IgM cold auto antibodies can spontaneously agglutinate causing false positive rxns
DTT breaks the disulfide bond in the IgM
- Equal part 0.01 M DTT + 50% RBC suspension (PBS)
- Incubate 37C for 15 min
- Wash cells w/ saline and make 2-5% cell suspension
- Include a 6% albumin or mono control in testing

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6
Q

What is the procedure to differentiate IgM and IgG?

A

DTT breaks the S-S bonds of monomeric bonds of IgM
-Equal parts of 0.01 M DTT + serum/plasma
-Dilution control = Equal PBS and Serum/Plasma
-Incubate 37C for 30-60 min
-Test DTT treated serum using standard methods.
If DTT treated plasma is neg and ctr is positive = IgM
If DTT is positive and ctr is positive = IgG
If both ctr and DTT are negative = Invalid (diluted)

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7
Q

What is the procedure for using DTT to denature antigens?

A

Useful to destroy k, Kpb, Jsb, Lub, and other high’s

  • 4 volume of 0.2 M DTT + 1 volume of washed Packed RBC’s
  • Incubate 37C for 30-60 min
  • Wash with saline
  • Resuspend cells in 2-5% cell suspension
  • Ctr = Treat K+ w/ DTT and test with anti-K
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8
Q

What is in ZZAP?

A

0.5 mL of 1% ficin or papain
2.5 mL of 0.2 M DTT
2.0 mL of PBS
AKA WARM made by imunocor
Store at 4C for 5 days

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9
Q

What is ZZAP used for?

A

Used for warm auto adsorption

  • DTT removes bound IgG; free up binding sites
  • Ficin/Papain removes part of they glycoprotein structure enhancing antibody binding and adsorption
  • Can be used in allo adsorption to enhance binding
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10
Q

What antigens are destroyed by ZZAP?

A

DTT destroys YT, DO, CR, IN, LU, JMH, KN, LW, KEL

Enzyme destroys Fya/Fyb, MNS(s), EnaTS, EnaFS, Ch/Rg, XG, JMH, YT, Ge2, Ge3, IN, KN (KN resistant to papain)

Chido and Rodgers eXcluded John MH Yet GE2 and Ge INcluded Knops

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11
Q

What is the procedure for using ZZAP in adsorptions?

A

2 vol of ZZAP + 1 volume of washed packed RBC’s
Incubate 30 min at 37C
Wash cells x3 and pack RBC’s
Add patient plasma to packed RBC’s and mix
Incubate for 30-45 min at 37C
Centrifuge and test plasma or repeat adsorptions if needed

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12
Q

What are examples of how ZZAP can be used?

A

Can be used in place of enzymes or DTT

  • Make K0 cells to ID high incidence KEL antibody
  • Identify Kel allo antibodies in AIHA
  • Characterize Kell auto antibodies
  • Characterize Kell antigens
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13
Q

What is AET used for?

A

Used like DTT

  • Destroy Kel antigens
  • Characterize New Kell antigens (if not destroyed = not Kell)
  • Determine if auto antibody has Kell specificity
  • Anti-Gerbich reacts strongly with AET treated cells
  • Kx is not destroyed, anti-Kx reacts strongly with DTT/AET treated cells
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14
Q

How does AET work?

A

No one knows!
AET treated cells look like PNH cell
PNH cells have increased sensitivity to C’ lysis but have normal K antigens

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15
Q

What is the procedure to use AET?

A

AET is a dry powder = 6% AET + distilled water and adjust pH to 8.0 with NaOH
One volume of washed packed RBC’s + 4 vol of AET
Incubate at 37C for 20 min
Wash cells 5-7x and test cells with anti-K as ctr

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16
Q

What is EGA?

A 
EDTA glycine-Acid
10% EDTA
0.1 M glycine HCL
1 M of TRIS (buffer)
EDTA is used instead of saline because saline would cause hemolysis
Buffer is used to bring pH back to 7-7.5
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17
Q

What is EGA used for?

A

Treating cells with low pH dissociates bound IgG for phenotyping with IAT
Inactivates antigens in the Kell, Bg, and Era
Does not affect Duffy or MNS antigens

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18
Q

What is the procedure for using EGA?

A

Wash cells 6x with saline
Mix Glycine and EDTA in a 20:5 ratio
Add 20 volumes of EGA to 10 volumes of RBC’s
Mix and incubate at RT for 2 min then add TRIS
Centrifuge and remove supernant
Wash RBC’s 4x with saline
DAT is done to verify IgG is removed

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19
Q

What are the limitations of EGA?

A

May not work (works about 85%)
May over treat cells and causing tanning or clumping of cells
Does not remove C so use anti-IgG

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20
Q

What is CDP used for?

A

Chloroquine Diphosphate
Dissociate cell bound IgG w/o damaging antigens
Allows for cell phenotyping
Also removes Bg antigens

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21
Q

What is the procedure for using CDP?

A

0.2 mL of wash packed RBC’s + .8 mL of CDP
Incubate for 30 min at RT
Do DAT on cells to ensure IgG is removed
IF DAT is positive incubate for another 30 min

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22
Q

What is the disadvantage of CDP?

A

May take 2 hours and may not completely remove antibody from DAT positive cells

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23
Q

What chemicals are commonly used to differentiate IgG and IgM?

A

DTT and 2-ME

*2-ME has strong odor so DTT is more common

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24
Q

What chemicals are used to disperse cold autoagglutinins from the RBC’s?

A

DTT and 2-ME

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25
What chemicals are used to destroy red cell antigens?
DTT and AET destorys KEL,LU, LW, DO, CR, YT, KN, IN, JMH?
26
Compare EGA and CDP?
EGA is more effective but destorys Kell and Era antigens | EGA is faster
27
Why must autologous cells be treated before adsorption procedures?
Autologous cells antigen binding sites may be blocked with IgG so antibody can't bind. DTT, enzymes, ZZAP, AET, CDP, and heat elution can be used to remove bound IgG
28
How can you enhance antibody uptake in adsorption procedures?
Using enzymes or ZZAP will enhance antibody binding by removing glycoprotein from the RBC membrane making antigens more accessible Can increase proportion of RBC to plasma Can increase the number of adsorptions but risk diluting allo antibodies PEG or LISS can be used to decrease incubation time
29
How do you determine if antibody has be adsorbed out?
Test the adsorbed plasma with group O cells or DAT neg auto cells to determine if antibody has been removed
30
What cells should be selected for warm auto adsorption?
Patients own cell if not recently transfused or pregnant If recently transfused do a phenotypically similar cell if possible. If phenotype not available use R1R1, R2R2, and rr cells that have at least one K-, Jka-, Jkb- if using enzymes to Destroy Duffy and MNS antigens.
31
Which auto antibodies will not be removed enzyme treated cells are used?
auto antibody specific for Duffy, MNS, Ena, Ge, JMH, Ch/Rg, Inb will not be removed with enzyme treated cells because the antigen is destroyed.
32
How does the acid elution remove bound antibody
It disrupts the antibody-antigen binding forces by lowering the pH Glycine acid dissociates the bound antibody
33
When should an eluate be preformed?
When patient has a positive DAT and has been transfused in the last 3-4 weeks if may detect a newly formed antibody or DHTR Can also be done on patients suspected of have immune mediated hemolysis
34
Can you use potentiators in the eluate testing?
LISS and bovine albumin is not necessary because eluate is prepared in a low ionic substrate PEG or GEL can be used if desired.
35
What is the purpose of testing the last wash?
The last wash should be tested to determine if all unbound antibody was washed away. A positive reaction in the last wash indicates inadequate washing or bound antibody became unbound during washing
36
How can you minimize dissociation of the bound antibody from the RBC's while preparing an eluate?
Using cold working wash (1-10C) may minimize disassociation of bound antibodyd
37
Can the patients red cells be used for phenotyping after an acid elution is preformed?
Red cells treated with acid elution should not be used for phenotyping or autologous adsorption because acid elution does not significantly reduce the strength of the DAT
38
During an acid elution, why must RBC's be centrifuged immediately after addition of eluting solution?
Hemolysis will occur if RBC's are not centrifuged after eluting solution added and supernant removed.
39
What is the clinically significance of the Thermal amplitude test?
Cold antibodies that are reactive at 30C or above may be clinically significant even if low titer
40
What is CAD?
Cold agglutinin disease is caused by cold reacting autoantibodies that are IgM (rarely IgG) and cause hemolysis
41
How does CAD cause hemolysis?
May activate complement causing intravascular hemolysis or may cause extravascular hemolysis by phagocytizing C3b coated cells.
42
How should a sample be collected for a thermal amplitude test?
The patients sample should be collected and maintained at 37C until serum is separated to avoid autoadsorption and cause false negative results An EDTA specimen can also be used if warmed and mixed prior to separating plasma
43
What are the disadvantages and advantages to using saline in serological testing?
Least sensitive technique Can incubate at RT or 1-6C to enhance IgM Can increase plasma to cell ratio Lengthy incubation (min 30)
44
What are the disadvantages and advantages to using BSA in serological testing?
Bovine Serum Albumin enhances direct agglutination Enhances Rh antibodies and second stage agglutination by reducing intracellular distance May increase plasma to cell ratio
45
What are the disadvantages and advantages to using LISS in serological testing?
Enhances first stage of agglutination by lower salt concentration and reducing zeta potential on membrane Increases rate of antibody uptake so reduced incubation time Ratio of 2:2:1 is critical for Low ionic environment
46
What are the disadvantages and advantages to using PEG in serological testing?
Polyethylene Glycol is a high molecular weight that excludes water from the cells Very sensitive Requires the use of anti-IgG for the AHG phase to avoid non-specific reaction due to complement May miss complement dependent antibodies In patients with high globulin levels such as in multiple myeloma a gel may form when peg is added
47
What are the disadvantages and advantages to using GEL in serological testing?
Uses a small volume of plasma Very sensitive Gel card can be kept for review and intreptation
48
How can antibody reactions be enhanced?
Increase serum to cell ratio (can't with LISS or PEG) Extend incubation time but not beyond plateau of RXN curve when antibody disassociates May lower incubation temp for detection of IgM
49
What antigens are enhanced by enzymes?
ABO, Rh, Kidd, Lewis, I and P1
50
What is ficin made from?
Figs
51
What is papain made from?
Papaya
52
What is Trypsin made from?
Bovine/Porcine source
53
What is bromelain made from?
Pinnapples
54
What is neurominidase made from?
Vibrio Cholera
55
What does neurominidase do?
It removes only the terminal NANA and results in artificial T activation of cells
56
How do enzymes work?
Removes sialic acid residue from RBC membrane to make antigens more accessible. Removes Glycophorin antigens and Duffy Enhance auto antibodies
57
What is the one-stage enzyme treatment?
Serum, Cells, and enzymes are incubated together Antibody may bind before enzyme destroys enzymes may denature antibodies
58
What is the two stage enzyme treatment?
Red cells are pretreated and enzyme is washed away before testing Can take longer unless using premade enzyme panel
59
How can we seperate donor cells from auto cells in a sickle cell patient?
0.3% hypotonic saline will lysis normal RBC's leaving sickle cells behind Hgb A cells lyse and Hgb S resist
60
Why should a DAT be ready at microscopic?
In a recently, transfused patient a positive DAT should be read at microscopic level for MF This can be first indicator of DHTR
61
How do you antigen a patient who has been recently transfused?
Use cell seperation to antigen type the patients retics | If patient also has WAA w/ DAT+ separate cells and remove autoantibody before antigen typing
62
How do neutralizations work?
Can be unknown antibody or unknown antigen Incubate antibody and antigen together and add antigen positive indicator cells If reaction occurs, indicate antibody and antigen did NOT bind If reaction doesn't occur, indicates antibody DID bind to antigen
63
What substance is used to neutralize Lewis antibodies?
Lewis antibodies can be neutralized with Lewis positive Saliva
64
What substance is used to neutralize P1 antibodies?
P1 substance is found in pigeon egg albumin, hydatid cyst fluid, and liver flukes
65
What substance is used to neutralize Chido/Rodgers antibodies?
Pooled plasma contains C4
66
What substance is used to neutralize I antibodies?
Human milk
67
What substance is used to neutralize Sda antibodies?
Pooled human urine or guinea pig urine
68
What is RESt used for?
Can be used to remove anti-I -IH, and-H in recently transfused patients. Also adsorbs anti-B so can't be used for ABO typing
69
How can we predict red cell survival in patients its difficult to find compatible blood?
MMA is an in-vitro way to predict clinical significance of an antibody 51Cr in vivo "crossmatch" is where a small amt of radioactively labeled blood is infused and radioactivity is measured at different intervals
70
How can Flow cytometry be used in the blood bank?
``` Measures residual leukocytes Detecting platelet antibodies Detecting drug induced platelet antibodies Detecting fetal-maternal hemorrhage Determine red cell survival HLA antibody testing Detection and quantification of red cell antigens Quantification of Red cells bound IgG ```
71
How does microhematocrit cell separation work?
Retics are less dense and will remain at the top after centrifugation
72
What are disadvantages of cell separation?
Sample should be collected at least 3 days after transfusion to avoid contamination with donor retics The patent should have a normal retic count If sickle cells or spherocytes present may not have normal separation of retics Observation of no mixed field or negative DAT may indicate good separation
73
What is the Freeze-Thaw method for concentrating antibodies?
Freeze sample at -20C until solid Remove from freezer and allow to thaw with out mixing There will be 3 layers with out clear lines Remove the top layer until antibody is concentrated
74
What can the capillary tube method be used for?
RBC antigen typing antibody detection IgG subclassing of antibodies
75
How sensitive is flow cytometry residual leukocyte measurement?
Using a detergent and propidium iodide solution to label the WBC nuclei, flow cytometry has reached the detection sensitivity of 0.1 WBCs per µL
76
How can flow cytometry be used to detect platelet antibodies?
One method is to incubate normal, washed platelets with the patient’s serum. These sensitized platelets are then washed and mixed with fluorescent-labeled (usually FITC) poly- or monoclonal antibody specific for human immunoglobulin (IgG). To detect IgM platelet antibodies a second fluorescent label (usually PE) can be used. Fluorescence is then measured and compared to normal platelets incubated with normal serum. Can also be used in XM PLT's. Measures platelet specific and non-specific antibodies
77
How can drug induced platelet antibodies be measured?
The flow cytometry test can be readily adapted to detect both IgG and IgM drug dependent platelet antibodies. In this case fluorescence of normal platelets sensitized with the patient’s serum in presence of the drug is compared to patient’s sample without the drug. Flow is recognized as superior to other assays when detecting quinine-, quinidine-, and sulfonamide-dependent platelet antibodies. Patient may not be sensitized to the drug tested but to a metabolite of the drug.
78
How can flow be used to measure FMH?
The assay determines HbF or Rh(D) antigen or a A combination of both using anti-HbF FITC versus anti-D PE.
79
How can flow be used to measure RBC survival?
Flow cytometry can detect and accurately quantitate mixtures of a minor population of red cells as low as 0.125%. This is as small as 10 mL of red cells. This has been useful in studying: - the amount of donor blood circulating after major organ transplants - determining autologous red cells in a transfused patient for antigen typing - Determine FMH
80
How is flow used in HLAantibody testing?
``` Flow cytometry is more sensitive than lymphocytotoxicity testing: Detects low titer HLA antibodies Detects B cell specific class II antibodies Identifies IgM autoantibodies (which often cause false positive reactions in cytotoxicity assays) Eliminates reactions attributed to immune complexes (also had caused false positive reactions) ```
81
How long to transfused cells survive in the patient?
Average life span of donor cells is 110-120 days | After about 30 days only 1/2 of transfused cells remain
82
What causes shorten RBC survival?
Immune RBCs destruction: Intravascular: IgM-complement mediated hemolysis Extravascular: IgG and/or complement mediated hemolysis (phagocytosis) Nonimmune RBCs destruction Increased age of donor RBCs unit Anticoagulant/preservative – RBCs ratio; inappropriate freezing Bacterial contamination RBC enzyme deficiency Improper blood administration, mechanical trauma, excessive heating
83
What is the principle of the MMA test?
Monocytes are incubated in a media (RPMI with FBS). The patient (or a phenotypically matched donor, NOT for RBCs antigens BUT for Fc receptors, is best source for monocytes. RBCs coated with antibody and suspended in a media, are then added to the monocyte monolayer Cells are stained and observed for adherence and phagocytosis
84
What does the MMA value mean?
MI values of ≤5% have indicated that incompatible blood can be given without the risk of an overt hemolytic transfusion reaction but it does not guarantee normal long-term survival of those RBCs
85
What other assays can determine the clinical significance of an antibody?
In vivo 51Cr survival assay: Determine the destruction rate of incompatible labelled RBCs aliquot In vitro Cheminulescence assay: Measure the metabolic activity of monocytes during erythrophagocytosis In vitro Flow cytometry assay: Measure the number of antibody attached to the RBC, using a monoclonal FITC- conjugated rabbit-anti-human IgG antibody In vitro Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) assay: Determine the capacity of the antibody to lyse antigen positive RBCs.

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