Special Techniques Flashcards
(163 cards)
1
Q
Donath Landsteiner Test
A
Detects presence of PCH causing anti-P. 9 tubes total 1a1b1c 2a2b2c 3a3b3c. 1’s are incubated with patient serum and rbcs, 2’S are incubated with patient serum and normal serum and rbcs 3’s are incubated with just normal serum and rbcs. A’s are incubated at 4 degrees, B’s are incubated at 37 and c’s are incubated at 4 degrees and then at 37 degrees. you check for hemolysis it should be present in 1c and/or 2c and none of the others. All rbcs must be P positive
2
Q
ZZAP
A
A reagent used primarily in reference laboratories composed of a mixture of a proteolytic enzyme (papain) and a sulfhydryl reagent (dithiothreitol, or “DTT“). It is used to remove immunoglobulins and complement from the surface of red blood cells, commonly when evaluating a potential autoantibody. ZZAP also deactivates a multitude of red cell antigens on the red cell surface. The most important antigens damaged/destroyed by ZZAP include all Kell antigens, M, N, and the two main Duffy antigens (Fya and Fyb), to name a few.
3
Q
2-ME
A
2-mercaptoethanol- disulfide reagent
4
Q
DTT
A
dithiothreotol- disulfide reagent- breaks disulfide bonds such as those in IgM and Kell antigen
5
Q
Anti-G adsorption/elutions
A
First adsorption with r’ (dCe) cells, anti-G and anti-C will bind but not anti-D. elute off and you will have anti-G and anti-C if present in the eluate. 2nd adsorption with R0 (Dce) cells, anti-D would bind and anti-G would bind but not anti-C, you then elute this off and test this, at this point after both elutions neither anti-D or anti-C would be present, if there is reactivity it is due to anti-G
6
Q
Minimum distance apart for two red cells for IgM and IgG to bind both
A
79 a
7
Q
How do enzymes work in RBC agglutionation testing
A
- Sialic acid provides negative charge, when removed net negative charge is gone, reducing zeta potential and allowing RBCs closer 2. glycoproteins attract water molecules, water molecules need to be shared by RBCs, RBCs get closer 3. glycoprotein structures protrude from the surface of the red cell, by removing and reducing steric hindrance, antigens are more accessible to antibodies
8
Q
Antigens that are enhanced by enzymes
A
Just kidding, Lewis rules!! JK, Le, Kidd
9
Q
Antigens destroyed by enzymes
A
M, N, S, s, Fya, Fyb (S s vary)
10
Q
Antigens not denatured
A
K,k Fy3, Kd, Rh, Le
11
Q
Antigens destroyed by DTT
A
L-DICK, JKML. Lutheran, Dombrock, Indian Cartwright, Kell, JMH, Knops, Mer2, LW
12
Q
Enhancement media
A
PEG, LISS, Albumin, not Saline
13
Q
Albumin, what it is
A
a simple form of protein that is soluble in water and coagulable by heat, such as that found in egg white, milk, and (in particular) blood serum
14
Q
Albumin what it does
A
Concentration typically used= 22%, reduces the zeta potential , does not destroy or inactivate antigens, allows red cells to get closer and enhances agglutination
15
Q
LISS
A
low ionic saline solution- 20% less NACL concentration. Formula: .17M saline .15M phosphate .3M sodium glassine , lower number of sodium and chlorine ions lowers the ionic layer around RBC. Promotes non covalent bonds that are dependent on distance between antibody and antigen sites on RBC membrane, does not increase reactivity strength, just allows less time for the agglutination to take place, due to ability of cells to get closer, by reducing the zeta potential
16
Q
PEG what it is
A
polyethylene glycol, water soluble linear polymer, prepared by polymerization of enthylene oxide. amphiphilic= soluble in water methanol benzene dichloromethane, insolubel in hexxan and ditethyl ether contains a hydroxyl group that provides a site for covalent bonds with other molecules
17
Q
Peg what it does
A
doesn’t denature antigens, contains a hydroxyl group that provides a site for covalent bonds with other molecules, doesn’t prevent the approach of other small molecules. REMOVES WATER FROM SURFACE OF RBCs increasing antibody concentration and promoting the binding of antibodies with antigenic sites
18
Q
Why enhance?
A
IgG molecules are unable to approximate RBCs without enhance substances that promote agglutination
19
Q
Violation of the package insert
A
Any violation of package insert must first be validated in the laboratory using the variation.
20
Q
Adsorptions-autologous
A
Removal of autoantibodies to determine if there are alloantibodies present in serum. Best option for untransfused patient with positive DAT and/or positive autocontrol.
21
Q
RBC treatment for auto-adsorption
A
Treat with either gentle heat elution (45C) ZZAP(DTT+Ficin), WARM
22
Q
How many adsorptions do you perform in an autoadsorption?
A
3 different aliquots
23
Q
Allogenic adsorptions
A
used to determine if there are alloantibodies, remove antibody of high prevalence antigen, RBCs or stroma (fluffy white clouds)
24
Q
3 cell types to use in allogenic adsorption
A
R1R1, R2R2, rr, at least one of them negative for Jka, Jkb, K, S, s
25
adsorbed cells treated with?
enzymes, ficin or papain, Fya, Fyb M and N will be destroyed variable on S/s
26
Acid elution
ELUKIT II - commercial kit used by decreasing the pH and making it more acidic to release the antibody
27
EDTA elution
glycine acid
28
Reasons to do titrations
1. Cold agglutinin disease screen and diagnosis (thermal amplitude study) 2.Maternal Fetal monitoring, titer increase 3. antibody identification -HTLA-like Knops 4. Plasma and platelet products- anti-A and anti-B titers
29
Thermal amplitude studies
for cold agglutinin disease, titiration with type specific type 0 autologous, cord or adult I RBCs warm seperated and warm washed RBCs (4C, 22C, 20C, 37C)
30
Titration methods
1. Point dilution 2. Master doubling dilutions
31
Master doubling dilutions
1:2 1:4 1:8 to 1:2048, maternal serum for possible HDFN prdeiction or indication for more invasive or expensive testing. HTLA-like reactiviide, inhibition studies for strong reacting antibody, cold agglutinin studies
32
Point dilution Titration
cold agglutinin screen, products Type O platelets, Type A plasma
33
Cell separations
reticulocyte- microhematocrit, phhthalate esters, percoll-renografin, silicone oil
34
Hemoglobin S positive RBCs separation
hypotonic saline wash-0.3% NaCl
35
ELISA uses
Detection of low level Ig on RBCs, IgA, IgM, IgG. Quantitation of RBC antiobdy
36
ELISA methods
antigen bound to bottom of well, add patient serum, antibody will bind, anti-human IgG antibody with enzyme linked is added, substrate for enzyme is added and color change occurs where enzyme/IgG is present
37
Inhibition/neutralization P1
hydatid cyst, pigeon egg whites
38
Inhibition/neut. Lewis
using serum with Lea and Leb antigen ( secretor has both, non secretor but positive for Lewis has Lea)
39
inhibition using Urine neutralizes what?
Sda
40
Inhibition/neutralization Chido/rogers/Knops a
use serum
41
Chloroquine diphosphate
can be used to weaken expression of HLA Class I molecules (Bg antigens) also weakens Rh antigens. Can also be used for removal of bound antibody
42
Ficin/papain destroys
M, N, S, Fya, Fyb, JMH, Ch, Rg, Xga
43
Ficin/papain enhances
Rh, P1PK, I, Kidd, Lewis
44
Trypsin
removes CD38 from surface of the cell, helps with daratumumab patients
45
Sulfylhydrl reagents
2ME- 2 mercaptoethanol, DTT dithiothreotol, AET 4-amino-ethylisothioronium
46
Used to remove CD38 from the cell
Trypsin and DTT
47
Glycine-HCL/EDTA
EDTA-glycine acid- can be used to remove antibody from red cells. However, destroys Bg and Kell system antigens and Era antigen
48
Problem with high protein reagents
can cause false positives, most anti-sera reagents used today are low protein reagents
49
No antisera for following antigens exists:
Doa Dob Jsa V VS
50
bystander hemolysis
hemolysis of own red cells upon hemolysis of incompatible donor cells so that new HCT is even lower than the original hct prior to transfusion
51
Kidd system genetic variation is higher in which population?
blacks
52
What percentage of D negative asians have an intact but inactive D
15-30%
53
Hybrid Rh D common example phenotype
RHDIII-(CE4-7)-D parital C, partial e, D negative, V- VS+
54
What to keep in mind if looking for a particular SNP
ethnicity of the patient, can narrow down the window knowing what is most common irregularities in different populations
55
microhematocrit cell separation
reticulocytes can be separated from more mature red blood cells based off density. This is useful in gaining a recently transfused patients phenotype. Spin down and take top 5mm of microhematocrit tube for testing. Reticulocytes are less dense than mature RBCs
56
Percoll-Renograf
continuous density gradient- centrifugation and separation of different components based off density. This is one form of this test from percoll company. can be used to separate mononuclear cells from red cells in peripheral blood
57
Proteolytic enzymes destroy which antigens
M,N FYA, FYB, Xga, JMH, ch, rg, Yta
58
Proteolytic enzymes enhance which antigens
Rh, P, I, Kidd, Lewis
59
what neutralizes I antibody
breast milk, REST rabbit erythrocyte stroma
60
P1 neutralization
hydatid cyst, pigeon egg whites
61
Sda neutralization
urine (human and guinea pig)
62
What does saliva neutralize
ABH and Lewis
63
What neutralizes Ch/Rg
Pooled AB plasma
64
what neutarlizes HLA antibodies (BG)
Humane platelet concentrate.
65
rBGA procedure and purpose
recombinant blood group antigen, by company imusyn, it can specifically inhibit certain antibodies, as in cases where a patient has multiple antibodies to isolate and differentiate. Idea is you put pt. plasma(which contains antibodies) and run through gel, you then take pt. plasama (with antibodies) and add recombinant blood group antigen in order to inhibit or neutralize the antibodies and run them again, this can help differentiate if there is a different antibody present- shows different reactivity in comparison to non-neutralized plasma
66
antigens neutralized by rBGA
Knops, Cromer, Dombrock, Fya/b Inb JMH, Kel, Lua/b Lwa, Sc1, Xga, Yta
67
Sephadex separation
cell separation through affinity chromotography, take sample and run through small column, sephadex ion exchanger. IgG is not retained (though you may need additional washes to remove all of it) IgM is retained (80% of original) this is important in the separation of IgG and IgM antibodies
68
Reasons for Mixed Field
HPC, Subgroup (A3,B3) Transplant, Chimerism, Mosaicism
69
Bombay Missense mutation
725 T>G in FUT1 and deletion in FUT2
70
LAD
leukocyte adhesion deficiency Type II, due to mutation in GDP-fucose transporter gene, recurrent infections severe mental/growth retardation, persistent leukocytosis, neutrophils deficient in expression of selectin ligand activity. Decrease expression in H and Lewis Blood Group
71
Parabombay
honozygous non functional FUT1, normal FUT2, therefore type 1 chains, present in secretions, not present on the RBC. Type 1 chains are passively adsorbed onto red cells- weak positive reactions (Ah, Bh, ABh) Anti-H is typically weaker and less clinically significant than bombay anti-HI and anti-A/B depending on blood type
72
Flow Cytometry
fluorescent "tagged" antibodies against cell surface molecules incubated with target population of cells, this is then passed through the flow cyclometer where a laser excites the fluorescent target causing emission of photons of specific wavelength which are detected by sensors. Fluorescence is measured on a cell by cell basis therefore can detect very small populations of cells,
73
Polybrene
hexadimethrine bromide- cationic polymer it neutralizes the charge repulsion between virions and sialic acid cells surface, causes non specif red blood cell aggregation
74
Plasma Treated we EDTA/Glycine Acid disrupts which antigen group?
Knops
75
What antibodies can be made by D--/D-- individuals
RH17 antibodies
76
Making Lectins procedure
grind up extract and incubate with 0.9% saline and then filter supernatant.
77
Monocyte monolayer assay principle
predicts in vivo response to transfusion of incompatible RBCs. predicts survival of antigen positive RBCs in host. (used if RBCs are unavailable or if there is an antibody to a high incidence antigen)
78
Monocyte monolayer assay cannot be used to predict:
HDFN, autoantibody significance in vivo or clinical significance of IGM antibodies (Gerbich and Cartwright)
79
Monocyte monolayer assay quantitates:
phagocytosis and adherence of antibody coated red cells.
80
Chemiluminescence assay what it does
measures respiratory release of oxygen in after phaogcytosis of antibody coated red cells.
81
Three tests helpful in understanding in vivo clinical significance of antibody
1. Monocyte monolayer assay 2. chemiluminescence assay 3. cellular cytotoxicity
82
Monocyte monolayer how it's performed
Donor red cells, recipients antibodies are mixed together. Monocytes are isolated and placed in a single layer on plate in lab. Incubate together looking under microscope for rosette formation- red blood cells bind to periphery of monocytes or are inside of monocytes-indication that moncyte recognized rbcs via Fc receptor on Fc antibody region. Calculate the "monocyte index" if less than 5% indication no rapid destruction of RBCs is predicted
83
Which cells destroy RBCs during a transfusion reaction
Monocytes
84
No anti-reagent for
Doa Dob Jsa Jsb and V/VS
85
2 drops of serum and 1 drop of 5% rbcs creates a dilution of what?
40:1 minimum ratio between serum:cells, can increase by adding additional drops 4:1(3% red cell) equals 133:1 ratio
86
Reaction Mediums (3)
albumin, LISS and polyethylen glycol
87
Albumin
macromolecules of albumin allow antibody-coated cells to come into closer contact with each other so aggregation occurs. Increases sensitivity!!
88
Percentage of alubumin added
22% bovine albumin is used (provided same sensitization in 30 minutes as it took saline only 60 minutes) Reason it's no longer used very much, could miss several clinically significant antibodies and is more expensive.
89
LISS
enhance antibody uptake, decease incubation times from 30-60 minutes to 10-15 minutes. REDUCES ZETA POTENTIAL. could either suspend red cells in LISS or use an additive LISS reagent (more common)
90
PEG
poly eythelyne glycol- water soluble, increases antibody uptake. Works by removing water molecule surrounding the RBC, effectivey concentrating antibody. Can cause aggregation after 37 degree incubation and cetrifugation-this step is skipped. In comparison with LISS, PEG increases sensitivity to clinically significant antibodies and decreases the sensitivity to clinically insignificant antibodies
91
LISS and PEG incubation time
10-15 minutes
92
Incubation time with saline
30-120 minutes
93
Check cells
Group O cells sensitized with IgG, in order to cause positive reaction after negative IAT/DAT
94
Why is it important to wash as quickly as possible and add AHG as quickly as possible in succession
because low-affinity antibodys may elute off of the red cell, after removal from the incubator
95
Saline pH
7.2-7.4
96
CBER recommended method for the evaluation of AHG RCF
1000 for 20 seconds, higher RPM=more sensitive results
97
EDTA should be used to collect for DAT why?
To avoid the in vitro complement attachment associated with refrigerated clotted samples
98
LISS disadvantages
inability to automated, many procedural steps requires highly trained staff, fewer method-dependent abs detected.
99
PEG disadvantages
same as LISS plus it detects more unwanted antibodies
100
Gel disadvantages
warm autos are enhanced, increased detection of unwanted antibodies
101
Gel vs. solid phase advantages
Gel- more sensitive DAT method SP- increased sensitivity for all antibodies. SP more sensitive at detecting anti-D than gel
102
SP disadvantages
increase sensitivity for all antibodies, warm auto abs enhanced, detects unwanted antibodies
103
Type 2 chain and Type 1 chain
1-4 Beta linkage on type 2 chain, type 1 chain has 1-3 beta linkage
104
Blood type A sugar
GALNAC
105
bgss
blood group specific soluble substances- increased amount in patients with carcinoma of the stomach and pancrease. Excess amounts of BGSS will neutralize the reagent (Anti-A or B) leaving no unbound antibody to react with cells. Causes weakly reactive or missing front types
106
Anastomosis
cross connection between adjacent channels- chimerism in twins develops from connections in utero, this causes "true chimerism"
107
Mosaicism
two cell populations in an indvidual without a twin, not true chimerism, comes from dispermy- two sperm fertilizing one egg
108
Plasma expanders
dextran and polyvinylpyrrolidone causes abnormal protein or plasma abnormalities and result in pseudoagglutination or rouleax, can interfere with blood typing.
109
Acriflavine
yellow dye used in some commerical reagents for anti-B, some patients can have an antibody against this, forming and anti-B anti-acriflavine immune complex which can then be adsorbed onto the red cells- cause false positive results
110
Pancreatic/stomach/ovarian cancer and relation to abo discrepancies
Can release additional soluble antigen in large amounts so when reagent anti-A (as example) is mixed with patient red cells, it may bind to the soluble antigen present and therefore there is no agglutination though the antigen is present on the red cell as well. This can be solved by washing patients red cells to rid them of excess soluble antigen. This would be shown when there is a backtype, but a lack of matching front type
111
HTLA like antibodies, name some, how they react and the problem with them
Csa, Ch, Rg, Kna, McCa, JMH, Yka, all have very high titer but low avidity, reacts with almost every cell, due to high frequency of antigen, reacts very weakly at AHG, but does not disappear with increased titers, some as high as 2048. These are typically not clinically significant but can cover up other alloantibodies that may be
112
Significant titer increase
two or more tubes (4 fold increase) in titer
113
Organic solvents-what they are, how they work, some examples and problems
Used in eluting off an antibody. Some examples are: dichloromethane, xylene, ether. Work by disrupting the lipids in the RBC membrane to reduce surface tension and reverse the van der Waals forces holding antigens and antibodies together. Issues with these step from the fact that they have unsafe tendencies ie. carcinogenic or flammable and are not kept in a lab routinely for testing.
114
Which antibodies, if proven to be clinically insignificant do not need antigen negative units provided but crossmatch compatible (5)
anti-N, anti-M, anti-P1, anti-Lea and anti-Leb
115
JMH destroyed by
both enzymes and DTT
116
When antibody screen is negative, and AHG crossmatch is incompatible what are the next steps:
1. verify the donor unit blood type is compatible with the patients
2. test the donor unit for positive DAT
3. start to suspect an antibody to a low prevalence antigen.
117
When testing for cold autoantibody what are good reference cells to test against
1. O adult cells (H and I)
2. cord cells (H and i)
3. A1 adult cells (I)- H is very few on these cells.
with an autocontrol tested at 4 degrees to detect specificity whether anti-I or anti-IH etc.
118
Warm auto antibodies may occur secondary to which disease states?
SLE, CLL, lymphoma, and other autoimmune disease, as well as certain drug therapies.
119
For WAA patients, when no alloantibody is detected after further testing (adsorption) what is necessary for the crossmatch?
An immediate spin ABO-compatible crossmatch is all that is needed with no underlying alloantibodies
120
Important consideration for autocontrol in antibody screening
Autocontrol should be negative if it is not an autoantibody. However, if patient has been recently transfused the antibody might react with donor cells still present in the patient, CHECK HISTORY!
121
Use of Peg in adsorptions
Peg can be used to enhance the antibody removal cutting processing time approximately in half, while still providing for adequate autoantibody removal
122
Acceptable for patient with WAA and anti-K in crossmatching standards
least incompatible is acceptable, if it appears less incompatible with the warm autoantibody than the patients own cells.
123
Gel technology disadvantage as it relates to ABO group
longer turnaround time for ABO determinations and less sensitive detection of ABO antibodies in patient serum or plasma when compared with the tube method.
124
Staphylococcus Protein A
has affinity for IgG 1,2,4 and IgG immune complexes, can be used in a membrane filter, when plasma pushed through this membrane the IgG is adsorbed onto the membrane with Staph Protein A, also removes lesser amounts of IgM and IgA -was originally used to treat ITP, however has since been no longer been manufactured in the US due to adverse effects.
125
Heat elution principle
antibody bound to the red cells, disrupting testing results, need to remove in order to obtain correct antigen typing results, this type of elution is not meant for antibody recovery and testing, simply to remove and discard the antibody. used to identify iGM antibodies present, will elute off for testing.
126
Heat elution procedure
wash red cells with saline, add saline 3:1 with red cells, incubate at 45 degrees C with gentle agitation for 10-15 minutes. Remove supernatant, wash with saline. perform a DAT to test that all antibody is removed, If not repeat. once fully removed may test for antigen
127
Heat elution control.
Perform simutaneously with red cells positive for antigen that needs to be tested for to ensure that the antigen is not destroyed during this process.
128
saliva treatment
acquire salive and incubate for 10 minutes to get rid of enzymatic enzymes. (place in boiling water bath
129
dilution in saliva secretor neutraization studies
serial dilutions of antisera- pick lowest dilution that provides a 2+ result as your dilution.
130
Procedure for saliva secretor neutralization test
add one drop of diluted anti-sera, add one drop of the saliva. Mix and let incubate for 10 minutes, then add 1 drop of appropriate red cells and incubate at 30-60 minutes RT to determine reactivity.
131
Controls for saliva scretor neutralization test
No controls should contain the antigens, saline for example. They should all be positive, i.e. not neutralized by the saline and therefore will agglutinate
132
Use of sufylhydrl reagents to disperse polyagglutination
When agglutination is caused by IgM antibodies on the surface of the red cell. Need to remove, wash red cells with saline, then add .01m dtt or 2-ME let incubate to dissociate IgM antibodies, can then test red cells.
133
Control for Use of sufylhydrl reagents to disperse polyagglutination
Test treated red cells with 6% albumin to see if polyagglutination occurs, if it doesn't IgM successfully removed.
134
Chloroquine diphosphate removal of antibodies principle
when DAT positive with IgG antibodies, can use chloroquine diphosphate for removal with little damage to RBCs
135
preperation of chloroquine diphosphate
20 g chloroquine diphosphate into 100mL saline. Adjust pH to 5.1 with NAOH
136
control in chloroquine diphosphate testing
must treat normal red cells positive for antigen (heterozygous) to ensure presence of antigen after treatment
137
procedure Chloroquine diphosphate
0.2mL of red cells (previously washed) 0.8mL of chloroquine diphosphate, mix and incubate at RT for 30 minutes. , wash with saline 3-4 times, then test red cells against IgG to deterine if removed, once this DAT is negative, you may use the treated red cells for antigen typing/testing
138
Acid/glycine EDTA principle
can be used in removal of antibody for adsorption or antigen testing of red cells, post removal
139
acid glycine/ EDTA reagent
0.1 M of acid glycing to 5mL of 10% EDTA
140
acid glycine/EDTA test procedure
10 volume of washed red cells, 20 volumes of acid glycine/ EDTA. mix well incubate RT 2-3 minutes. Add 1.0 M tris-Nacl. centrifuge, remove supernatant wash 4 times with saline. Test with IgG to ensure removal of antibodies.
141
problems with acid/glycine EDTA
if acid is left on red cell too long, can cause irreversable damage to red cell, can also destroy Bg antigens, Kell antigens, and Er antigens.
142
separation of transfused from autologous red cells for phenotyping procedure
wash with saline, fill microhematocrit tube, centrifuge tube down remove top 5 mm of the tube for red cell testing.
143
idea behind separation of red cells
new red cells are less dense will be at the top of centrifuged set of cells.
144
separation of sickle cells from normal
sickle cells more resistant to hypotonic solutions, add 0.3% saline solution, normal cells will lyse, sickle cells remain intact for testing. must wash cells in .9% saline to return to normal tonicity.
145
LISS testing variety
can be added to the reaction, red cells can also be suspended in LISS concentration instead of normal.
146
preparation of ficin
100mL PBS pH 7.3 1 g of ficin powder mix very well, filter or centrifuge to obtain clear sample
147
preparation of papain
Add 2g of papain to 100mL of PBS pH 5.4, agitate 15 minutes, filter or centrifuge to obtain clear solution, add L-cysteine hydrochloride and incubate at 37 for 1 hour, add PBS pH 5.4 to obtain total amount of 200mL
148
GPI linked Antigens
JMH, EMM, DAF, cartwright, Dombrock
149
HTLA-like antibodies
CH/RG, JMH, Cartwright, Knops, Dombrock, Cost
150
Procedure for treatment with enzymes
15 minutes, 10 minutes 5 minutes, three tubes, add .1 % ficin stock and .9 parts PBS, add 1 drop of ficin preparation and equal amounts of RBCs (previously washed), incubate at 37 for each time listed. Once incubation is complete, wash the red cells three times with saline. Add one drop of red cells and 2 drops of serum to a test tube for each of the three time lines as well as a control with the original untreated red cells. incubate 37 degrees for 15 minutes, wash add AHG and read the reactions. looking for difference in reaction strength over time intervals.
151
control for enzyme treated cells
Need to find an antibody that reacts with enzyme treated cells, but only reacts normally at IAT. anti-D is a good option for a positive control. Negative control should be serum believed negative for all any antibodies
152
no agglutination in positive tube and agglutination in negative tube.
1. treatment is inadequate. 2. overtreated with enzymes.
153
1 stage method of enyzme treatment
add 2 drops of enzyme solution, 1 drop of red cells and 2 drops of patient plasma, incubate, centrifuge, shake out for aggutination, then wash and add AHG
154
problems with enzyme testing
1 stage not sensitive 2 stage very sensitive but immense qc required, must have QC for every cell tested both positive(anti-D) and negative (AB plasma) to ensure that appropriate treatment occured.
155
what do enzymes do
strip of final NeuNac (Nana) from surface of red cell allowing closer contact.
156
2 stage method
1 drop RBCs and 2 drops of enzyme, incubated 37 for 15 and then washed three times, then serum is added 2 drops and incubated, washed, and AHG added. Also centrifuge after incubation and before washing step.
157
how much IgG does monospecific and poyspecific antisera detect
as little as 150-500 molecules of IgG per cell.
158
Reasons for performing titer (4)
1. prenatal 2. determination of HTLA-like reactivity 3. elucidating autoantibody specificity 4. observing the effect on sulfylhyrdyl reagents on certain antigens activity
159
DTT treatment in determing IgM vs. IgG presence
two test tubes, both with patient reactive serum, one DTT and second with PBS solution. mix incubate 37 for 1 hours, wash both and test with reagent cells, test reactivity with both samples if positive with DTT treated- IgG antibody potential, if negative IgM was present and not IgG component is present. Control is comprised of serum and saline, therefore should be positive if IgM was present in solution, compare the two tubes
160
anti-Sda urine test
collect urine, and boil for 10 minutes. Mix equal volumes of serum and urine. dilution control: serum and urine. negative control tube: urine and PBS. the urine control tube should be negative as there is no anti-Sda present when red cells added. The anti-Sda plus urine sample should be negative when reacted with red cells because Sda is neutralized, the dilutional control should still be positive to rule out that the negative result from the patient is due to the inhibition and not the dilutional effect.
161
Cold acid elution procedure purpose
used in recover of warm reacting auto and allo antibodies, causes disruption of the antigens, and destruction of their tertiary structure.
162
Cold acid elution procedure
place Glycine-HCL and saline into ice water bath. add 1mL of red cells to a test tube and chill in ice water bath for 5 minutes before adding glycine-HCL. add 1 mL of chilled saline and 2 mL of the glycine-HCL to the red cell tube. incubate in ice 1 minute, centrifuge and remove supernatant, test in parallel with eluate as control should be negative.
163
prevention of hemolysis during cold acid elution
stabilize using 22% albumin
