The crossmatch Flashcards
1
Q
crossmatch general
A
- serum from the potential recipient is reacted in vitro with lymphocytes from the potential donor
- A positive crossmatch is predictive of actue or hyperacute rejecttion of the transplanted organ
- A negative crossmatch decreases the lielihood of an acute rejection but cannot predict long-term fraft outcom3e when there are HLA differences between the donor and recipient
2
Q
HLA antibody is most commonly formed from:
A
- multiple pregnancies
- organ transpant
- transfusion of cellular blood prducts containing leukocytes
3
Q
Hyperacute rejection
A
- occurs within minutes of transplantation and results in diffuse thrombotic destruction of the transplanted organ
- was fairly common until adoption of pretransplant lymphocyte crossmatch
- today usually the result of clerical errors including transplantatin of blood croup ABO-incompatible orgrans
4
Q
Early crossmatch
A
- HLA antibodies were discovered before HLA complex identified
- earliest methods involved agglutination of peripheral blood leukocytes and distinguishing the patterns of agglutination and nonagglutination in both autoimmune and alloimmune situations
- white blood ceels initially prepared from mechanicaly defibrinated blood (raotation or agitaiton of whole blood with glass beads)
- Later preparations of white blood cells came from EDTA anticoagulated blood
- different methods led to different results, less than 90% reproducibility
- Most antibodies discussed in terms of in-tro function or activity rather than the antibody class or subclass
- “partial” or “incomplete” antibodies when the likely antibody is IgG
- IgM is complete (cross links 2 cells)
- IgM temperature optimum is closer to room termerature, while IgG is most reactive near 37 degress celcius
- Leukocyte-poor blood eliminated the transfusion reactions associated with leucoagglutinins
5
Q
Microcytotoxicity
A
- Leukoagglutination methods consumed significant amounts of relatively rare serum and large amounts of peripheral blood for each test
- Microcytotoxicity method attempts to reduce reagent use
- Became known as the NIH Standard
- Large now replaced by flow cytometric methods
- 1 microliter per test compared to 0.1 mL
- 200-250 cells per reaction well
- Density gradient is used to isolate mononuclear cells (primarily lymphocytes)
- seperates neutrophils, erythrocytes, and platelets from mononuclear cells with a single cntrifugation step
- Commerical kits are available for ioslation of lymphoctes
6
Q
Microcytotoxicity Method
A
- incubation of isolated lymphocytes from periphereral blood (of lymph node or spleen from a deceased organ donar) with serum, usually at room temperature.
- After incubation, a source of complement is added (most frequently, diluted rabbit serum)
- After a soncd incubation, the cells are stained, usually with qa fixative (glutaraldehyde)
- Dye updake or exclusion can be used to identify the killed versus unkilled cell populations
- Complement fixation results in “hiles” in the cell membrane
- large dye molecules can pass through
- Chemical fixation stabilizes (preserves) the reaction so that it can be more conveniently interpreted
7
Q
Microcytotoxicity modifications
A
- methods to improve sensitivity or specificity
- Sensitivity may be increased by extending the incubation periods (NIH extended) and specificty increased by wash steps
- Antihuman glubulin can increase sensitivity
- in cases where HLA antibodies are deected but are too weak to induce cytotoxicity or do not activate complement binding)
- 4 to 64 fold increase in sensitivity
- AHG initially prepared by immunivizing rabbits with fractionated or whole human serum
- The globulin franction (enriched by ammonium sulfate or alcohol precipitation) generally gave the most acceptable and consistent reaction
- current practive uses Ig of specific subblasses such as IgG.
- The more broadly reactive “Coombs” reagents (AHG) react with multiple classes and complement protents
- AHG technique remains a standard method (but not required)
8
Q
Crossmatching by microcytotoxicity
A
- nearly replaced by flow cytometric methods
- Crossmatches for living donors are usually between a single potential recipient and a single potential donor
- For deceased donor crossmatches, there are usually multiple potential recipients for the orgran
- As a result, multiple donor to recipient combinatinos are usually set up for crossmatchng at the same time
- For microcytotoxicity crossmatching of multiple organ recipients against a single donor, the donor cells are loaded into each well of the tray, and serum from each potential recipient is added
- The trays may also be preloaded with sera from potential recipients to reduce time and was a common practice in labs supporting large numbers of potential recipients (called the “wait” list)
- Current rules from nthe United Network for Organ Sharing (UNOS) and the Organ Procurement Transplantation Network (OPTN) require donor/recipient combinations (match run) be derived from a national database and single national computer system which negates the efficiency of preloaded trays.
- A positive controll with pan-reactive serum well is used
- A nagative control well uses autologous serum from the donor or prescreened, antibody negative srum
- Standard 0,1,2,4,6,8 scoring method is used
- A positive reation (greater than 10% cell killing; score of two or greater) is considered contraindicative for transplantion.
- Reproducibility is poor when less than 20% of the target cells are killed
- Common practice to score crossmatches as neagtive when less than half of the replicates show more than 10% cell death
9
Q
ABO and solid organ transplantation
A
- major markers for immunologic compatibility in transplantation, not just for blood transfusions
- A, B, H antigens are probadly present on cell surfaces, including the endothelial linings of blood vessels
- ABO mismatched solid organ transplants are rapidly rejected (destroyed) by antibody-mediated complement deposition and thrombosis of the blood vessels
- Blood group 0 individuals are universald donors
- AB individuals are universal recipients
- Blood groups A and B must receive organs from donors with the same blood group or blood group O
- RhD is not present in transplant organs
- ABO groups not evenly distributed in populations
- In USA, 44% are group O, 42% group A, 10% group B, 4% group AB
10
Q
T and B cell isolation
A
- 65-85% of peripheral blood lymphocytes are T cells
- But HLA Class II antigens are not normally expressed on T cells.
- And therefore antibodies to HLA Class II antigens are unlikely to be detected by microcytotoxicity without isolating or enriching for B cells
- B cell enrichment methods include nylon wool (B cells adhere, T cells do not), sheep red blood cell (SRBC) rosetting (T cells rosette and can be removed by density-gradient isolation, and positive seletion using antibody to B cell CD19 linked magnetic beads
- flow cytometric methods have eliminated the need for isolation of T and B cell franctions for most assays
11
Q
B cells
A
- more difficult to work with than T cells:
- lower number in peripheral blood
- lower initial viability and viability during preservation
- lower density of HLA Class II antigens on cell surface relative to Class I
12
Q
Immunofluorescence
A
- Antibody to HLAs can be detected by antihuman immunoglobulins conjugated to fluorescent dyes.
- requries specialized microscopy
- increased senstivity
- can use additonal dyes to identify subsets of cell markers, subpopulations of cell types, and evaluation of cell viability
- combining antibody specificity with distinct fluorescent dyes has allowed detection of antigens that are present only in specific subsets of cells
- for example, an antibody covatlently linked with rhodamine isothiocyanate (RD) with specificity to a B cell antigen such as CD18 can be use to identify B lymphocytes, and a second antibody labeled with fluorescein isothiocyanate (FITC) with specificity to human immunoglobulins can identifty the presence of the anti HLA antibodies.
- Disadvantages include complicated and expensive microsopy with flluoresent light source and filters
- Compare with negative controls to compensate for autofloresence
- dyes remain active for short time, and must be performed in darkened rooms.
- this method being supplanted by methods that give more stable reactsions and are less subnjectively evaluated
13
Q
Flow Cytometry
A
- originally called “pulse cytophotometry”
- use a laser of other intense monochromatic light source (mercury ar lamp) to interrogate and analyze cell populations that have been treated with dyes, lectins, or antibodies with specificities for nuclear, cytoplasmic, of membrane targets
- rely on detection of the energy released from excitation of electrons in florochromes covelently attached to antibodies (in most cases) to cell target antigens or immunoglobulins, such as anti_HLA antibodies bound to the surface of the cell
- target cells are suspended in a rapidly moving column of fluid called “sheath fluid” past the lisght source
- advantage over flouorescence microscopy methods is that light emissions can be readily and objectively quantified
- photomultiplier tubes are optically arranged to detet selected wavelength of emiited light
14
Q
Flow Cytometry measurements
A
- foward scatter (FSC or FS) is light that is refracted toward the detector in line with the primary laster
- light is normally blocked by an obscurator bar fixed in line with the light source
- light that is refracted by a cell passing through is scattered in the foward direction proportion to the size (volume) of the cell.
- Side scatter (SSC or SS) , scattered light is detected and indicates cellular complextity
- combination of FS and SS is used to group blood cells into populations that correlate well with morphology
- “Gating” is using software to select cell populations for analysis
- For HLA testing, the gated population is usually the small, nongranular population that corresponds to lymphoctyes
- FS and SS can be combined with other cellular parameters to construct automated differentials of white blood cells, and combined with vital dyes and impedence measurements
15
Q
Histocompatibility testing with Flow Cytometry
A
- normally performed on lymphocyte samples that have been isolated by denstiy gradient sedimantation or magnetic bead isolation
- light scatter gating should still be used to prevent inadvertent analysis of nonlymphocyte cell populations and debris
- (eg Monocytes and neutrophils nonspecifically bind immunoglobulis through Fc-receptors and false-positive binding of antibody can be expected
- Nonspecific binding also due to B cell Fc receptor binding, reduced by pretreatment of lymphocytes with proteolytic enzyme Pronase
- Additoinal gating can analyze only cells marked as T cells by antibodies to the pan-T cell marker CD3 labeled with peridinin chlorophyll protein complex (PerCP) and B cells using CD19 (or CD20) labeled phycoerythrin
