Pre-Transfusion Testing Flashcards
What is the principle of the Antiglobulin test? What is this also known as?
Based on the principle that AHGs from immunized nonhuman species bind to human Immunoglobulins like IgGs or complement either free, in serum, or attached antigens on RBCs. This test is also known as Coomb’s Test.
Discovery of AHG lead to the finding of what blood group systems?
- Duffy
- Kell
- Kidd
What are the two classes of AHG sera?
- Polyspecific
- Monospecific
What is the difference between monospecific and polyspecific AHG production?
- Monospecific - Hybrodoma technology; fuses murine spleen lymphocytes with myeloma cells to create immortal cell line. this make a specific antibody directed at a single epitope
Polyspecific- inject animals with human globulin components and collecting the antihuman antibodies. more than one spleen lymphocyte wille produce immunoglobulins. Sera directed at multiple epitopes.
What is contained in Rabbit Polyclonal?
What part of the immunoglobulin chain is it specific to?
What is contained in Monoclonal IgG?
- Contains anti-IgG with no complement activity through hybridoma technology.
- IgG heavy chain
- Murine Anti-IgG
All of the following are advantages of Monoclonal AHG except?
- Higher titre of Ab than polyclonal AHG
- No batch variation
- Not subject to cross reactivity
- Over-specificity
- Over- specificity (disadvantage)
What are some disadvantages of Monoclonal AHG?
- Over-specific
- only one epitope
- may need to blend serval monoclonal antibodies to ensure variants detected
- overly sensitive
- complement fixation may not start
What are the advantages of Polyclonal AHG?
More likely to pick up variants
cheaper
What are the disadvantages of Polyclonal AHG?
Low specificity
Low titre
What is contained in the following?
- Rabbit Polyclonal
- Rabbit/murine polyclonal blend
- Murine monoclonal
- anti-IgG and anti-C3d
- blend of antihuman IgG and murine monoclonal anti-C3b and anti-C3d
- Murine monoclonal anti-IgG, anti-C3b and anti-C3d
What class of antibodies make up a majority of the mixture in AHG?
IgG1 and IgG3 subclasses
Although rare, why might IgM antibodies be found in AHG?
Can fix complement so detected by anti-complement
What is the principle of IAT?
-
Detect in-vitro sensitized cells. This is a two stage process.
- Cells (antigens) exposed to antobodies via antisera / serum
- antobodies or complement sensitize RBC during invitro incubation phase; 37C in LISS/albumin
- Cells tested with AHG to check for sensitization and strenght of reation is graded
What is the principle of DAT?
- Detect in-vivo sensitized red cells with IgG and/or complement components.
- RBCs mixed with AHG to determine if sensitization has occurred.
Identify some of the causes of in-vivo antobody-antigen complexes? (4)
- AIHA
- HDN
- Drug-induced hemolytic anemia
- HTRs
When might a DAT be required?
- If patient has benn transfused over the last 2 weeks
- has a history of antibodies
- is pregnant
Why does the DAT not require an incubation phase?
Because the antigen-antobody complexes have occured in-vivo.
How are positive DAT results monitored?
DAT panel usisng monospecific anti-IgG and anti-C3d to determine the specific type of protein sensitizing the cell
Interpreting the significance of a postive DAT requires?
- Knowlege of patient’s diagnosis
- drug therapy
- recent transfusion therapy
- all of the above
All of the above
DAT can detect what level of IgG and C3d molecules per RBC?
- IgG = 100 - 500
- C3d = 400 - 1100
IAT can detect what level of IgG and C3d molecules per RBC?
- IgG and C3d = 100 - 200
What factors influence IgG molecules sensitized on RBC and rate of sensitization?
- Serum : cell ratio
- Reaction medium: Albumin, LISS, PEG
- Temp.
- Incubation time
- Washing RBCs
- Saline used for washing
- Addition of AHG
- Centrifugation for reading
What can cause false positives with the AHG test? (6)
- Bacterial contamination causeing T antigen activation (caused agglutination)
- Saline/glassware contamination
- Extreme reticulocytosis
- AHG contaminated with Rabbit-transferrin
- Positive DAT cells cannot be used in IAT as test would always be positive
- refridgerated specimens - cold auto abs sensitize cells
What can cause false negatives?
- Anticoagulated plasma / old serum
- poor washing can neutralize AHG
- Contamination with human globulins
- Elution of Ab during washing
- incorrect incubation temp.
- cell:serm ratio
- undercentrifucation
- Forgetting to put in AHG
Who is antigen typing performed on?
Patients with a history of clinically significant antobodies
Blood untis for this group for transfusion should be?
Antigen negative to clinically significant antibodies.
How are the number of units to be transfused calculated for Antigen typing?
The number of units requested is divided by the frequency of the antigen-negative individuals.
- Example: Crossmatch request for 2 untis of RBCs for a patient anti-E positive.
- 2 units/ 0.7 (freq. of E -ve people) = 2.8 (3 units must be typed to find 2 E -ve units)
- If multiple abs; E-ve and c-ve
- 2/(0.7x0.20) = 14.3 (14/15 units tested to find 2 units E and c -ve)
Why is knowing the donor’s race useful?
Antigen incidence varies between races
For whole blood transfusions - when antibody(ies) are identified the patient’s red cells should be phenotyped for corresponding antigen gto confirm the validity of the test results.
Patient should lack the antigen with the following exceptions?
- Autoantobody and there is a positive DAT
- Antigen present of previosly transfused cells
- For red cell transfusion Donor cells should lack corresponding antigen to patient’s abs.
What factors are valuable in the abs identification process?
Why is knowing if a patient has been previosly transfused or pregnant important?
- Pt hx
- age
- sex
- race
- diagnosis
- transfusion history
- pregnancy
*transfused or prego Pts may have created an ab as they were exposed to non-self antigens.
What is the best approach for narrowing down you antobodies(y) possibilities on an Ab ID panel?
- Exclusion method
- based on negative results obtained against a panel of 11-20 manufactured O cells, in all 3 phases of testing using homozygous expressed antigens.
- If antigen present on panel cell and patient’s serum did not react it can be ruled out.
How is the inclusion method carried out?
After negative cells evaluated, remining positive antigens examines to see if pattern of reactivity mathces a pattern of antigen positive cells.
- If multiple abs are present how might you seperate specificities and enable idetification?
- How does this affect the RBC membranes?
- What is the purpose of doing this?
- Treat panel cells with enzymes
- modify RBC surface by removing salic-acid residues and denaturing or removing glycoproteins.
- To enhance expression of certain antigens and destroy others.
If the Ab panel does not ID one specific Ab, what may be done?
Additional panels cells tested to veriry or ID multiple Abs. Ensure selected cells have minimal overlap.
When might selected cell panels also be useful?
Selected cell panels also useful when patient has a known Ab and technologist is trying to dermine if additional Abs present.
Describe the principle of Adsorbtion?
- Antibodies removed from serum by addiung target antigen and letting antobody bind to antigen.
- Solid precipitate formed (antibody-antigen complex)
- removed by centrifuge
In adsorbtion the adsorbent is typically composed of?
RBCs
What is the adsorbed serum tested against and for what?
RBC panel for unabsorbed alloAbs
What Commericial Reagents are available for adsorbtion?
- Human Platelet concentrate
- Rabbit Erythrocyte Stroma (RESt)
What is Human Platelet concentrate used to adsorb?
How does this happen?
- Bg-like antibodies from serum
- HLA antigens on PLTs bind the HLA related bg-like Abs, leaving oth. specificities in serum.
- Antibody ID performed on adsorbed serum
RESt is used to adsorb what Abs?
What structures does RESt have?
How does RESt adsorbtion carried out?
- Some cold reacting Abs.
- H, I and IH-like structures
- Incubate at 4oC will remove insignificant Abs, which may interfere w/ significant warm-reating Abs
Most oth. Ab specificities remain unaffected by by RESt, however, RESt possesses what kind of structures?
The structure similarity means RESt adsorbed serum should NOT be?
- Similar to B and P1 antigens
- RESt ,may adsorb anti-B
- NOT used for Reverse grouping and crossmatching
What antibodies are commonly removed via Autoadsorbtion?
Autoabs
What does the simplest method of autoadsorbtion use to remove abs?
Patients own RBCs
What is the FIRST STEP in autoadsorbtion?
Wash patient cells to remove unabound Ab
What is the second step in autoadsorbtion?
May treat cells to remove any autoAb coating cells
What is the THIRD step in autoadsorbtion? How long?
What does the temp depend on?
- Incubate patient cells w/ patient serum for up to ONE HOUR
- thermal range of autoab being removed
What is the FOURTH step in autoadsorbtion?
Look for agglutination in sample (+ve agglutination = RBC binding sites saturated with autoAb)
What is the FIFTH step in autoadsorbtion?
What happens if not agglutination present?
Harvest serum and incubate with new aliquot of autologous cells.
- Harvested serum tested against patient’s RBCs, if no reaction happens adsorbtion is complete.
- If reactivity happens = further adsorbtion needed
How many aliquots are needed for autoadsorbtion to occur?
3 - 6 aliquots
Describe when homologous adsorbtion may be used?
- Patient seveerly anemic so not enouch autologous RBCs for adequate adsorbtions.
- Patient recently transfused; donor RBCs may adsorb alloAbs
How is homologous adsorbtion carried out?
- Patient phenotyped
- RBCs matched to phenotype and used for adsorbtion in place of autologous cells
In homologous adsorbtion, if an exact match cannot be made what is the focus?
Finding cells that lack antigens to which patient may form alloAbs
When might Differential Adsorbtion be used?
Phenotyping patient is Hard because of DAT or recent transfusion.
Describe the method of Differential Adsorbtion?
Patient serum split into 3 aliquots
Eacg aliquot adsorbed using a different cell
What cells are normally used in Differential Adsorbtion?
What must the cells be negative for?
One cell is usually R1R1 and another is R2R2 and the third is rr
one for K, one for Jka, and one for Jkb
In Differential Adsorbtion the cells are treated with enzymed to render which systems antigens negative?
MNS and Duffy
In Differential Adsorbtion, antibody panels are performed on the 3 aliquots together (T/F)
FALSE
Ab ID is performed on each aliquot seprately
Differential Adsorbtion; adsorbtion may be performed when multiple Abs are present? (T/F)
True
Differential Adsorbtion; an adsorbing cell must be?
Antigen positive for one suspected specificty, but negative for others.
Differential Adsorbtion; following adsorbtion what is the cell tested for?
Too see if any additional Abs have been unmasked.
What is Elution used to do?
Used to relased, concentrate and purify Abs
What do the different Elution methods do?
- Change thermodynamic environment,
- changing attractive forces between antigen-Ab complexes and break bonds for dissociation,
- or change structures of RBC surface.
Describe the two types of elution and the difference between them?
Total elution - Ab is released and RBC antigens destroyed.
Partial Elution - ABs removed, RBC antigens stay intact
What is partial elution useful for?
RBC prep for phenotyping and autoadsorbtion
What reagents are used in Partial Elution?
Chrolroquine Diphosphate
EGA
ZAPP
Temp. dependent elution methods are best for detecting?
IgG Abs directed at ABO system antigens
What do the simplest elution methods involve?
What are the RBCs washed with and put in?
changing the temperature of the Ab-Antigen environment
Wash with saline and put in equal volume of saline or albumin
What temp is the gentle heat method performed at?
How does this affect the RBC?
What type of elution method is this?
45oC
RBC intact
Partial elution
What temp is the Total Elution method done at?
RBCs?
What does this enable
56oC
Destroyed
Ab ID
The Lui Freeze method is what type of elution?
Total Elution
Decribe the Lui Freeze method?
- Wash RBCs
- Suspend in Saline/ albumin
- Freeze at -18oC or colder until solid
- rapidly thaw = RBCs burst = free Ab
What is a common quick easy method for total elution to detect non-ABO antigens?
pH methods
Describe Acid Elution?
- Wash
- mix Ab coated RBCs w/ glycine acid pH 3.0
- Disrupts Ab-antigen bond
- Ab released into supernantant
- Harvest supernantant
- neutralize pH
- ID Ab
organic solvents are used in what type of elution method?
Total elution
All of the following are solvents used in total elution methods EXCEPT?
- Dichloromethane
- Xylene
- Elthere
- Chloroquine diphosphate
Chloroquine diphasphate
What part of the RBC do organic solvents affect?
Acts on fats (lipids) = reduce surface tension = reverse van der waals forces that hold Ab-antigen complex
T/F - Organic solvents used for total elution are
- very potent
- time-consuming
- Best for ABO Ab detection
- Most CRITICAL STEP is washing to remove unbound Immunoglobulins
All are correct
If FIRST step washing in use of organic solvents is not done correctly and unaboud Ab remains in test system, what can happen?
What is tested in parallel with supernatant to detect unbound Ab?
The last wash should be?
Contaminate final eluate and cause FALSE positive
Control
Non reactive or eluate results invalid
Nuetralization; other stuctures in the body and in nature have similar_____ ______ similar to RBCs that can be used to neutralize Abs?
- Antigenic structures
What is the First step in neutralization?
Incubate patient’s serum with neutraizing substance to bind Ab
What is the second step in nutralization?
AB ID using treated serum
What is used in neutraization to confim loss of reactivity is due to neutralization and not dilution?
control
When it appears multiple antibodies are present in a sample, the panel can be treated with _____ to help seperate specificities and enable Ab ID?
Enzymes
How do enzymes affect RBCs?
Modify RBC surface by removing syalic acid residues or denaturing/ removing glycoproteins
Enzymes may be used in pace of? In what kind of method?
LISS and PEG in a ONE STEP test method
Describe a second more sensitive method using encyme treated panel cells?
- Treat panel
- peform ID
- Compare reactivity of treated panel to untreated panel reactivity/results
- identiy those that gave weaker reaction/ not react w/ treated panel and those w/ strionger reactivity follwing treated panel.
Dilution is indicative of?
relative concentration
What is a Diltuion factor used for?
The result using the dilution is calculated by?
- to correct for using diluted sample in determination rather than undiluted sample
- multiplying by the reciprocal of the diltuion made
Sinle dilutions are used when?
A dilution refers to?
T/F dilutions can be made single or in a series?
Concentration of a substance is top great to be accruartely calculated
The number/volume of parts to be dilted in the total volume of final solution EX - 1/2, 1/4, 1/8 etc.
To calculate the concentration of a single ditultion you?
Multiply the original concentration by the dilution factor as a fraction.
EX 1:5 (1mL sample in 4mL diluent) dilution of 500 mg/dL solution = 500 mg/dL x 1/5 = 500x0.2 = 100 mgdL
When Ab testing in disease states what must be considered?
- Phase of disease
- Patient condition
When should blood be drawn is testing serum for Abs for infectious organism?
Acute Phase
Convalescent phase (2 weeks later)
* Samples called acute and convaescent serum
What may be noted whent acute and convalescent serum are detected?
Difference in Ab titer
Which diseases may not manifest a rise in Ab titre until months after the acute infection?
Leigionnair’s disease
Hepatitis
What statement best defines Ab titer?
Reciprocal of the highest diltuion of the patients serum in which the Ab is still detectable
A high titre indicates?
High amount of Ab present
What techniques are precisely quantify amount of Ab present?
Flow cytometry
ELIZA
Radioimmunoassay
What menthod can be used to quatify Ab present?
Ab titration can determin Ab concentration
What diltions are used for Ab titration?
Two fold serial diltions of serum containing Ab
What is done with the specimen after initial Ab titre determined?
Why?
Frozen
To compare current speciment results with previos specimen
What is considered a significant score in Ab titre comparison?
A fourfold increase (reactivity in 2 more tubes) or increase in score on 10 >
Tire-leve studies are useful for which patients?
Obstetric w/ IgG that may cause HDFN
An increase in Ab titre during pregnancy is indicative of?
- Antigen positive fetus at risk of HDFN
- Need for intrauterine transfer
Ab titre is useful to differentiate?
the titre level for RhIG is rarely above what?
Immune anti-D from passive aquired anti-D due to RhIG administragtion.
4.4
Performing a titre is one way to confirm prescence of ______ previosly known as ____ ___, ____ ____.
Abs
high titre, low avaidity (HTLA)
high titre, low avaidity (HTLA) are oserved during which pahse of testing?
AHG, weakly positive reactions
high titre, low avaidity (HTLA) exmples? (7)
What is their clinical significance?
- Anti-Ch
- Anti-Rg
- Anti-Csa
- Anti-Yka
- Anti-Kna
- Anti-McCa
- Anti-JMH
NO Clin Significance, may MASK other Abs
What is the primary purpose of crossmatch (compatibility testing)?
To prevent transfusion reaction
What is the secondary purpose of compatibility testing?
Select blood products w/ acceptable survival rates
What are the major cause of transfusion related fatalities and the percentage?
- Clerical errors
- 47% misID at bedside
To prevent collectio of smpe from wrong patient what two items are compared?
SF 518
patient wrist band
What specimen requirement is strictly enforced with pregnant patients?
3 day old
The 3 day rule is enforced with specimen collection for which patients?
Pregnant
Transfused w/i last 3 months
What is the preferred specimen for transusion services?
Why?
Plasma
No waiting time to clot
What is the pr4eferred speicmen for complement ID?
Why?
Serum
EDTA can neutralize complement
What specimens are required for nonates?
Less than 4 months use infant and mothers serum for intial testing
What pre-transfusion testing is required? (4)
What testing is not required?
ABO Rh typing
Ab screen unexpected Abs
Tests to prevent disease transmission
Confimatory testing
Repeat weak D
What is the difference between T & S and T and C testing?
T& S - routine
T and C - routine and emergency
- All tests the same
- ABO Rh
- Ab ID
- XM
What is the difference between T & S and T and C testing if Ab screen is negative?
- T&S - XM not set aside for patient
- immediate spin XM quickly if blood needed
- T&C Follow MBOS to determine # of units to set aside
- XM and set aside blood if req by MBOS
hat is the difference between T & S and T and C testing if Ab screen is Positive?
T&S - ID Ab, AHG XM set aside antigen Neg blood
T&C - ID Ab (unles emergency), AHG XM (always), set aside antigen Neg blood.
What must you do before XM units?
Select appropriate blood or blood component most compatible to patient.
What is the most common donor unit contaminant?
Yersinia enterocolitica
When is matchin of donor and recipient with respect to other blood group antigens NOT necessary?
When the recipient does not have additional serum Abs
Donor units must lack the antigen to serum in patients serum Abs, how is this done?
Phenotype blood with antisera for specific antigen
When must the ABO and Rh be the same as the recipient?
Whole blood - NO ALTERNATIVE ALLOWED
Whole blood is ABO Rh specific
What is the last blood bank test performed before component is issued?
testing patient serum w/ donor RBCs
What is the final check on ABO blood grouping?
What may this detect?
XM
Abs in patient serum that may react with donor antigens that may not have present during screen.
In extreme situations with no time for pre-transfusion testinf what blood type is given?
O negative
When large amounts of donor units other that recipients ABO blood group is given ( ex O given to A) what must be tested and why?
Serum sample for unexpected anti-B or Anti-A before additional blood given
tranfusion of which product doen NOT require compatibility testing?
Plasma
Blood units for intrauterine transfusion must be compantible with?
Maternal Abs capable of cross placenta
If ABO Rh of fetus is determines, group specific blood can be given provided?
if ABO unknown what bloog is given?
there is no fetomaternal incompatibility
O Neg
For neonate transfusion (<4months) blood should be compatible with?
What grouping is required for Neonate transfusion?
- maternal Abs
- Abs in infants circulation reactive at 37oC and AHG
Forward grouping
What is defined as a massive transfusion?
8-10 RBC units in 24hrs
4-5 units in 1 hr
What is properative autologous blood?
Is ABO and Rh tested?
What test is not reuired?
What must they be labelled with?
Blood units for own use by donor
Yes
Unexpected Abs and disease transmission
For autologous use only
What is the primary objective of the XM test?
to ID abs in rrecipient serum that could (inc. anti-A and anti-B) that could destroy transfused RBCs
A recipient will not recive a transfusion until cause of?
incompatibility is dertermined.
What owas developed to ensure effective use of blood?
Why?
Maximum Surgical Blood Ordering Schedule (MBOS)
To enhace effectiveness of blood inventory
How many patient identifiers are required to accurately ID intended recipient?
minimum of 2
What 3 items are required on the donor blood unit label?
- Intended recipients 2 identifiers
- Donation ID number
- Compatibility test result/interpretation
What may alsi be performed after all information has been verfied before issuing blood unit?
Computer XM
