1308- lecture 3: blood bank reagents and equipment

What can strength of agglutination be affected by? Name 3 things

Antibody concentration (titre)

Reagent preservation

Storage conditions/expiration

What is potency?

Strength of a reagent, how strong it'll react to an antigen/antibody

Potency is measured by?

strength of agglutination

Serological agglutination in BB is measured/reported as?

4+, 3+, 2+, 1+ (depends on button size)

0 for neg rxns

Specificity is?

The ability of a reagent to react only w/ its intended antigen/antibody

E.g. anti-A antisera will ONLY agglutinate A-antigens

Why is specificity important?

prevents false+ results/esures accurate antigen typing/antibody ID

High specificity= interpretable rxns

How often is reagent quality control performed?

daily, must meet min reactivity standards set by manufacturer and regulatory bodies

Why is reagent quality control performed?

It verifies potency and specificity

Explain potency vs specificity

Potency: expected rxn strength w/ known positive cells

Specificity: no rxn w/ known neg cells

What agglutination results indicates that reagent potency is deteriorating?

<3+

If the reagent strength is poor, why is that bad?

loss of potency = detection of antigens in PT samples compromised

reagent is now unacceptable for PT testing and you need to correct/doc this

Anti-A/B commercial reagents are AKA

antisera aka known antibodies

What are Anti-A/B commercial reagents used for?

used to determine the prescence/abscnece of the A/B antigens of the ABO blood group system

What is the process of using reagents for ABO testing?

PT/blood donor cells (unknown antigens) are added to the known antibody (A/B)

Cells + antisera are gently mixed, centrifuged, and observed for presence of agglutination

Agglutination=specific antigen is present bc it reacted w/ its specific antibody

ABO forward typing is aka

ABO forward grouping aka front testing

What does forward testing determine?

determines which ABO antigens are present/absent on donor/PT RBCs (this determines blood group!)

Blood group is aka

blood type

ABO reverse typing is aka

ABO reverse grouping aka back testing aka serum ABO typing

What does ABO reverse typing do?

Confirms the forward typing

How does ABO reverse typing confirm forward typing?

Determines which ABO antibodies are present in PT/donor cells based on which antigens they have on their RBCs

They cant have the antibody to an antigen they possess on their RBCs (or their antibodies would destroy their own cells)

Name 3 reagents used for ABO forward typing:

Anti-A

Anti-B

Anti-A,B

2 reasons why we used pooled cells in reverse ABO testing?

Stronger antigen expression = enhances detection of weak or low titre antibodies

Improved sensitivity and reliability = reduces the risk of false - rxns

Landsteiners rule

"Same" antigens and antibodies CANNOT coexist

People without group A-antigens on their RBCs will have?

WILL have anti-A antibody in serum/plasma

People without group B antigens on their RBCs will have?

WILL have anti-B antibody in serum/plasma

People without group AB antigens on their RBCs will have?

WILL have both anti-A antibody and anti-B antibody in serum/plasma

People WITH both AB antigens on their RBCs will have?

will have NO ABO antibodies in their serum/plasma

What is the theory behind reverse testing?

PT possess the antibody directed against the antigen of the ABO system that is LACKING on their RBCs (landsteiners rule!!!)

What does a serofuge do?

Separates cells from serum, plasma, or from BB reagents using centrifugal force (which seperates components by density)

If the serofuge isnt fully seperating its components, what could be the possible cause?

Low speed, short spins, tubes arent balanced

If the serofuge is hemolyzing its specimens, what could be the possible cause?

Excessive speed, old blood, vigorously handled

If the serofuge is breaking tubes, what could be the possible cause?

Wrong rotor, uneven balancing

If the serofuge is vibrating or making noises, what could be the possible cause?

Rotor imbalance, work bearings

If the cell washer is hemolyzing RBCs, what could be the possible cause?

Excess RPM speed, prolonged spinning, improper mixing

If the cell washer isnt fully washing specimens, what could be the possible cause?

Too little saline, wrong cycle settings

If the cell washer is stopping or having error codes, what could be the possible cause?

Mechanical failures, clogged tubing

If the cell washer is recovering low amounts of cells, what could be the possible cause?

Cells could be stuck in tubing, improper spin

Is it monoclonal antibodies or polyclonal that react with a specific epitope on a given antigen?

Monoclonal!

Polyclonal can react with various epitopes on a given antigen

5 steps to producing monoclonal antibodies

1) inject animal (or tissue culture) w/ antigen to stimulate antibody production

2) isolate antibody-secreting B cells (which are specified for that specific epitope)

3) fuse the b-cell w/ a myeloma to make hybridomas

4) select specific hybridomas

5) clone hybridomas to produce monoclonal antibodies

6) harvest monoclonal antibodies to make you monoclonal antisera

5 steps to producing polyclonal antibodies

(go look at the rabbit diagrams yall)

1) inject antigen into animal

2) antigen activates b cells

3) plasma b cells produce IgG immunoglobulins

4) obtain serum from animal containing polyclonal antibodies

5) antisera can now be prepped w/ polyclonal antibodies

Do polyclonal or monoclonal antibodies require a hybridoma to create antibodies?

MONOCLONAL to create antibodies and then you can harvest them

Poly vs monoclonal antibodies: which has a hetero antibody population and which has a homo?

Poly: heterogenous

Mono: homogenous

Poly vs monoclonal antibodies: cross reactivity

Poly: stronger possibility of cross-reactivity w/ similar antigens

Mono: less likelihood of cross-reactivity

Do polyclonal antibodies have a collection or a single IgG antibody?

They have a collection of IgG antibodies that bind to diff epitopes on a single, target antigen

Which antibody has a single IgG antibody? Mono or polyclonal?

Monoclonal has a single IgG antibody that bonds to one epitope on a single, target antigen

Epitope

the part of an antigen molecule to which an antibody attaches itself

Antisera

serum that contains antibodies for one or more antigens

Reverse ABO antibodies are aka?

isoagglutinins (naturally occurring antibodies)

What antibodies dont require previous exposure from transfusions or pregnancy to be demonstrated?

Reverse ABO antibodies aka isoagglutinins aka naturally occuring antibodies

Name a few blood group antibodies that ARENT isoagglutinins

Anti-D

anti-K

anti-Fya

anti-C

What does buffer solution in reverse ABO testing do?

Preserves the cells

Minimizes RBC hemolysis

Maintains antigenicity during shelf life of product

Antigenicity

property of behaving as an antigen

The Rh system determines the prescence/absence of which antigen?

the D-antigen

myeloma

a malignant (cancer) cell that can divide indefinitely

Why do we use myeloma and B-cells to make hybridomas in monoclonal antibody production?

Myeloma live forever + B cells provide specificity (and only the fused cells will survive, so then you have cells that produce the same antibody forever)

What is the purposose of BB reagent QC?

to ensure accuracy, potency, and specificity

in commercial antisera and RBCs

What are 3 common reagent QC problems?

Weak or missing rxns

Unexpected positive rxns

Variable rxns

What could an unexpected positive rxn be caused by (relating to reagent QC)?

contamination or improper storage

What could weak or missing rxns be caused by (relating to reagent QC)?

reagent degradation

What could variable rxns be caused by (relating to reagent QC)?

poor antigen expression or technical error

How often must preventative maintenance of automated cell washers be done?

Daily

Weekly

Monthly

Quarterly

How often must serofuge/centrifuge maintenance be done?

Daily

Weekly

Monthly

When troubleshooting reagent RBCs, what evidence could point to reagent RBC deterioration?

Spontaneous agglutination

Significant hemolysis

Loss of agglutination strength overtime

What information do QC logs include?

Date and time

Reagent name

Lot # and expiry date

Tests performed

Controls used (+/-)

Results observed (agglutination strength)

Initials of individual performing test

Comments or corrective action if needed

What do you do if QC fails?

Repeat w/ new reagent or cells

Record investigation and action taken in the log

DONT release PT results until QC is acceptable

QC documentation is comprised of what 6 steps?

1) Prepare QC log

2) Perform QC testing- record graded rxns including +/- controls

3) Record results immediately

4) Take corrective action as needed

5) You/another review and sign/date log

6) Maintain records

Hybridoma

an immortal cell line that can produce the exact same specific antibodies indefinitely

Polyclonal antibodies are aka

polyclonal antisera

Antibodies are aka

immunoglobulins

Antigens are aka

immunogens

What are polyclonal antibodies?

Antibodies that have an affinity for the same antigen but different epitopes of that antigen

Basically: they're all specific for that single antigen, but not for the same epitope on that antigen

What are monoclonal antibodies?

Are identical antibodies produced from a single clone of a B cell, all with the same specificity for one epitope

What is the purpose of QC documentation?

Ensure traceability of QC results

Confirm most recent date of testing

Confirms reagents perform as expected

Provides evidence for auditing/accreditation

Supports PT safety by verifying test reliability

Name types of reagents used for typing the D-antigen?

high protein based, low protein based, saline based, chemically modified, monoclonal or blends of monoclonal antibodies

Saline based reagents (for Rh antigen typing) contain what immunoglobulin?

IgM immunoglobulin

What advantage does a asline-based reagent provide for Rh antigen typing?

they are low-protein based and it can be used to test cells already coated with IgG antibody (e.g. someone with an autoantibody attached to their red cells)

High-protein based reagents for Rh antigen typing are primarily made of what?

primarily IgG anti-D and contain potentiators and high protein [ ]

What is one downside to high-protein based reagents for Rh antigen typing?

can result in false + rxns, so an Rh control must be run in parallel to the anti-D testing

BB equipment has its own dedicated logbook, which can ID the unit with what info?

Equipment name

Equipment ID #

Manufacturer

Model #

Serial #

Date unit was placed into service

Antisera is?

blood serum containing antibodies against specific antigens

Anti-AB reagents for ABO forward typing is primarily used to do what 3 things?

Confirm weak subgroups of A (A2, Ax)

Test neonatal samples

Resolve discrepancies when forward/reverse results don't correlate

Anti-AB reagents for ABO forward typing is primarily used to do what 3 things?

Confirm weak subgroups of A (A2, Ax)

Test neonatal samples

Resolve discrepancies when forward/reverse results don't correlate

What does reverse ABO testing determine?

Determines the ABO antibodies in PT blood

When do ABO antibodies develop in humans?

between age 3-6 months

At what temp do ABO antibodies react?

at RT or colder, because they are IgM antibodies which are cold-reacting

What does it mean if ABO forward and reverse typing dont coorelate?

results are deemed discrepant and the ABO cannot be interpreted (in an emergency, O-neg will be transfused)

What type of cells are used for ABO reverse testing?

commercially prepared cells of KNOWN antigen content (A1/B pooled cells)

Pooled cells

the manufacturer mixes cells from multiple donors that are either group A1 or group B

How would an individual get anti-D to be present in their serum/plasma?

they have to have been previously exposed to the D-antigen through pregnancy or transfusion, and then an immune response is responsible for the anti-D

Is anti-D naturally occuring?

NO

Anti-D is aka

anti-Rh

How is Rh typing different from ABo typing

when an individual lacks the D-antigen, it does not correspond w/ the antibody present/absent in a person's plasma

If a person is B-neg, what antibody do they posses? A, B, D, etc?

they possess the A-antibody but they DON'T possess the D-antibody (as it isnt naturally occuringggg)

Relating to reagents for ABO forward typing: what is the direct testing method?

RBC added to antisera

spin

read for prescence/abscence of agglutination

When doing the direct testing method with Anti-A reagent, what does it mean if there IS agglutination?

A-antigen present on RBC (graded 1+ to 4+)

When doing the direct testing method with Anti-B reagent, what does it mean if there IS agglutination?

B-antigen present on RBC (graded 1+ to 4+)

When doing the direct testing method with Anti-A,B reagent, what does it mean if there IS agglutination?

A/B-antigen present on RBC (graded 1+ to 4+)

When doing the direct testing method with Anti-A,B reagent, what does it mean if there IS NOT agglutination?

negative for presence of both antigens

When doing the direct testing method with Anti-B reagent, what does it mean if there IS NOT agglutination?

negative for presence of B Antigen

When doing the direct testing method with Anti-A reagent, what does it mean if there IS NOT agglutination?

negative for presence of A Antigen (graded as 0)