Blood Bank unit 1

What is an ABO discrepancy?

occurs when a reaction pattern, other than one of the four expected reaction patterns, is displayed during testing

what are the two main types of ABO discrepancy?

missing reaction and extra reaction

what are the strategies for working through ABO discrpancies?

1. repeat ABO typing with new RBC suspension using same sample

2. test a new sample

3. review patient’s medical history

4. look at ABO typing reaction

What do you look for in patient history?

• previous BB records

• diagnosis

• medications

• age

what do you do if discrepancy still remains?

• determine if there is a missing reactions or extra reactions

• perform appropriate resolution techniques on current sample

• repeat testing on new sample

what happens if ABO discrepancy still remains after troubleshooting?

transfuse O cells (may require physician approval/signature

what are examples of identification technical errors?

• current blood type does not match historical blood type

• wrong blood in tube

  • when suspected, collect and test new sample or test another in lab sample collected at a a different time

what are examples of documentation of technical errors?

• inaccurate recording of results (incorrect reactions, incorrect interpretation)

• inaccurate recording of patient history (transfusion history, pregnancy history, medication list)

what are examples of reagents and equipment technical errors?

• invalid daily/weekly/month calibration and/or QC

• reagents are contaminated/neutralized

• reagents are expired

• improper centrifugation

• daily/weekly/monthly/preventative maintenance not performed

what are missing or weak reaction in the forward typing caused by?

• age

• disease

• subgroups of A or B

• transplants

what is the least common form of discrepancy?

forward missing or weak reaction

what are resolution techniques for forward missing or weak reactions?

• incubate at room temp for 15-30 minutes

• incubate at 4C for 15-30 minutes

• use a different anti-sera

• read microscopically

• treat RBCs w/ enzymes

• subgroup detection

what are forward extra reaction caused by?

• rouleaux

• wharton’s jelly

• cold agglutinins

• polyagglutinable cells

• acquired B phenomenon

• B(A) phenotype

• chimerism

what are extra reactions caused by rouleaux?

abnormal amounts of plasma proteins

what does extra reaction caused by rouleaux appear as?

loose agglutinates in test tube

-microscope presentation of stack of coins

what is the resolution for rouleaux?

wash RBCs with saline multiple times

what are extra reactions in forward caused by wharton’s jelly?

gelatinous tissue contaminant found in cord blood

-cause RBCs to stick together

what is the resolution for wharton’s jelly?

wash RBCs 3-4 times with saline

what are extra forward reaction caused by cold agglutinins?

cold autoantibodies that bind to and clump RBCs

what are resolution to cold agglutinins?

• wash RBCs using warm (37 C) saline

• perform prewarm techniques

  • perform partial elution

what is the process of performing partial elution?

treat RBCs with chemicals such as EDTA-Glycine-Acid or chloroquine to remove autoantibodies

what are extra reactions caused by polyagglutination and acquired B?

bacterial enzymes may alter the RBC membrane leading to polyagglutination and acquired B

what happens if you suspect extra reaction caused by polyagglutination and acquired B?

check the patient’s diagnosis for evidence of sepsis

what is extra reactions in the forward reaction caused by polyagglutination?

a hidden antigen on the RBCs is exposed and reacts with most human sera

-”T” antigen exposed by bacterial or viral enzymes

-T antigen activation —> polyagglutination with most anti-seras

what does the patient appear as in polyagglutination?

AB positive

what is the resolution to polyagglutination?

test patient’s RBC with a control (monoclonal, albumin, etc.)

-control += polyagglutination

-control negative= AB +

what are extra reactions in the forward reaction caused by acquired B antigen?

patient is group A, but alteration in immunodominant sugar by bacteria results in sugar that resembles group B (D-galactose)

what does acquired B antigen resembles?

group B and cross-reacts with some types of monoclonal anti-B

what are the resolutions to acquired B antigen?

• test with acidified monoclonal/polyclonal anti-B reagent

• test with different monoclonal anti-B reagent

• perform autocontrol

• incubate patient RBCs with acetic anhydride

• perform secretor studies

why do you perform an autocontrol for acquired B?

it is important for distinguishing acquired B from cold agglutinin

what are the forward extra reaction caused by B(A) phenotype?

autosomal dominant phenotype that occurs when B individual have elevated levels of galactosyltransferase

-transfers smaller amounts of the A sugar onto the red cells

-patient RBCs agglutinate with anti-A reagent containing murine monoclonal antibody MHO4

what is the resolution for the B(A) phenotype?

test the patient RBCs with alternate monoclonal anti-A reagent that does not contain MHO4

what are extra reactions in the forward caused by chimerism?

two different RBC populations (from two different genetic sources) present in one individual (dimorphic RBC population)

when can chimerism be seen in?

• transfusion with type compatible RBCs

• transplantation with human progenitor cells of a different ABO group

• fraternal twins

  • large fetal-maternal hemorrhage

what is the resolution to chimerism?

there is no solution

what is the mixed-field reaction?

contain both agglutinated and unagglutinated cells

what can mixed field reactions caused by?

• two distinct cell populations

• bone marrow transplant

• stem cell transplant

• A3 phenotype

• T-polygglutinable RBCs

what are reverse missing or weak reactions caused by?

• age

• disease

• dilution w/ IV fluids

• plasma exchange

• dimorphic RBC populations

what are resolution techniques for reverse missing or weak reactions?

• incubate at RT for 15-30 min

• incubate at 4 C for 15-30 min

• increase serum:cell ratio

• read microscopically

• treat with enzymes

• test plasma against two different manufacturer’s lot numbers

what are reverse extra reaction caused?

rouleaux, subgroups of A with anti-A1, cold autoantibodies, cold alloantibodies, intravenous immune globulin (IVIG)

what are extra reaction caused by rouleaux?

false positive agglutination caused by excess protein

-more likely to affect reverse grouping than

-easier to see under microscope

what does it mean when you do the saline replacement for rouleaux and there’s no agglutination?

rouleaux present

what does it mean if you do the saline replacement for rouleaux and there’s agglutination?

true agglutination

what are extra reactions in reverse caused by subgroup of A w/ anti-A1?

groups as A in forward and O in reverse (typically a weak 1+ rxn with A1 cells)

-check for presence of rouleaux 1st

what is used to differentiate A1 subgroup from non-A1 subgroups?

anti-A1 lectin (dolichos biflorus)

Are subgroup of A with anti-A1 clinically significant?

not clinically significant

what is the resolution for subgroup of A with anti-A1?

subtypes with A1 lectin and testing with other cells

How do you subtype with anti-A1 lectin?

subtype with anti-A1 lectin

negative: patient is a subgroup of A

positive: patient is type A1

additional testing of pt plasma with A2 and/or O cells

neg for both: anti-A1 present

pos for one or both: another cold alloab present (not anti-A1)

what are extra reactions of reverse caused by cold autoantibodies?

antibodies specific for autologous antigens that react at room temp or below

-if tested, all screen cells and auto control are positivewhat

what are resolutions to cold autoantibodies?

prewarm techniques, cold autoadsorption, DTT treatment

what are extra reactions caused by cold alloantibodies?

antibodies specific for human RBC antigens that react at room temperature or below

-if tested, one or more screening cells positive, autocontrol negative

what are resolutions to cold alloantibodies?

prewarm technique

-ABID at room temp or 4C

what are extra reactions in reverse caused by IVIG?

intravenous immune globulin (IVIG)- broad collection of pooled antibodies collected from many donors

-used to treat certain diseases

-during infusion of IVIG to the pt, passive anti-A and anti-B can be acquired, causing extra reactivity in the back type

what are the resolutions to IVIG?

focus on patient history, absorption S

what is weak D phenotype?

cause weakened expression of the D antigen

-reactions are negative or <2 at IS/initial phase of testing

what are the 4 possible mechanisms for D antigen variability?

• weak D

• partial D

• Del

  • nonfunctional RHD

what is inherited mutations weak D?

genetic mutation by amino acid substitution

what types of weak D are inherited mutations?

1, 2, 3

what are the mechanisms of weak D inherited mutation?

intracellular or within the transmembrane region of the protein

-affects the insertion of the D antigen into the membrane

-decrease the number of D antigens, normal expressionw

what are inherited mutation considered?

weak D positive or weak D positive

what is the infusion of inherited mutations?

D positive

what is the position effect (ceppellini effect)?

suppressing effect of C in the trans haplotype position to D (weaker expression of D antigen)

-inheritance of C and D on opposite chromosomes

what is the mechanism of the position effect?

change in amino acids —> conformational change of D antigen —> weakened expression

-normal amount of D antigen, decrease expression

what should you transfuse with someone with weak D position effect?

D positive RBCs

what is partial D?

RBCs lacks parts of the D antigen

-produced by amino acid changes or replacement of parts of RHD with RHCE

what is the clinical significance of partial D?

usually types as D positive at IS but can make an alloanti-D to the part of D antigen that they do not have

when should you suspect partial D?

if a presumed D positive pt forms an anti-D (auto neg) or if monoclonal reagents with different compositions/manufacturers yield conflicting interpretations

what should you transfuse when someone is partial D?

D negative RBCs

Can you distinguish the difference between weak D and partial D through testing with anti-sera?

no

-can only determine through molecular testing

what are the reactions of weak D person?

• reactions are negative or <2 at IS/initial phase of testing

  • reactions are >2 at AHG of IAT

what is Del?

extremely weak D expression, detectable only by absorption/elution of anti-D

what do Del type as?

D neg on routine IS & IAT testing ( can be weakly pos through molecular or adsorption/elution techniques)

what is nonfunctional RHD?

RHD alleles that do not code at full-length polypeptide

-no D antigen is expressed

what do nonfunctional RHD type as?

D negative

what are some anti-reagents sensitives and limitations of older polyclonal-based?

• some are anti-M only —> unsuitable for weak D (IAT) testing

  • IgM reacts with partial DIV and DV RBCs, but not with DVI RBCs

why is it important to know that IgM reacts with partial DIV and DV RBCs, but not DVI RBCs?

DVI variant can result in formation of anti-D

-could lead to HDFN in infants with mothers who have DVI variant

what are some anti-reagents sensitives and limitations of modern monoclonal blends?

• blends of IgM and IgG monoclonal anti-D, licensed for both IS and IAT

• different clones target different epitopes

• higher sensitivity monoclonal blends —> many samples D positive at IS that once required IAT

what is the procedure of weak D testing of IAT method?

1. label 2 tubes anti-D and control and add 1 drop of anti-D and 1 drop of control to the labeled tubes

2. add 1 drop of 2-5% suspension of patient or donor RBCs to both tubes and incubate 37C and wash 3x

3. add 1-2 drops of anti-IgG reagent and add IgG sensitized control RBCs to negative

what is the critical role of control in IAT?

weak D must include control with every test

-control determines whether there was prior sensitization of pt RBCs with IgG antibodies

what happens if the control is positive for IAT of weak D?

results are invalid/inconclusive

when is weak D always performed?

• blood/organ/tissue donors

• RH neg infants born to Rh neg mothers

  • RH positive pt who now type as Rh neg

when is weak D might be performed?

• all infants/neonates who are Rh negative at IS

• maternal samples with pos fetal screen

what are the AABB standards for blood suppliers?

-testing is mandatory

-if initial anti-D is negative, weak D testing is strictly required

-if either test is positive, the component must be unequivocally labeled Rh positive

what are the AABB standards for transfusion services?

-routine weak D testing is optional

-mandatory if assessing maternal risk

-mandatory if you are resolving D typing discrepancies

what are clinically significant antibodies?

react at 37C are associated with hemolytic transfusion reactions or HDFN, and require antigen-negative blood

what are examples of clinically significant antibodies?

Rh (D, C, E, c, e), Kell (K), Duffy (Fya, Fyb), Kidd (Jka, Jkb)

what are clinically insignificant antibodies?

typically IgM, react best at room temp/immediate spin, and rarely cause in vivo hemolysis

what does clinical significance depend on?

reaction phase, temperature, and complement activation- not antibody alone

what does clinical significance determine?

antigen-negative units and a full AHG crossmatch are required

what should you at in patient history?

transfusion history, pregnancy history, diagnosis & medications, prior antibody records, race/ethnicity

what does it mean when you rule out?

cross off antigens carried by panel cells that show a negative reaction with the patient’s plasma

-never rule out based on a positive reaction

-use homozygous

what is the rule of 3?

3 antigen-positive cells must react and 3 antigen-negative cells must not react with the patient’s plasma

what are techniques for multiple antibody identification?

• test selected cells, keeping antigen dosage in mind

• test enzyme-treated panel cells

• treat cells or plasma with DTT

• phenotype the patient

what antigens are destroyed by enzymes?

duffy, MNS, Xg

what antigens are enhanced by enzymes?

ABO, Rh, Kidd, P1, Ii, lewis

what is used specifically to denature Kell antigens?

DTT

when should you suspect a high-frequency antibody?

most or all panel cells react and autocontrol is negative

when would you suspect a low frequency antibody?

antibody screen is negative but a cross match is unexpectedly incompatible

the patient should be ____ for the corresponding antigen when confirming a suspected antibody

negative

what does performing and confirming an antigen type require?

a positive and negative control day of use

-positive control must be heterozygous