Carol A Starks MS, MLS(ASCP), CLS A Starks MS, MLS(ASCP), CLS castarks Brief review of the common...

Carol A Starks MS, MLS(ASCP), CLS

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Brief review of the common blood groups◦ ABO

◦ Rh

◦ Kell

◦ Duffy

◦ Kidd

◦ P

◦ Lutheran

◦ MNS

◦ Lewis

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Basic Blood Bank workups

◦ ABO discrepancies

◦ Single antibody not demonstrating

◦ Single antibody

◦ Multiple Antibodies

◦ Cold

◦ Warm Auto Antibodies

◦ High Frequencies

◦ Low Frequencies

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Genotype

Phenotype

Homozygous

Heterozygous

Dosage

AHG (aka IAT)

IS

Clinically Significant – (HTRs or HDFN)

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ABO Antigens begin to appear on fetal RBCs at weeks gestation; reach adult levels by age 4.

ABO antigens are found on endothelium, kidney, heart, lung, bowel, and pancreatic tissues.

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CLINICALLY SIGNIFICANT

Naturally occurring

Appear at age 4 months; and reach adult levels by age 10.

Levels may fade with advanced age.

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Generally IgM class antibodies

For Group A and Group B persons the predominantantibody class is IgM

For Group O people the dominant antibody class is IgG (with some IgM)

React best at room temperature (22-24oC) or below in vitro.

Activates complement to completion at 37oC

Can cause acute Hemolytic Transfusion

reactions

RBC Immune form: Predominantly IgG

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They both react strongly with reagent anti-A.

80% of group A individuals phenotype as A1

20% phenotype as A2

Reagent anti-A is a mixture of two Abs ;

anti-A which react with both A1 and A2 cells.

anti-A1 which reacts with A cells but not with A2cells in simple testing .

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Differentiation between the A blood subgroups

Reagent anti-A is a mixture of two Abs

The two Abs can be functionally separated by adsorption with A2 cells.

Anti-A1-lectin: is another source of anti-A1.

lectins are seed extracts that agglutinate human cells with some degree of specificity.

The seeds of the plant Dolichos biflorus serve as the source of the anti-A1 lectin; this reagent agglutinates A1 or A1B cells but does not agglutinate A2 or A2B cells.

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Other A subgroups: RBC of the A int, A3, Ax, Ay or A cl. are only rarely seen in transfusion practice.

Subgroup of B: infrequent than the weaker subgroup of A, identified by anti-B and anti-A,B. Subgroups B3 , Bx , Bm and Bcl .

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ABO discrepancies happen when there is no match in results between forward and reverse grouping.

ABO discrepancies are usually technical in nature and can be simply resolved by correctly reporting the testing and carefully checking reagents with meticulous reading and recording of results.

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There are some ABO discrepancies that can happen due to technical errors and may lead to false positive or false negative reactions.

False positive reactions are due to;

Un-calibrated centrifuges

Contaminated reagents

Dirty tubes or glassware

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False negative reactions can be due to many causes

Failure to add serum or reagents

Use of incorrect reagents or samples

Cell suspension is too heavy or too light

Inadequate identification of samples or test tubes

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Group I discrepancies

These discrepancies are between forward and

reverse grouping due to weak reaction or missing antibodies.

These kind of discrepancies are the most common.

The reason for the missing antibody or weak reaction is that the patient has depressed antibody production or cannot produce the ABO antibodies.

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Resolving discrepancies

Eliminate all technical errors

Enhancing the reaction in reverse grouping

Incubate the patient’s serum with reagent cells at room temp. for 15 mins.

Increase serum to cell ratio in the reverse typing.

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Anti-A Anti-B Anti-D A cells B cells Interpretation History

0 0 4+ 0 0 ? O pos

This type of discrepancy can be seen in new born infants, elderly patients.

Patients with lymphoma.

Patients using immunosuppressive drugs.

Patients with immunodeficiency disease, BM transplant.

Group II discrepancies

These discrepancies are between forward and reversegrouping due to weak reaction or missing antigens.

These discrepancies are the least frequent. Can be caused by some subgroups of A or subgroups of B or both.

Also it can be present in patients with leukemia and Hodgkin's disease.

To resolve the problem wash the patient’s cells with saline.

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42 year old man, with a historical type AB+

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4+ 0 4+ 0 0 ? AB Pos

Where is the problem here?What do you think went wrong?What do you do next

Missing Antigens!

I made a mistake, repeat

Get a history

History told us this patient was recently transfused with multiple units of O positive and A positive RBCs. There were a total of 12 units over the last 6 days.

This would be considered indeterminate due to recent transfusions.

What would future transfusions look like?

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Group III discrepancies

These discrepancies are between forward and reverse grouping due to protein or plasma abnormalities.

These can be caused by elevated levels of globulin from certain diseases such as multiple myeloma, Hodgkin's lymphoma. Some are caused by (Rouleaux formation).

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Rouleaux or red cells result from a stacking of erythrocytes that adhere in a coin-link fashion giving the appearance of agglutination.

To resolve this kind of problem, washing the patient’s red cells with saline or adding a drop or two of saline to the tube in case of rouleaux formation.

If the agglutination is true red cell clumping will remain.

Cord blood must be washed 6-8 times in forward grouping ONLY. This is to remove the Wharton’s jelly.

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4+ 2+ 4+ 4+ 4+ ? A pos


4+ 0 4+ 0 4+ A pos A pos

Saline Replacement

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Group IV discrepancies These kind of discrepancies are between forward

and reverse groping due to miscellaneousproblems.

Polyagglutination can occur due to exposure of hidden erythrocyte Ag. (T antigen) in patients with bacterial or viral infection.

Bacterial contamination in vitro or vivo produces an enzyme that alters and exposes the hidden Ag. on red cell leading to T activation.

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Second most important blood group after the ABO system.

Terminology◦ Fisher Race (DCE or CDE)

◦ Wiener (R1, r1, etc.)

D type is determined by the presence of the D antigen.

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Require exposure

Warm Reacting

D induces the most antibodies, then c and E.

HTR with extravascular hemolysis

Severe HDFN with anti-D, anti-c, and mild HDFN with anti-C, E, e.

Due to the mosaic and weak features of the D antigen, can have some weird stuff.

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Clinically and serologically important group.

Antigens in the system:◦ K, k, Jsa, Jsb, Kpa, and Kpb.

Low frequencies: K, Jsa, and Kpa

High frequencies: k, Jsb, Kpb

Kell antigens destroyed by Thiol reagents.◦ DTT

◦ ZZAP

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Require Exposure◦ Typically transfusion

Warm reacting IgG

Can cause severe HTR

Severe HDFN, with lower bilirubin and retic levels. Attacks early RBC precursors.

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Consist of Fya and Fyb antigens.◦ FYA high frequency in the Asian population

◦ FYB high frequency in the Caucasian population.

Fya neg and Fyb neg most common phenotype in African Americans. (gives protection from malaria(vive))

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Exposure required

Warm reacting; IgG

Decreased activity with Ficin (enzymes).

Marked dosage and can show variability in reactions.

Severe HTR, delayed and extravascular usually

Often mild but can be severe HDFN.castarks

Antigens Jka, Jkb, Jk3.

Jk3 very high frequency

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Require exposure.

Warm reacting, IgG, often with an IgM component as well.

Can fix complement with the IgM component.◦ Severe acute HTN possible.

Demonstrate marked dosage.

Variable expression, and will disappear with time/storage.

Known for delayed HTN, that can be severe and intravascular.

“Kidds play hide and seek”.

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Antigens P1 and Pk

Antibodies◦ Cold reacting, IgM

◦ Naturally occurring

◦ Insignificant

◦ Neutralized by H. cyst fluid, or pigeon egg whites.

Auto anti-P (PCH)◦ Biphasic IgG that binds in the cold and then

hemolyzed when warmed.

Give full crossmatch compatible typically.

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Antigens Lua and Lub

Lua is a low frequency

Lub is a very high frequency

Antibodies

Uncommon, may be naturally occurring

Not usually significant

Decreased activity by enzymes

Typically give full crossmatch compatible.

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Antigens M, N, S, s.

M roughly equals N in prevalence (≈75%)

s is more frequent than S.

S-s- individuals may also be U-. U is a high frequency antigen. These individuals can make Anti-U.

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M and N are almost the opposite of S and s.

M and N can be ignored unless they react at 37.

Rarely associated with severe HDFN.

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Anti-M and Anti-N Anti-S and Anti-s

Naturally occurring Require exposure

Cold reacting, IgM Warm IgG

Dosage Minimal Dosage

Insignificant Significant

Coded for one gene Le. (like D)

Le will add a fucose to the GlcNAc resulting in Lea.

“Secretors” will add another fucose resulting in Leb

Formation of Leb is preferential in secretors.

Antigens are not tightly bound, rather they are absorbed onto the service of the RBCs.

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Pregnancy◦ Antigen levels decrease due to change of

composition of patients plasma. Can give rise to transient Lewis antibodies

Lea-Leb+ do not develop Anti-Lea

Children’s Lewis typing will change as chains are converted. Lea+Leb+ will change to Lea-Leb+

Associated Infections:◦ H Pylori attaches via Leb

◦ Lea-Leb- more susceptible to Candida and E coli.

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Antibodies are typically insignificant.

Rare HTR

Minimal to no HDFN (does not cross placenta)

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When do we perform an antibody identification.

When testing suggests a new antibody (warm autos)

To confirm a previously identified antibody (per facility SOP)

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Basically just a larger antibody.

Uses reagent O cells from 8 – 20 donors

Results are documented on a sheet called an antigram. The antigram is a “roadmap” that outlines every cell’s phenotype.

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Antigram Geography

Having a CONSISTENT approach to antibody workups minimizes error.

Most workups are simple but cutting corners increases risk for dumb mistakes.

Use this or your own system, but to the same approachEVERY TIME!

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Check history

Check Autocontrol

Look at general pattern

Look at what is NOT there(cross outs)(rule outs)

Look at what is there

Use special techniques if needed

Ensure statistical significance.

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History can give you a clue and keep you from doing something dumb.

Up to 70% of cases are impacted by history.

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Anti-D in a pregnant patient

Recent bacterial infection treated with antibiotics that cause warm auto

Recent transfusion

ITP

Racial significance◦ African Americans lack Duffy Ags

◦ Asians: RARELY D-

◦ Caucasian: May lack high freq. Ags

Previous Ab and/or phenotyping.(Kidds)

Transplants

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