Blood Bank Fundamentals 2 Immunohematology Methods and ...

Blood Bank Fundamentals 2

Immunohematology Methods and Blood Groups

October 2021

Program Director: Jayanna Slayten, MS, MT(ASCP)SBBcmSupervisor, Indiana University Health Blood BankAnd Adjunct Faculty, University of Texas Medical Branch SBB Program

Faculty Disclosure

• None

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In compliance with ACCME policy,

AABB requires the following

disclosures to the session audience

Objectives• Explain the American Society of Clinical Pathologist (ASCP) SBB and BB

exam requirements

• Review and explain the topics outlined on the ASCP BB/SBB Exam Content

Outline

• Define and relate pertinent information from the ASCP Content Outline that

will be on these exams to aid in preparing for the BB or SBB exam.

• Discover helpful hints for studying for and taking these exams.

• Apply knowledge for interactive question and answer polling session.

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Blood Bank Fundamentals 2021

Session 1 Session 2 Session 3 Session 4

Blood Bank Fundamentals 1: Immunohematology Building Blocks

Blood Bank Fundamentals 2: Immunohematology Methods and Blood Groups

Blood Bank Fundamentals 3: Transfusion Medicine Physiology and Pathophysiology

Blood Bank Fundamentals 4: Donor Collection, Blood Products, Laboratory Management and Blood Bank Quality

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Session 2 Speakers

Michelle Lodermeier,

MBA, MLS(ASCP)CMSBBCM

American Red Cross

SBBT Program

Minneapolis, MN

Paul Mansfield,

MT(ASCP)SBBCM

American Red Cross

Immunohematology Reference Lab

Philadelphia, PA

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What Blood Bank Tools to Use: When and Why

Michelle Lodermeier, MBA, MLS(ASCP)CMSBBCM

American Red Cross, SBBT Program

Read, know, and understand…

• Technical Manual Methods (current edition available online)

– Principal (includes application)

– Interpretation

– Notes (includes limitations)

Antigen and Antibody Reactions

• Agglutination - crosslinking of antibody coated RBCs

resulting in visible clumping

– Stage I: Sensitization of antigen with antibody

• Affecting Factors: Temp, pH, antigen distribution, incubation

time, antigen/antibody ratio, ionic strength, steric/chemical fit

– Stage II: Formation of bridge between sensitized RBCs

causing lattice formation

• Affecting Factors: # of antibody binding sites, antibody size

(IgM vs IgG), location and # of antigen sites, zeta potential,

centrifugation, degree of water extrusion from RBC membrane

Picture from: http://learnserology.ca/LearnSerology_content/LS1_Serological_endpoints_Agglutination_Stage_2.html

http://learnserology.ca/LearnSerology_content/LS1_Serological_endpoints_Agglutination_Stage_2.html

• Hemolysis - release of intracellular hemoglobin from ruptured

RBCs

– Requires activation of complement

• Does not happen in plasma (EDTA binds Ca++ and Mg++)

• Complement fixation test - antigen & antibody incubated in the

presence of complement; indicator cells pre-sensitized with

antibody added

– Positive: lack of hemolysis (classic pathway activated)

– Negative: hemolysis

Antigen and Antibody Reactions

Picture from: https://www.shutterstock.com/image-photo/red-color-

serum-involved-blood-cell-1496710691

https://www.shutterstock.com/image-photo/red-color-serum-involved-blood-cell-1496710691

Antigen and Antibody Reactions• Precipitation - visible formation of an insoluble

complex– Lattice formation occurs when antibody = antigen

– Basis of immunodiffusion (radial and Ouchterlony) and immunoelectrophoresis

• ELISA – quantitate antibody or detect low-level immunoglobulins on RBCs– Direct

– Indirect

– Sandwich

– Competitive

Picture from: https://www.biosciencenotes.com/elisa-enzyme-linked-immunosorbent-assay/

Picture from: http://technologyinscience.blogspot.com/2012/01/ouchterlony-immunodiffusion-ouchterlony.html#.YS-OnOhKjIU

https://www.biosciencenotes.com/elisa-enzyme-linked-immunosorbent-assay/

http://technologyinscience.blogspot.com/2012/01/ouchterlony-immunodiffusion-ouchterlony.html#.YS-OnOhKjIU

Flow Cytometry

• Quantitative and qualitative data on cell populations– Stream of individual cells pass laser in a flow chamber;

scattered light creates electrical signals for analysis • Forward scatter = cells size• Side scatter = cell internal structure• Fluorescent labeled antibodies to detect targets• Cell populations can be separated by “gating” on software

• Applications – Define cell markers and antibodies

• RBC, platelet, and neutrophil • Antigen zygosity

– Detect minor cell populations (FMH, survival times)– Detection and/or measurement of low-level bound IgG, IgA,

IgM • DAT negative AIHA

Picture from: https://www.abcam.com/protocols/introduction-to-flow-cytometry

Neutralization

• Specific blood group soluble

substances (antigen) combine

with antibody, preventing

reactivity with added antigen

positive indicator cells

– Requires a dilutional control

• Applications

– Confirm specificity

– Reveal other non-neutralizable

antibodies (masked)

Sources and Specificity

(some available commercially)

ABH saliva of secretor individuals

Lewis saliva

P1 hydatid cyst fluid, pigeon ovalbumin

I breastmilk

Sda guinea pig urine (+ individuals)

Ch/Rg plasma (+ individuals)

Cromer human urine and plasma (conc.)

Various

Blood Groups Recombinant blood group

antigens (rBGA)

Predicting Red Cell Survival…• Sometimes incompatible blood is transfused, or clinical

significance of an antibody is unknown:

– Rare blood is needed and not available

– Antibody is unidentified

• Monocyte monolayer assay (MMA)

– Monocytes incubated with antibody and/or complement coated

RBCs

– Phagocytosis predicts in vivo antigen-positive RBC survival

– Not applicable: HDN, autoantibodies, clinical significance of

IgM

• In vivo crossmatch

– 51Cr-labeled RBCs transfused

– Radioactivity in recipient measured to predict RBC survival

Photograph by Sandra Nance

Adsorption• Removal of antibody from serum by incubation with cells or stroma

– Testing variables: cell phenotype, temperature (warm/cold), incubation time,

serum/cell ratio, enhancement, pretreatment of cells

– Commercial reagents

• Rabbit erythrocyte stroma (anti-B, -P1, -H, -I, -IH)

• Human platelet concentrate (HLA antibodies)

• Applications

– Remove “masking” antibody: autoantibody, separate multiple antibodies,

antibody to high prevalence antigen

– Confirm antigen or antibody specificity

– Reagent preparation

Autologous Adsorptions for Warm Autoantibody

• Autologous (patient’s cells + patient’s plasma)

– Limitation #1: not transfused in past three months

– Limitation #2: antibody may be diluted out

– Requires a large volume of patient RBCs

– May need to prepare DAT negative cells (ZZAP, W.A.R.M.)

**BEST PRACTICE: Prove WAA by testing autologous DAT negative cells with plasma /eluate**

Allogeneic Adsorptions for Warm Autoantibody

• Allogeneic (donor cells + patient’s plasma)

– Limitation #1: can only rule out alloantibodies to common antigens (routinely)

– Limitation #2: antibodies to high prevalence antigen may be adsorbed

– Limitation #3: antibody may be diluted out

• Known phenotype adsorbing cell: phenotypically similar for commonly clinically

significant antibodies

– May need to perform cell separation/hypotonic wash, removal of immunoglobulins, DNA analysis

– Limitation #1: Antibodies may be missed in patients with unknown variant antigens

• Unknown phenotype adsorbing cells: R1R1, R2R2, rr

– Untreated: negative for combination of common clinically significant antibodies

– Enzyme treated: one must be K-, Jk(a-), Jk(b-)

• Pretreating cells destroys antigens = easier to find an adsorbing cell and allows for the

detection of more alloantibodies

**BEST PRACTICE: DNA analysis is recommended to

predict antigen types whenever possible**

Chloroquine Diphosphate &

EDTA Glycine-Acid (EGA)• Applications

– Both dissociate IgG from RBC; DAT negative RBCs may be used for phenotyping– Chloroquine denatures Bg/HLA related-antigens (used for confirmation)

– EGA denatures KEL system, Bg antigens, and Er antigens

• Limitations

– Over incubation can cause irreversible membrane damage resulting in hemolysis or weakened RBC antigen expression

– Do not use polyspecific antiglobulin reagents – Chloroquine does not dissociation complement proteins

Elution• Remove and recover antibody from sensitized RBCs (heat, lui-freeze thaw, cold acid

elution)

• Limitations: reactivity in last wash (invalid), antibody binding to tube, disassociation of

antibody prior to elution, unable to phenotype antigens destroyed by treatment

• Applications

– Investigate positive DAT• Nonreactive: Drug, antibody to low prevalence antigen

• Pan-reactive: Autoantibody, antibody to high prevalence antigen

• Alloantibody: Transfusion reaction, HDFN

– Purifying and/or concentration

– Adsorption/elution • Reagent preparation

• Detect weak antigens using polyclonal antisera

• Identification of anti-G

Titration

• Semi-quantitatively determine amount of antibody present or strength of antigen

expression on RBC

– Interpret (reciprocal of highest dilution with 1+ reactivity) and score titration scheme

– Cell selection: consistency and antigen expression (single vs. double dose, phenotypes with

weaker or stronger expression)

• Applications

– Serial dilutions

• Prenatal studies

• Antibody identification - “HTLA” reactivity or separate specificities

• Cold agglutinin

• Patients receiving non-ABO identical solid organ transplant

– Point dilutions

• Isohemagglutinin titers (donor products)

• Cold antibody screen

**CAUTION: manual titers are subject to tech variation in dilutional preparation and grading**

Cell Separation • Separate autologous RBCs from transfused RBCs

– Microhematocrit • Reticulocytes are less dense & have lower specific gravity than older

transfused RBCs• Limitations: fresh sample that is >3 days since last transfusion,

reticulocyte production (note underlying disease state or chronic transfusion), antigen expression on retics

– Hypotonic Wash• Hemoglobin S RBCs (SS or SC) don’t lyse with when washed with

hypotonic saline (0.3% w/v diluted BB saline)

• Application– Phenotyping– Testing with autologous serum or eluate (WAA)– DAT

**BEST PRACTICE: DNA analysis is recommended to predict antigen types whenever possible**

Proteolytic Enzymes

DESTROYED: Fya Fyb M N S s(S s reported to vary)

ENHANCED: RH System, LE System, JK System

• Sialic acid (net negative charge) and glycoproteins (hydrophilic) removed, allowing RBCs to be close and antibodies to better access antigens

• Can use one or two stage method

• Standardization procedure – Stages of unlimited enzyme modification – When optimum enzyme modification occurs– Serological standards

• Application– WAA react better with enzyme treated cells

– Denature antigens– Antibody Identification

• Enhance reactivity of antibodies• Investigation of AB to high prevalence antigen• Separate multiple antibodies

https://borneorainforestinformation.weebly.com/parts-of-a-rainforest.html

Sulfhydryl Reagents

• AET, DTT(0.2M), and 2ME irreversibly reduce protein disulfide bonds to free sulfhydryl groups, disrupting protein tertiary structure

• Applications– Antibody Identification

• Investigation of antibody to high prevalence antigen

• Separate multiple antibodies

• Anti-CD38

– RBCs treated to remove antibody

– Serum treated to destroy IgM reactivity (0.01M) • Requires a dilutional control

DESTROYED: KEL, LU, KN, LW,

YT blood group system antigens

Picture from: https://www.quora.com/How-do-reducing-agents-denature-proteins

https://www.quora.com/How-do-reducing-agents-denature-proteins

DARA Mitigation Techniques• Pretreatment

– Determine antigen and antibody status

• During treatment (interference from anti-CD38)– DTT treated RBCs– Trypsin treated RBCs– Phenotyped cord RBCs– Phenotyped DAT negative RBCs from

patients treated with anti-CD38– Preselected AA donors lacking CD38 on

RBCs

Picture from: https://onlinelibrary.wiley.com/toc/15372995/2015/55/6pt2

https://onlinelibrary.wiley.com/toc/15372995/2015/55/6pt2

ZZAP Reagent• Proteolytic enzyme and sulfhydryl mixture removes

immunoglobulins coating the RBCs, uncovering antigen sites

to increase adsorption

• Applications

– Denature antigens

– Treat RBCs • With a positive DAT prior to autoadsorption

• Prior to alloadsorption

Picture from: https://www.thoughtco.com/mixture-definition-chemistry-glossary-606374

https://www.thoughtco.com/mixture-definition-chemistry-glossary-606374

Enhancement Media• Albumin (typically 22%) – reduces zeta potential around red blood cells,

enhancing agglutination

• LISS –contains fewer Na+ ions than “normal” saline, resulting in less zeta

potential, increasing the rate of association between antibodies and antigen – Decreases incubation time

• PEG – removes water from the system allowing RBCs to get closer together

and concentrates antibodies around the red cell surface

– Do not interpret at 37C (false positives); read only after washing at AHG phase

• Polybrene – prompts nonspecific agglutination that is not dispersed when

sodium citate is added if an antigen-antibody bridge was formed.

– Rapid method to detect antibody

Techniques for Antibody Detection

• Test tube “gold standard”

– Direct agglutination: Immediate spin (IgG or IgM)

– Indirect agglutination: AHG (IgG)

• Column agglutination technology - red cell agglutinins trapped in dextran acrylamide

gel (buffered or anti-IgG) at centrifugation; there is no washing or check cells

– Positive: agglutinates on top or within gel column

– Negative: RBCs not agglutinated centrifuge to bottom

• Solid phase - antigen bound to solid support (microtiter plate), incubated with

antibody. After wash step, indicator red cells with anti-IgG added and centrifuged

– Positive: Indicator RBCs adhere to side of well

– Negative: Indicator RBC settle to bottom of well (button)

– Other uses: platelet, HLA, infectious disease testing

Less Sensitivity TUBE > GEL COLUMN > SOLID PHASE More Sensitivity

TIP: know advantages & disadvantages: sample volume, automation, subjective grading, rouleaux

Antibody to High Prevalence Antigen

• Clues:

– Reacts with all cells (typically same strength) and negative auto control (if not recently

transfused)

– Reacts with phenotypically matched cell

• Problem Solving:

– Ethnicity

– Serological phenotype or genotype

• What antibodies can the patient make

• Test patient for high prevalence antigens

• DNA analysis can expedite workup

– Chemical/enzyme treatment (Papain, ficin, trypsin, DTT)…..get it into a system!

– Rare and null cells

• Source of units: autologous, siblings, ARDP

**BEST PRACTICE: rule out underlying

alloantibodies prior to testing rare cells**

Miscellaneous • Saline Replacement

– Rouleaux (microscopic “stacked coins”) will disperse, and true agglutination will remain

• Seen in patients with abnormal albumin:globulin ratios (multiple myeloma, globulinemias) or infusedwith volume expanders (dextran, hydroxyethyl starch)

• Paroxysmal nocturnal hemoglobinuria (PNH)– Acquired intrinsic RBC membrane abnormality resulting in

sensitivity to complement mediated lysis • Cells lack GPI anchored protein (CD 59 & CD 55)

• Sucrose lysis (screening)

• Ham’s test (confirmation)

Molecular Methods • Predict the phenotype of donor/patient RBCs or platelets

• Polymerase Chain Reaction (PCR) - specific DNA

sequences multiplied rapidly (billionfold amplification)

– Requires two primer: forward (5’) and reverse (3’) to flank

the region of interest

• Transcription-Mediated Amplification (TMA) - specific RNA

sequences to be multiplied rapidly (billionfold amplification)

– RNA must be first converted to DNA to produce RNA

amplicon (common in donor screening for viral markers)

Picture from: https://genius.com/Biology-genius-the-central-dogma-annotated

https://genius.com/Biology-genius-the-central-dogma-annotated

Direct Antiglobulin Test

• Antiglobulin reagent added to RBCs sensitized cells in vivo

– Strength of agglutination related to amount of bound IgG/complement

– Used to determine if hemolysis is immune mediated

• Autoimmune: WAIHA, CAS, Mixed-Type, PCH

• Alloimmune: transfusion reaction (IgG and/or complement) , HDFN (IgG)

• Drug-induced

– Positive DAT ≠ hemolysis: correlate lab values with clinical presentation

• Healthy individuals can have positive DAT

• IVIG and RhIG can cause positive DAT

• When to investigate a positive DAT (classify, ABID, eluate)

– Evidence of hemolysis, recent transfusion, drugs, HPC transplant, IVIG, therapeutic agents

Picture from: https://www.sciencedirect.com/topics/immunology-and-microbiology/elution

https://www.sciencedirect.com/topics/immunology-and-microbiology/elution

Auto Immune Hemolytic Anemia• Warm auto immune hemolytic anemia

– DAT: IgG and/or C3 positive; positive eluate

– Antibody: “spill-over” may be free floating in plasma, reacts with DAT negative

autologous cells, broadly reactive or specificity

– Recent transfusion? Stop and consider multiple alloantibodies or antibody to

high prevalence antigen

– Resolving serologic issues: adsorptions, may need to remove IgG or perform

DNA analysis

• Other considerations

– Blood selection

– DAT negative AIHA: low affinity, IgM, IgA, below detection limit

Picture from: https://www.youtube.com/watch?v=WjYDsOYX2wc

https://www.youtube.com/watch?v=WjYDsOYX2wc

Auto Immune Hemolytic Anemia• Cold Agglutinin Disease

– DAT: C3 positive; nonreactive eluate

– Antibody: IgM (high titer & broad thermal amplitude); usually anti-I (Mycoplasma

pneumoniae)

– Resolving serological issues: warm washing, saline/albumin controls, DTT treat plasma, may

need to remove IgM, DNA analysis, adsorptions if masking alloantibodies at 37C

• Mixed-type AIHA

– DAT: IgG and C3 positive or C3 positive; positive eluate

– Antibody: IgG and IgM (broad thermal amplitude); specificity unknown

• Paroxysmal cold hemoglobinuria

– DAT: C3 positive; nonreactive eluate

– Antibody: negative IAT; biphasic auto-anti-P binds at low temps & activates complement at

37C

– Donath-Landsteiner

Picture from: https://www.youtube.com/watch?v=WjYDsOYX2wc

https://www.youtube.com/watch?v=WjYDsOYX2wc

Drug Associated Hemolytic Anemia

Common culprits: Pipercillian, Piperacillan in Zosyn, Cefotetan, Ceftriaxone

1. Drug dependent - reacts with drug treated cells – DAT: IgG positive; eluate: nonreactive eluate; IAT negative

2. Drug dependent – reacts with untreated cells in the presence of the soluble drug

– DAT: C3 positive; eluate: nonreactive; IAT negative

3. Drug independent – cannot be distinguished serologically from WAIHA – Serum/eluate reacts with all cells – Difficult to prove; drug history helps support

4. Non-immunologic protein adsorption - drug cause RBCs to be sticky – DAT: weak positive; eluate: nonreactive– Rarely associated with DIIHA

**TIP: refer to table in Technical Manual for drug names and known mechanism**

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Blood Groups

Paul Mansfield, MT(ASCP)SBBCM

American Red Cross

Immunohematology Reference Lab

Philadelphia, PA

General Blood Group info

Read and know:

• Technical Manual- Current Edition

• The Blood Group Antigens FactsBook

According to the International Society for Blood

Transfusion there are currently 43 recognized

blood group systems containing 345 red cell

antigens as of June 2021. The 43 systems are

genetically determined by 48 genes.

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General – Antigens

• Genetics

• Biochemistry

• Null phenotypes

• Effect of

chemical

treatments

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• Prevalence

• Racial variation

• Cord cell expression

• Soluble antigens

Reid, M.E., Lomas-Francis, C., & Olsson, M.L. (2012). The Blood Group Antigen Facts Book (3rd ed.). San Diego, CA: Elsevier, Academic Press

General – Antibodies

• Characteristics reactivity

• Techniques for detection/confirmation

• HTR

• HDFN

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ABO• Gene interaction - A, B, H, Se

• ABO gene produces glycosyltransferases– Adds sugar to paragloboside (precursor molecule)

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Gene Transferase Sugar added

H -2-L- fucosyltransferaseL-fucose

(to Type 2 chains)

Se -2-L- fucosyltransferaseL-fucose

(to Type 1 chains)

A-3-N-acetyl-

galactosaminyltransferaseN-acetyl-D-

galactosamine

B -3-D-galactosyltransferase D-galactose


ABO• Antigen structure

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ABO• Antigen expression

– Soluble antigen: saliva (secretors), body fluids• Built on Type 1 precursor chains

– RBC membrane antigen: platelets, lymphocytes, endothelial and epithelial cells

– Built on Type 2 precursor chains

• Cord cell expression weaker than adults• Fully developed by 2-4 years of age

• Reaction with enzymes/chemicals

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Harmening, D.M. (2012). Modern Blood Banking and Transfusion Practices (6th ed.). Philadelphia: F.A. Davis Company.

ABO• Subgroup characteristics

– Subgroups of A (A1, A2, A3, Am, Ax, Ael)• A1 and A2 account for 90% of group A individuals

– Subgroups of B (B3, Bm, Bx, Acquired B)

– Characteristic reactions with antiserum• A3 and B3 – mixed field

• Ax stronger with anti-A,B than anti-A

– Reaction of serum with reagent RBCs

– Saliva of secretors

• Bombay, parabombay– No H

– Forward and reverse looks like an O!!!

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ABO• Acquired B antigen

– Found in group A1 individuals• Transient discrepancy as individual type weakly with some anti-B

– Due to deacetylation of A antigen

– Associated with colorectal carcinoma and bowel obstructions

• Important lectins– Ulex europaeus

– Dolichos biflorous• Appropriately diluted, reacts with A1 cells

• ~80% of A and AB persons are A1+.

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ABO• Antibody Characteristics

– Naturally occurring usually IgM with some IgG

• Occasionally anti-A1 is seen in A2 and A2B individuals

– Antibodies detected in an infant are maternal

– Anti-A and anti-B production begins after the first few

months of life

– Production peaks at 5-10 years of age

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ABO Discrepancies • Causes of ABO discrepancies

– Technical errors• Specimen mix-up

• Inappropriate cell suspension

• Incorrect interpretation

– Specimen issues: RBCs• Weak Subgroup, transfusion, transplant, spontaneous

agglutination due to cold agglutinates, aby to dyes

– Specimen issues: serum• Weak subgroup, BMT, immunodeficient patients, detection of

other alloantibodies

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ABO• Resolution of ABO discrepancies

– Repeat test (same and new sample)• The best first step and include a clerical check of reagents, patients

and history

– Wash pt and reagent RBCs

– Incubate at RT and down to 4

– Treat pt cells with enzymes

– Ads/elution studies

– Saliva studies

45


Lewis

• Soluble antigens Lea and Leb

– Adsorbed onto the red cell

• Gene interaction - Le, Se creates these antigens

• -4-L-fucosyltransferase adds L-fucose to type 1 precursor chains

• Antigen structure includes Lea being the building block for Leb

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I System/Collection• Soluble antigen

• Adult and cord cell expression

– Neonates have the i pheno and with increasing age

develop the I pheno by age 2

• Disease associations

– Most often seen as auto specificities in Cold Agglutinin

Syndrome which can be seen in Mycoplasma pneumoniae

– Also can be seen in other hemolytic disorders

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P1PK and Globoside• P1 and Pk are in the P1PK and P is in the Globoside

• Antigen Structure similar to the ABO as adding sugars to precursors

• Soluble antigen

– Neutralized Pigeon egg white, hydatid cyst fluid (P1, Pk)

• Autoanti-P and PCH

– Donath-Landsteiner test, biphasic hemolysin

• Anti-PP1Pk and spontaneous abortion

• P antigen receptor for Parvovirus B19

48


MNS• A highly complex system with about 46 antigens

• Glycophorin A – M/N– 5 terminal amino acids for M and N specificity

• M: serine-serine-threonine-threonine-glycine

• N: leucine-serine-threonine-threonine-glutamic acid

• Glycophorin B – S/s– Amino acid residue at position 48 (prev 29)

• S – methionine

• s – threonine

49


MNS• Antigen

– Prevalence in White and Black populations• Prevalence are similar with the exception of about 2% of African Americans are S-

s- and lack U.

– Enzyme and chemical treatment

– Hybrid SGPs• Highly homologous and genetic material is exchanged from one gene to another.

Many Lows!

• Antibodies varies a great deal!– Immunoglobulin class

– Optimal technique

– Clinical significance

50


Rh• Over 50 antigens in this complex system

• Rh complex is the basis– Rh Associated Glycoprotein (RhAG) and the Rh proteins

– Essential to the RBC membrane and formed through:• CD47

• Integrin associated protein

• Glycophorin B

• Fy5

• LW glycoprotein

51


Rh• Rhnull types

– Extremely rare and often result because of changes in RhAG

– Lack LW and Fy5 antigens

– Anti-Rh29 (total Rh)

• Prevalence of 5 major antigens

• Most are very antigenic!

• Antibodies are often clinically significant and cause – HTR

– HDFN

52


Rh• D is the most important !

– responsible for the Rh Pos/ Neg in your blood type.

• Types of Weak D– Quantitative: inherited gene, encodes less than normal number of D

antigen sites

– Position effect: weakening of D antigen by a C gene in trans to D gene• Dce/Ce (Ro/r’)

– Partial: lack part of the D antigen complex

• Standards for D typing– Donor vs. patient

53


Rh• Antigens associated with D variants that cause other antigens

to be expressed– Goa associated with DIVa

– Dw associated with Dva

• Compound antigens/antibodies which are a result of antigens on a single polypeptide protein– ce(f), Ce(rhi), CE (Rh22), cE (Rh27)

• Anti-G Study adsorption/elution– Prenatal cases to determine the need for RhIg

54


Kell• 36 antigens

• Gene interaction – XK1 gene– Kell glycoproteins are linked to the XK protein which is an integral protein

that expresses the Kx blood group antigen

• Racial differences– Js(a+) and Js(b-) very common in AA population

• Chemical treatment

• KEL3 (Kpa) in cis position causes weakened Kell antigens.

• Ko is the null and patient make an anti-Ku

• Kmod expresses weak Kell antigens

55


McLeod phenotype

• Associated with McLeod Syndrome

– No Kx or Km antigens

– Depressed Kell antigens and will make anti-Km

– If patient has Chronic Granulomatous Disease (CGD) the

patient can make an anti-Kx and an anti-Km.

56


Duffy• 5 antigens

• Racial differences– Most notably Fy(a-b-) is common in Africans

– Fya is a high prevalent antigen in Asians.

• Chemical treatment

• Anti-Fy3 vs Anti-Fy5– Rh null cells (Fy3+, Fy5-)

• Association with Malarial resistance– The glycoprotein is a receptor for Plasmodium vivax – receptor

of a parasite which causes a type of malaria

57


Kidd• 3 antigens

• Inheritance for Jk(a-b-)– Typically, a result of a silencing allele which is seen in Polynesians

• Jk(a-b-) resistant to lysis in 2M urea– Kidd antigens are urea transporters

• Delayed HTR’s– Notorious for these as ab’s tend to drop to low or undetectable levels in

the plasma

• Antibodies– Often show dosage and need enhancements to detect

– May activate complement

58


Lutheran• 24 antigen and polymorphic

• Lu(a-b-) inheritance– Recessive: LuLu

• 2 rare silencing alleles

– Inhibitor: In(Lu)• Rare dominate gene In(Lu)

– X-borne

• Lu linkage to Se– First example of autosomal linkage in humans

59


LW• Association with D

– Initially thought to be anti-D as it reacts stronger with D+ cells

• Cord cell expression – Very strong on D+ and D- cord cells

• Distinction from anti-D– Antigens are sensitive to 0.2M DTT

• Absent on Rhnull cells

60


HTLA Chido/Rogers• Chemical treatment- destroyed by enzymes

• Soluble antigen

• The antibody can agglutinate C4 coated cells

Knops• Located on Complement Receptor 1

• Hegleson phenotype is the null

• Soluble antigen

• Chemical treatment- destroyed by DTT• CR1 is a malaria receptor, AA population has a noted difference in

prevalence of many Knops antigens.

61


Others• Diego- many antigens most common Dia (low) and Dib (high)

• Cartwright- Yta is a high and Wra is a low

• Scianna - very rarely seen

• Dombrock - Gya is the null

• Colton - Coa is the high and Cob is the low

• Indian- Inb high seen lacking in western Asia

• Xg- Partly on the X chromosome

62


63

Test Your Blood Bank Knowledge

Q and A Session

BB Fundamentals Session 2

BBF2.00: The following reactions indicate which type of

immune mediated hemolytic anemia?

A. Hemolytic transfusion reaction

B. Warm autoimmune hemolytic anemia

C. Cold agglutinin disease

D. Drug induced immune hemolytic anemia

64

Anti-IgG/C3d Anti-IgG Anti-C3b, C3d Saline ABID Panel Eluate

3+ 3+ 0✔ 0Reacts with

all cells tested

Nonreactive with

all cells tested

BBF2.01 Which methodology(s) are based on

agglutination?

A. Tube Indirect Antiglobulin Testing (IAT)

B. Gel Column Technology

C. Solid Phase

D. Both A and B

E. All of the above

BBF2.02 A child with a viral infection has recently

experienced hemolysis. The child has a negative DAT

and a negative antibody screen. What test would be

appropriate for the child?

A. Antigen typing for I/i antigens

B. Donath-Landsteiner Test

C. Eluate

D. Auto Adsorption

66

BBF2.03 A patient’s serum reacted with cells I and II of

a three cell screen and three group O, Rh-negative cord

cells. When the cells were DTT treated, the serum was

negative. The most likely specificity is:

A. Anti-Csa

B. Anti-Dib

C.Anti-JMH

D. Anti-LW

67

BBF2.04 A patient’s serum reacts microscopically to 1+ with

8 of 11 cells tested with no apparent specificity. The

antibody was non-reactive with the same panel of cells that

were ficin treated. This antibody was titered and tested with

one of the incompatible cells. The titer was 64 with a score

of 15. Which of the following specificities should be

considered?

A. Anti-Yka and anti-Rg

B. Anti-Ch and anti-JMH

C. Anti-Kna and anti-Csa

D. Anti-Ch, -Rg, and -Csa

68

69

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Transfusion Medicine Physiology and Pathophysiology

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