Blood Bank Exam #1

Natural/Innate immunity: What/function, mechanism, response

• what you are born with; recognize invaders & eliminate on their own

• primary lines of defense

• non-specific (no memory)

• immediately available

• mechanism doesn’t change on repeated exposure to any specific antigen

• immune system responds with: fever (cytokines), some WBCs, macrophages

Acquired immunity: Specific

• specialized & acquired by contact with specific foreign substance

• initial contact with foreign substance → synthesis of antibody proteins → reactivity to that particular foreign substance

Acquired immunity: Memory

• response improves with each successive encounter with same pathogen

• remembers infection agent & can prevent it from causing disease later

• immunity to withstand & resist subsequent exposure to same foreign substance is acquired

Cytokines: What/function & immunity

• soluble proteins secreted by particular cells types & mediate inflammation and immune response

• cytokines can build up in unit of red cells during storage

  • Some white cells in unit → can cause transfusion reaction by stimulating patient’s immune system

• Natural immunity: macrophages, neutrophils, etc.

• acquired immunity: T & B cells

Complement: What/function (3) & immunity

• proteins in circulation that promotes acute inflammatory response

  • can alter surface of cell to enhance phagocytosis or lysis

• helps with inflammation, phagocytosis, and create membrane attack complexes (MACs)

• can be activated by antibodies or can directly cause lysis of red cells

• natural immunity: directly attacks pathogens

• acquired immunity: antibody-mediated activation

Immunogenicity

• ability of an antigen to stimulate antibody production

• larger & more numerous antigen is more likely to stimulate an immune response → antibody production

Antibody/Immunoglobulin vs Antigens/Immunogen (& epitope)

• antibody: Y-shaped protein made in response to a foreign antigen

• antigen: foreign molecule capable of triggering immune response in body → production of specific antibody types

• epitope: portion of antigen molecule that is directly involved in interaction with antibody

Heavy vs Light chains of Antibodies

• heavy: 1 of polypeptide units that makes up immunoglobulin molecule (2 heavy chains per IgG antibody)

• light: small chain in an immunoglobulin molecule that is bound to the larger chain by disulfide bonds (2 light chains per IgG antibody)

Fab vs Fc regions of Antibodies

• Fab (fragment antigen-binding region): portion that binds to the antigen’s epitope

  • the area that has the light chains & binds fragments

• Fc (fragment crystallizable region): part that binds to complement

Constant vs Variable region of Antibodies

• Variable: unique to each antibody molecule & allows molecule to bind specifically to a particular region

• constant: same in each immunoglobulin

Paratope (3)

• antigen binding site; hypervariable region of antibody

• small amount of amino acids at end of heavy & light chains can create diversity of antigen binding sites

• antigen binding sites are very specific

Hinge region & Hemagglutination

• flexible portion of heavy chain located between 1st & 2nd constant regions

• allows molecule to bend to let 2 antigen-binding sites operate independently

• held together by 2 disulfide bonds

• Hemagglutination: flexibility of antibodies being able to hold on to epitopes of 2 different antigens

Antigens for destruction

• are flags of destruction

• once antibodies bind to antigen, antigen is now marked for immune system to destroy

Antibody types: IgG & IgM (size, temp, subclasses)

• IgM is too large to pass placenta, but IgG can easily cross

• IgG is clinically significant due to optimum temp is 37C (body temp)

• IgM is less clinically significant except ABO antibodies

  • optimum temp: 4-25C

• IgG has 4 subclasses (1-4)

  • IgG subclass 3 is best at binding complement

IgG vs IgM: Total serum concentration, MW, serum half-life

• IgG: 70-80%; 50000; 23 days

• IgM: 5-10%; 900000; 5 days

IgG vs IgM: Complement, clearance of red cells, optimum medium

• IgG: activates complement not as efficient; extravascular; high protein/AHG

• IgM: activates complement very efficiently; intravascular; saline/immediate spin

IgG levels according to age

• maternal antibodies start to increase between 3-6 weeks of age

• baby’s own antibodies show up at 2 months

• at 10-18 months: levels are 60% of adult levels

Primary vs Secondary Immune response (antibodies, peak, lag, antibody affinity)

• 1st response to an exposure to an antigen → mostly IgM

  • there’s a lag period from exposure & level of antibodies we can detect (5-10 days after immunization)

  • antibody affinity: lower average, more variable

• next time: immune system sees antigen again → production of IgG

  • lag time is much shorter & # of antibodies is much larger (1-3 days after immunization)

  • antibody affinity: higher average affinity

RBC antigens (4)

• have various functions and some are transport structures

• >1 million antigen sites on surface

• 45 recognized blood group systems; 362 red cell antigens

• slight difference in red cell antigen → immune system recognize the donor cells as foreign

Red cell Antibodies (4)

• red cell antigens that are different from recipient → immune response → antibody production to donor antigens

• immune system will continue to make antibodies

• 2nd time: recipient’s immune system sees antigens → lag time for making antibodies shorter

• antibody is clinically significant if destroys red blood cells in vivo (in body)

Mismatched transfusion

• will cause transfusion reaction (can be fatal)

• free-floating Hb can lead to severe organ damage, inflammation, & vascular complications

Gene

unit of inheritance encoding in a chromosome

Chromosome

double stranded DNA structures that carry genetic information

Alleles

alternate forms of a gene at a given locus

Heterozygous vs homozygous

• 2 different copies of the same gene (ex: Bb)

• 2 identical copies of the same gene (ex: BB or Bb)

Dominant vs recessive

• gene production expressed over another one

• trait only expressed when inherited from both parents

Phenotype vs genotype

• observable trait; trait that is expressed (think: phenotype is a photo of what is present)

• genetic-up, the genes that are present; genes may be present but might not be expressed (think: genotype = genes)

Polymorphic vs Amorph

• >2 alleles at the locus

• gene doesn’t express detectable antigen

Incomplete dominance

neither allele for a trait is fully dominant; heterozygous offspring with intermediate or blended phenotype

Codominance

equal expression of 2 different inherited alleles

Type O (3)

• there is no “O gene”

• think of O as a zero; absence of the A & B genes

• O is an amorph: a gene not expressing a detectable trait

Codominance in blood types (3)

• A & B are codominant

• O is recessive because A or B are dominantly expressed or homozygous O genes make up type O

• type O is the absence of A or B, there is no O gene

BB Pedigree

family tree diagram showing the inheritance of traits or genetic conditions across generations

5 patterns of inheritance

• autosomal dominant

• autosomal recessive

• autosomal codominant

• X-linked dominant

• X-linked recessive

Autosomal dominant inheritance

• trait or disorder is passed down when a single altered gene from one parent is enough to cause the condition

• affects males & females equally; each child having 50% chance of inheriting it from an affected parent

• ex: the Rh factor (positive is dominant)

Autosomal codominance inheritance

• pattern where both alleles on a pair of autosomal chromosomes are fully & equally expressed in the offspring

• creates a distinct trait from either parent

• ex: almost all blood groups are codominant like AB blood type

Autosomal recessive inheritance

• shows a trait that skips generations

• affects males & females equally & appears in children carriers (heterozygous)

• ex: group O phenotype

Sex linked inheritance (3)

• involves genes located on sex chromosomes, causing traits to appear at different rates between males & females

• males have only one X chromosome → more susceptible to recessive X-linked disorders inherited from their mothers

• females require 2 copies to express the trait

X-linked dominant inheritance (4)

• a gene on the X chromosome causes a disorder, requiring only one copy of the gene for offspring to be affected

• females more likely to have it

• affected fathers passing the condition to all daughters but no sons

• affect mothers passing it to 50% of children → more females affected overall

X-linked recessive inheritance (4)

• affecting males much more often because they have only one X chromosome

• females have a healthy second X to compensate, making them carriers

• carrier mother passes trait to half her sons (who get disease) & makes half her daughters carriers

• affected father passes the trait to all daughters (making them carriers) but none of his sons

Cis vs Trans position (gene locus position)

• 2 or more genes on the same chromosome are inherited together

• 2 or more genes on opposite chromosomes

• During crossover in meiosis: a chromosome may receive another A or B gene → one chromosome may have both A & B

8 major blood types (most to least common)

O+ → A+ → O-/B+ → A- → AB+ → B- → AB-

What makes a blood type positive or negative?

• D antigen

• if have D antigen → blood is “+”

ABO Antibodies

• Anti-A & B are only natural occurring

• A makes Anti-B

• B makes Anti-A

• AB makes no antibodies

• O makes Anti-A & B

Landsteiner’s Law

• if antigen is present in RBCs, corresponding antibody will be absent from serum

• if antigen is absent from RBCs, antibody will be present in serum

• Type A (RBC) will have anti-B in plasma

• Type B will have anti-A in plasma

• Type O will have anti-A & B in plasma

H red cell antigen

• H must be expressed on RBC before any A or B antigens

• O group most amount of H antigen; O has only H

• H gene & L-fucose must be present for other sugars to attach and form other blood types

Type A coding

• genes that code for A type codes for an enzyme (A-transferase)

• enzyme acts on H antigen & converts it to A antigen

• A-transferase adds N-Acetylgalactosamine to H antigen → A antigen

Type B coding

• The gene that codes for the A blood type actually codes for an enzyme

• A-Transferase can act on the H antigen and convert it to a B antigen

• A-transferase adds galactose to H antigen → B antigen

Type AB coding

• The genes that code for the A and B blood types codes for both enzymes

• A and B enzymes compete for H antigens

• A enzyme is more efficient than the B enzyme at converting H into A

Amount of H per blood type

• O > A2 > B > A2B > A1 > A1B

• O: only has H; no efficiency

• A2 = least efficient enzyme

• B = avg enzyme

• A2B = 2 enzymes; least efficient & avg one

• A1 = most efficient enzyme

• A1B = most efficient enzyme & avg one

What if there is no H?

• bombay: 0.001% of population

• testing looks like O

• genotype hh = bombay

• genotype Hh/H = not affected

Antibodies expected to see in Bombay genotype

• Anti-A, B, H, & A,B

• O vs Bombay = patient plasma with O cell reagent

  • O = (-)

  • Bombay (hh) = 4+

Plasma transfusions

• Rh factor doesn’t matter

• if donor has Anti-D → cannot donate

• remember: Anti-D is not naturally occuring

• A = anti-B

• B = anti-A

• O = anti-A & B

Antibody and antigen testing

• use antibody as reagent to test for unknown antigens

• use red cells with known antigens to test for unknown antibodies

Antibody reagent tests for antigens

• Anti-A = blue; A antibodies used to find A antigens

• Anti-B = yellow; B antibodies used to find B antigens

• Anti-D = colorless

Red cell antigen reagent tests for antibodies

A1 cells (A antigens used to detect anti-A) & B cells (B antigens used to detect Anti-B)

Reading & Grading reactions

• tube method: 0 = (-); 1+, 2+, 3+ , 4+ = (+)

• 0 = smooth suspension, no agglutination or clumps

• 1+ = small agglutinates, clumps; cloudy/turbid background

• 2+ = medium sized agglutinates; clear to slightly cloudy background

• 3+ = several large agglutinates; clear background

• 4+ = red cell button; clear background

ABORh testing: Front type

• using known antibodies to test for unknown antigens

• 4% cell suspension → anti-A in one tube, anti-B in second → centrifuge

ABORh testing: Back type

• used to confirm Front type; use known antigens to find unknown antibodies

• A1 red cells in patient plasma → B reagent red cells in patient plasma → centrifuge

Rh control

• ABORh testing must have Rh control when front type is AB+

• make sure there’s no interference

• AB+ front type is antibody reagents all reacting (+)

• could be sign that patient has cold agglutinin

• control must be (-) to validate test

  • 1 drop of patient 4% suspension & 1 drop of patient plasma

  • Anti-A = 4+, Anti-B = 4+, Anti-D = 4+

Agglutination: Define & Factors

• antibodies can connect to more than 1 cell, forming a clump of cells that can be seen macroscopically

• sensitization: 1st stage antibody binds to antigen, no agglutination visible; fast & reversible

• lattice formation: after sensitization, random collisions between antibody-coated red cells develop cross-linkages

Agglutination: Sensitization

• factors influencing:

  • serum-to-cell ratio (prozone, postzone, zone of equivalence)

  • temp: IgG (37C) & IgM (RT)

  • incubation time

  • pH (6.5-7)

  • ionic strength (zeta potential)

Steric hinderance affecting binding

when different antigens are located close to each other, antibody binding is blocked

Agglutination: Lattice formation (factors)

• factors influencing:

  • distance between red cells (zeta potential)

  • serum-to-cell ratio

  • centrifugation

    • over & high speeds → false (+)

    • under & low speeds → false (-)

What affects the different stages in binding & agglutination of antigen & antibody in lattice formation? (2)

• size of antibody: IgM (large) & IgG (small)

  • IgG takes 2 to activate vs IgM takes 1

• binding sites of antibody: IgM (10) & IgG (2)

  • increasing binding site = quicker agglutination

Agglutination: Lattice formation (waters of hydration & zeta potential)

• acts as insulating bubble around RBC; makes it more difficult for binding & lattice formation to occur

• cell gets closer together if low potential

Van der Waales forces vs hydrogen bonds

• disruption of electrons causes formation of 2 dipoles which exhibit attraction to each other

• weak, reversable hydrogen bridges between hydrophilic groups which are stronger at lower temps

Zeta potential

degree of (-) charge on the surface of a red cell; potential difference between (-) charges of red cells & cations in fluid portion of blood

22% albumin

• reduces Zeta potential

• increased incubation time is needed

LISS

• contains glycine in an albumin solution

• reduces ionic strength in testing sample

• reduces zeta potential

• increase antibody uptake

• decreases incubation time

Polyethylene Glycol (PEG)

• removes H2O molecules from testing sample

• it competes with H2O for space around red cell → cells can come closer together → concentrating antibodies around red cells

• most sensitive than albumin or LISS

• reading only done at AHG

• can cause nonspecific aggregation → false (+)

Serum-to-Cell ratio (zones)

• max amounts of agglutination are observed when concentrations of antigens & antibodies

• zone of equivalence: antibody & antigen are equal proportions → lattice

• prozone: excess antibody; all binding sites are taken can’t form lattice

• postzone: excess antigen; too many antigens can’t see agglutination

Anti-human globin (AHG) reaction: Temp, vivo, significance, cells, phase

• optimum reactive temp: 37C

• destructive in vivo

• clinically significant; usually IgG

• if (-): add 1 drop of check cells

• only antibody reaction phase reading required by AABB

“Cold reactive”

• temp: <37C

• class = IgM

• ABO antibodies re IgM also but can be hemolytic in vivo/in vitro → serious transfusion reactions can occur

“Warm reactive”

• temp: 37C

• class: IgG

• can be involved in transfusion reactions & HDN

Direct antiglobulin test (DAT)

• detects coating on red cells which occurred in body

• (+) due to coating of either antibody and/or complement or drugs

• performed when auto control is (+)

• ex: HDN, autoimmune hemolytic anemia, investigation of transfusion reactions, etc.

Indirect antiglobulin test (IAT)

• detects antigen-antibody binding in lab setting (in vitro)

• specific antigen binding with specific antibody

• performed: procedure testing that involves incubation & adding AHG reagent

• ex: antibody screen, antibody ID, weak D

AHG: What & Importance of use

• rabbit antibody directed to human antibodies

  • rabbit antibody will bind with any human antibody

• AHG step: enhance to see agglutination (especially with IgG)

  • clinically significant antibodies (ex: IgG) are smaller & can’t overcome (-) charge that keeps RBC apart in saline

  • they bind to RBCs but no visible agglutination

• bridge gap between red cells to create visual agglutination

DAT reagents: Types & Process

• monospecific: contains anti-IgG or anti-C3d

• polyspecific: contains anti-IgG & anti-C3d

• patient’s red cells sensitized in body (no incubation) → AHG added then centrifuged → observe for agglutination

• (+) result: indicates immune mediated RBC destruction due to complement or immunoglobin (IgG)

IAT: Example tests & Process

• compatibility testing (detect abs to donor RBC or screen cells)

  • titration of antibodies & ID of antibodies (antibody panels)

  • RBC phenotype determination using known serum

• patient plasma that contains abs has screen cell reagent (known antigen) added → incubation → sensitized RBCs & washed → AHG added → agglutination observed

Sources of error in AHG testing

• false (-): washing, delays, contaminated reagent, omission of AHG, centrifugation, antigen excess, etc.

• false (+): improperly stored saline, dirty glassware, over-centrifugation, refrigerated & clotted sample, clot-activating or serum-separating silicone gel

Tube reagent QC

• test reagents with other reagents; done daily

• Anti-A with A1 cells → A1 = (+); B = (-)

• Anti-B with B cells → B = (+); A1 = (-)

• Anti-D with A1 cells = (-)

  • Anti-D with check cells = (+)

Unacceptable QC

• patient transfused with O units until QC issue resolved

• reagent red cells looked hemolyzed & antiserum looks cloudy

Rh blood group

• only 2 genes responsible for antigens

• major: D, C, E, c, e

• largest blood group; over 50 antigensRh

Rh group: Antibodies

• highly immunogenic properites

• Rh antibodies cause hemolytic transfusion rxns & HDFN (hemolytic disease of the fetus/newborn)

• D+ (>/=200 mL) red cells are transfused to D- recipients, ~85% responded & produced anti-D (not naturally occurring)

Hemolytic disease of newborn/fetus (HDFN)

• mom (-) for D antigen

• baby is D+

• Mom is exposed to baby’s red cells at birth or in a trauma situation

• Mom has another D+ baby: her Anti-D can cross the placenta & have an affect on the fetus.

Rh group Antigens

• D (85%) > c (80%) > E (30%) > C (70%) > e (98%) (highest to lowest immunogenicity)

• Fully expressed at birth (strong)

• Antigen detection at 8 weeks gestation

• On red cells only

Rh genetics

• 2 genes on chromosome 1 code for D & CcEe antigens

• RHD = codes for D antigen

• RHCE = codes for C,c,E,e antigens

• genes are codominant

RHD

people who have a deletion of this gene or have a defective EHD gene do not express the D antigen → D neg (Rh neg)

RHCE

• Antigens C,c,E,e are all on the same protein

• The difference between the antigens:

  • Big C and little c is one amino acid

  • Big E and little e is one amino acid

RHAG

• 3rd gene on chromosome 6: codes for the RhAG antigen similar to RHD & RHCE in structure

• RhAG is important in the expression of the Rh antigens; needed to express the DCcEe antigens

• Without RhAG Rh antigens are not expressed → “regulator type” of the Rh_null phenotype

Fisher-Race nomenclature

• represent both genes inherited from both parents, separated by “/” (forward slash)

• Antigens are represented by their common letter names; always in D, Cc, Ee order

• d (little d) does not represent an antigen it is a place marker for the absence of the D antigen

  • no little d antigen; no Anti-d

Fisher-Race: + vs -

• Rh+ (DD or Dd)

  • D antigen is expressed on the red cell

    • At least 1 D antigen is inherited from the parents

    • We don’t always know if Rh pos is homozygous or heterozygous for the D antigen.

• Rh- (d/d)

  • No D antigens on the red cell

    • No inherited D antigens from parents

Wiener nomenclatures

• R = D present

• r = no D antigen

• order: Rz, R1, R2, Ro, r^y, r’, r”, r

Wiener shorthand

• R₀ = Dce

• R1 = DCe

• R2 = DcE

• R_z = DCE

• r = dce

• r’ = dCe

• r’’ = dcE

• r^y =dCE

G antigen

• ~85% population

• needs amino acid serine to be expressed; D & C antigens have serine

• G antigen is present on any red cell that carries either the D and/or C antigen

• antigen not on the same protein, but RHD & RHCE are very close to each other on the red cell because they are on the RHAG protein

G antibody

looks like the patient has Anti-C and Anti-D

Compound Rh antigen

• epitopes created only when 2 specific antigens are inherited together on same chromosome (cis position)

• C/c and E/e are on the same protein

  • antibodies may form to react with the antigens in the cis position

• C/c & E/e need to be on the same chromosome to make a compound antibody

• ex: Rh6 (ce) = f antigen; Rh7 = Ce, Rh22 = CE, Rh27 = cE

Rh_null Phenotype

• has 0 Rh antigens → D-C-c-E-e-

• no D, C/c, E/e → lacks all 61 possible antigens with Rh system

• due to mutation or deletion of genes

• 2 different genetic backgrounds

  • regular

  • amorph

Rh_null Regulator mutation in RHAG (genotype)

• has a mutation in the RHAG gene → cannot express the Rh antigens

• each parent has complete deletion of 1 haplotype & both pass to offspring