WEEK 1 BLOOD BANK

how many alleles does an individual inherit?

• 2 total alleles, one at the same locus on each chromosome

what chromosome is the ABO blood group expressed on. What chromosome is the H gene located on?

• chromosome 19 - ABO

• chromosome 9 - H

What is the precursor substance for ABO antigens

• a chain of carbohydrates ending with a galactose sugar

  • called terminal oligosaccharide or the terminus

discuss the construction of the H antigen

• first antigen that must be constructed

• must inherit at least 1 functional H gene on chromosome 9

• encodes for the enzyme alpha-2-L-fucosyl transferase that attaches to the sugar L-fucose to the terminus

discuss the construction of the

• A antigen 

• B antigen

• O antigen

Must inherit at least 1 functional A or B on chromosome 19

Must inherit two copies of the O gene

- The H antigen must be attached to the precursor terminus

• A = N-acetylgalactosamine sugar

• B = D-galactose sugar

• O = amorph ( no sugar )

State the landsteiner’s rule

• presence of antigen = absence of antibody

• absent antigen = presence of antibody 

What immunoglobulin are the ABO antibodies?

• they are pentamers (IgM)

list the ABO group incidence % for European descent

• O - 45%

• A - 40%

• B - 11%

• AB - 4%

list the ABO group incidence % for African decent

• O - 49%

• A - 27%

• B - 20%

• AB - 4%

list the ABO group incidence % for Asian decent

• O - 40%

• A - 28%

• B - 27%

• AB - 5%

list the two outcomes from an agn-aby rxn

• visible agglutination

• hemolysis

discuss zeta potential and IgM/IgG 

• IgM

  • larger = overcomes zeta potential

  • direct agglutination

  • quicker testing

• IgG

  • smaller = cant overcome zeta potential

  • needs reagents (AHG) to cause agglutination

  • delayed testing

What is the purpose of the grading scale?

• consistency

• can be ± 1 from another tech

• can detect dosage

• can detect the presence of more than one aby in a patients serum

define rouleaux and how to resolve it

• pseudoagglutination caused by excess serum proteins

• resolve with saline replacement

what happens with a heavy or light suspension

• may stray from the zone of equivalence (80 serum: 1 packed RBC) and cause prozone or postzone

• prozone

  • excess aby

• Postzone

  • excess agn

What is antisera

• reagent that contains a known aby concentration to a specific agn

• can be monoclonal or polyclonal 

discuss polyclonal antisera

• made using different lines of B cells from multiple human donors

• creates a heterogenous mix of IgG abys able to recognize different epitopes of a single agn

• Pros

  • high affinity for target agn

• Cons

  • high chance of cross reactivity

Discuss monoclonal antisera

• made from clones of a single B cell clone to create IgM abys that recognize one epitope of a single agn

• pros

  • long term abys w/ no cross reactivity

  • IgM monoclonal reagents and direct agglutination = short testing time

• Cons

  • oversensitivity might cause false positives

discuss the antisera used for ABO blood group

• Anti-A and Anti-B

  • monoclonal IgM antisera

  • 3+-4+ = positive RXN

Discuss antisera used for Rh testing

• anti-D

• monoclonal IgM/monoclonal IgM blend antiserum

• 3+-4+ = positive RXN

List the percentages for Rh agns in the US

• European

  • 85%

• African

  • 92%

• Asian

  • 99%

discuss weak D

• weak expression of D antigen

• patient suspension is combined with Anti-D and incubated at 37 C to give IgG and anti-D to interact. suspension is washed, AHG is added, spun, and read. add check cells if negative. 

• invalidated by a positive Rh control or a positive DAT 

  • impossible to distinguish if its a weak D rxn or pre-sensitization to another aby

• Weak D positive women of child bearing age

  • further testing performed to see if they can make abys to D agn

what is the purpose of aby screen?

• Used to identify IgG aby to other blood groups

  • Rh

  • kell

  • Duffy

  • Kidd

  • MNS

  • P

  • Lewis 

• reagents must be group O to avoid reacting with anti-A/anti-B

• associated with the antigram

What is the next step after determining someone is AB pos from a front and back type?

• must used control gel card to distinguish between a true AB pos and a pan-reactive patient

  • control = neg (pos = discrepancy)

what is required for all inpatient and ED blood draws? 

• typenex system 

  • third unique identifier for confirmation

  • not used for ABO type and screens w/ no transfusion

discuss antiglobulin testing (direct (DAT) and indirect (IAT))

• used to identify antiglobulins or complement (globulins) that are absorbed on to patient RBCs

• Requires AHG (polyclonal or monoclonal)

• requires washing to remove excess, unboud globulins = false neg

discuss polyspecific AHG reagents

• contains IgG and anti-complement (anti-cd3)

• cant distinguish between two positive results

discuss monoclonal AHG reagents

• a specific aby against a single epitope on IgG

• highly specific

What class are most clinical significant ABYs (not ABO)

• they are IgG

discuss DAT

• test detects RBCs coated w/ abys or complement in vivo

  • autoimmune hemolytic anemia

  • drug induced hemolysis

  • hemolytic disease of the newborn

  • transfusion reactions

• patient cells are washed and tested directly with AHG reagent

Discuss IAT

• detects reactivity between reagent and abys in patient plasma in vitro

  • detecting unexpected abys in patient plasma (like ABY screen)

  • determines patient plasma and donor blood compatibility (crossmatch)

  • detecting agns on red cells using antisera detected against a particular agn (red cell phenotyping)

• reagent and plasma is combined, incubated, and washed prior to AHG

what blood groups do enzymes (enhancement media like PeG) enhance and destroy

• enhances

  • Rh

  • Kidd

  • Lewis

• destroy

  • Duffy

  • MNS

what is the purpose of check cells after the addition of AHG

• used on negative reactions to determine functional AHG reaction

• used to combat false negs

list false negative and false positive results in an antiglobulin test

• false negative

  • inadequate washing

  • delayed AHG addition

  • contamination of AHG reagent

  • AHG not added

  • under centrifugation

  • over centrifugation

  • heavy cell suspension

  • inadequate incubation

• false positive

  • cold autoantibody (already agglutinated)

  • dirty glassware

  • over centrifugation

what is an alloantibody

• development of an antibody to a red blood cell antigen after exposure to that antigen

  • pregnancy

  • transfusion

  • injection for research

  • environmental exposure (IgM)

  • dialysis (anti-N)

• Detected by an antibody screen

list clinically significant antibodies

• D

• C

• c

• E

• e

• f

• K

• k

• FyA

• FyB

• JkA

• JkB

• M

• N

• S

• s

• LeA

• LeB

• P1

things to watch for in aby identification to help determine aby specificity

• Temp

• Media (gel or tube)

• Reaction strength? (dosage)

• is there hemolysis

• which aby panel cells are reacting

• was there recent transfusion

discuss a cord blood sample

• whole blood obtained from the umbilical vein or artery after delivery

• requires 4x manual washing to remove jelly

• tested when there is a chance for hemolytic disease of the new born

• tested for ABORH forward type and polyspecific DAT (no type repeat)

  • typing determines incompatibility between mom and baby

  • A group O mom can create IgG anti-A,B antibodies

  • positive DAT means mom has a clinically significant IgG aby and the babys RBCs possess the agn

    • EX: mom = Rh neg (anti-Rh) and baby is Rh pos

what happens if a cord blood sample is AB pos?

• a monoclonal control will need to be run to rule out spontaneous agglutination or pan reactivity with antisera

list the most common cause of type discrepancies

• tech error

  • under centrifugation

  • not adding reagent/serum

  • wrong reagent/serum ratio

  • incorrect temp (cold reagents) 

list cell typing discrepancies and how to fix them

• Mixed field

  • recent transfusions

    • check history

  • BM transplant

    • check history

  • chimera

  • weak subgroup like A3

    • Type patient RBCs with anti-A1 lectin

• Polyagglutinable cells

  • abnormalities with RBC membrane with exposure to cryptic autoantigens (agns sequestered during initial immune response by exposed during disease and are seen as foreign). results in agglutination to A and B wells 

• Acquired B antigen

  • seen in group A patients with bowel infections. bacterial enzymes cause A-antigen to look like B antigen

    • Acidify to detect true B antigens (acquired B wont react)

    • run autocontrol 

• cells coated with antibody (positive DAT)

  • a strong DAT can cause front type discrepancies. zeta potential is lowered if cells have enough aby, causing direct agglutination that reacts with any front typing sera regardless of blood type

• ABYs to dyes

• Weak expression of subgroups (A or B antigen)

  • if reaction is weak (< 3+) 

    • RBCs are coated with both anti-A and anti-B and eluted off to see which sticks. Run with an autocontrol at 4 C for cold alloantibody activation)

Discuss plasma type discrepancies

• if weak or missing

  • perform in tube

  • incubate at RT for 5-15 min

  • use 4 drops plasma 

  • incubate at 4 C for 5-15 w autocontrol

• Immunodeficient, elderly, neonate

  • backtype may not appear

    • incubate at RT or 4 C with autocontrol with 4 drops plasma 

• If extra reactivity

  • follow below

• rouleaux

  • pseudoagglutination

    • saline replacement 

• unexpected abys

  • patient may have aby that react at 4 C but may react at RT. alloantibodies such as anti-M or anti-P1, cold autoantibodies such as anti-I and anti-IH

    • warm reagents and sample

    • perform aby screen

• A2 subgroup with anti-A1 aby

  • appears as normal A pos but backtype reacts with both. 

    • test patient RBCs with anti-A1 and plasma with A2 and O cells. (pos with A1 cells negative with A2 cells and o cells = anti-A1 aby)

    • aby screen

if cord blood is Rh positive what do you do?

1) interpret ABORH type 

2) complete

what do you do if Cord blood is Rh negative

1) check mothers Rh status to determine if weak D is required

what do you do if cord blood is Rh negative and mom is RH positive?

1) if mom is Rh positive, interpret ABORH and you are done

what do you do if cord blood is Rh negative and mom is RH negative?

1) perform weak D

what do you do if cord blood is Rh negative and mom is RH negative and the weak D is negative?

1) if weak D is negative, you just ABORh type and is done

what do you do if cord blood is Rh negative and mom is RH negative and the weak D is positive?

1) perform DAT on cord blood (if not already done)

what do you do if cord blood is Rh negative and mom is RH negative, the weak D is positive, and DAT is negative?

1) if DAT is negative then you are done after interpreting ABORH

what do you do if cord blood is Rh negative and mom is RH negative, the weak D is positive, and DAT is positive?

1) weak D test is INVALID. results cant be interpretated without consultation. test is complete

what do you do if the cord blood is DAT positive and there is incompatibility with mom and baby

1) perform Cord serum screen

what do you do if the cord blood is DAT positive and there is incompatibility with mom and baby, and only the A1 or B cells react (not screening)

1) interpret “passive ABO aby from mother”

2) complete

what do you do if the cord blood is DAT positive and there is incompatibility with mom and baby, and the A1 or B cells and/or screening cells react?

1) interpret “passive IgG aby aby from mother”

2) perform elution

• if positive perform aby panel