Review Objectives: Part 4

What are the main antigens in the Kell blood group system?

• K, k, Kpa, Kpb, Jsa, Jsb

• K = 9% frequency

Describe the Kell Blood group system.

• resistant to enzyme treatment

• sensitive to DTT treatment

What antibodies are associated with the Kell Blood group system?

• IgG

• severe transfusion reactions

• severe HDFN

• no dosage

What null/ alternate phenotypes are associated with the Kell blood group system?

• rare Kell-null from silent alleles

  • K0 → makes anti-Ku

• Mcleod phenotype - weakened antigen expression

  • seen in chronic granulomatous disease

  • males

  • missing XK protein

What antigens are in the Duffy blood group system?

• Fya

• Fyb

• Fy3

• Fy5

• Fy6

Where are duffy blood group system found?

• antigens on multi-pass glycoprotein

  • expressed on tissues as well as RBC

What duffy blood group system members are sensitive to enzyme treatment?

• Fya

• Fyb

Describe the Fy(a-b-) phenotype

• GATA box mutation: stops Fyb from expressing on RBCs

  • present elsewhere in body

• more resistant to malaria infection

  • merozoite can attach but not invade RBC

• prevalence in black individuals is 68%

  • NOT seen in other populations

What antibodies are associated with theDuffy Blood group system?

• IgG

• clinically significant

• demonstrate dosage

• anti-Fya more common

What are members of the Kidd Blood Group System?

• 3 antigens: Jka, Jkb, Jk3

• resistant to enzyme treatment

Describe the null phenotype for Kidd Blood group.

• VERY rare

  • found in polynesians/ pacific islanders

  • HK(a-b-) = Jk3-

    • anti-Jk3 reacts with all cells that are positive for Jka or Jkb

• individuals can not concentrate urine

What antibodies are associated with the Kidd blood group system?

• IgG

• reactivity enhanced with enzyme treated cells

• ALWAYS clinically significant

  • can bind complement

• exhibit dosage

• amount varies

What are the members of the MNS blood group system?

• M,N,S,s,U

  • antigens on single-pass sialoglycoprotein

What does Glycophorin A do?

• carries M or N on top of protein

What does Glycophorin B do?

• carries s or s partway down the protein

• carries U close to cell membrane

What are members of the Lutheran glood group system?

• Lua and Lub

Describe the anti-Lua antibody.

• IgM

• most likely found by crossmatching

• usually not clinically significant

• most are naturally occuring

Describe the anti-Lub antibody.

• most are immune stimulated, IgG

• can cause transfusion reactions and HDFN

What is IN(Lu)

• dominant inheritance pattern - supressor

  • Lumod

What is LuLu

• recessive null allele

• Lu null

What is an X-linked inhibitor in the lutheran blood group system?

• trace antigens present

• found only in males in one australian family

What antigens are in the P1PK blood group system?

• P1, Pk, NOR

  • P1 - strength of expression changes person-person

  • Pk - high frequency

  • NOR - low frequency

What is the p phenotype?

• null

• can make anti-PP1Pk

  • associated with spontaneous abortions

Describe Anti-P1

• IgM - react best at RT or 4 C

• reactivity enhanced with enzyme treated cells

• usually NOT clinically significant

• can be neutralized by P1 substance or pigenou egg whites

• may not react with all P1+ cells

I blood group attegents are present on:

• RBC

• PLT

• leukocytes

• in secretion

Describe the i-I pathway for the I blood group

• birth: fetal cells have mostly i antigen

• converts to I during first 18 months of life

• adult RBC have lots of I

  • rare: i remains after birth

What disease is associated with anti-I?

• auto anti-I associated with Mycoplasma pneumoniae infection

What disease is associated with anti-i?

• infectious mononucleosis

What antibodies react primarily at 4C/RT

• Lewis

• M

• N

• O

• P1

• I

What antibodies react at AHG?

• Rh

  • C,c,E,e

• Kell: K

• Duffy: Fya / Fyb

• Kidd: Jka/Jkb

• MNS: S/s

• Lutheran: Lub

What antibodies show dosage?

Rh + Duffy + Kidd+ MNS + Lutheran

• Rh: C/c/E/e

• Duffy: Fya/Fyb

• Kidd: Jka/Jkb

• MNS: M/N/S/s

• Lutheran: Lue/Lub

What antibodies are enhanced by enzymes?

• Rh

• Kidd

• P1

What antibodies are destroyed by enzymes?

• M

• N

• Fya

• Fyb

What antibodies are resistant to enzymes?

• Kell

• Lewis

• U

• Kidd antigens

What antibodies are clinically significant IgG antibodies?

• Rh

• Kell

• Duffy

• Kidd

• S/s

• Lub

What antibodies are usually NOT HDFN?

• lewis

• M

• N

• P1

• I

What causes delayed hemolytic transfusion reactions?

• KIDD

  • anti-Jka and anti-Jkb

  • the antibody may become difficult to detect or even disappear after initial immune response

  • after reexposure

    • anamnestic response → Ab rises → transfused antigen positive RBCs are destroyed

Which antibody does NOT SHOW DOSAGE?

• KELL

  • IgG + AHG + clinically significant + no dosage

What antibody is resistant to malaria invasion?

• Duffy: Fy(a-b-)

What antibody is associated with Mycoplasma pneumoniae?

• Anti-I

What antibody is associated with Infectious Mononucleosis?

• Anti-i

If a women is pregnant, what can happen to her lewis antigens?

• she can temporarily become Le(a-b-) and develop lewis antibodies

• lewis antibodies do NOT cause HDFN

What is Antibody detection (screening)?

• test designed to detect unexpected red cell antibody in serum / plasma

  • allo / autoantibodies

What can stimulate antibody production?

• nature

• response to foreign antigen exposure

  • transfusion or transplantation

  • pregnancy

Why do we perform antibody screening

• make sure donor doesnt have alloantibodies

• pretransfusion testing

• prenatal/postpartum test for HDFN

• newborn

• Autoimmune process evaluation

What are screening cells?

• provide representation of common clinically significant red cell antigens in the homozygous state

  • want at least one homozygous expression of each antigen between the screening cells

• all are group O

What are characteristics of reagent antibody screening?

• single donor source for each vial

  • donor testing ONLY

• phenotypic expression of common red cell antigens for each vial is established / provided in form of an antigram

  • all cells have been phenotype

• screening cells are used

Describe Enhanced Media: Albumin

• reduces zeta potential

Describe Enhanced Media: LISS

• low ionic strength solution

• reduces zeta potential

• increases rate of antibody uptake

Describe Enhanced Media: PEG

• excludes water

• allows Ab and Ag to get closer together

• NOT read after the 37 C incubation, before washing

What are the steps in the tube method of the antibody detection procedure?

• Immediate spin

• 37 C

• AHG

What occurs in the immediate spin phase of an antibody detection test?

• add 2 drops of plasma + 1 drop reagent RBC

• spin / read for agglutination

• record results

What occurs in the 37 C phase of an antibody detection test?

• add 2 drops enhanced media (LISS)

• incubate at 37 C for 10 minutes

• spin and read for agglutination

• record results

What occurs in the AHG phase of an antibody detection test?

• wash 3x with isotonic saline

• add 2 drops AHG reagent (anti-IgG)

• spin/read for agglutination

  • read all macroscopically negative results microscopically

• record results

• to all negative tubes - add 1 drop of check cells

During the AHG phase of an antibody detection test, what happens if you get negative tubes?

• add 1 drop of check cells

  • if pos = valid result

  • if neg = invalid, repeat test

What does it mean during an Antibody detection test if you have positive results at the IS phase?

• IgM antibody

What does it mean during an Antibody detection test if you have positive results at the 37C phase?

• IgM and/or IgG antibody

What does it mean during an Antibody detection test if you have positive results at the AHG phase?

• IgG antibody

  • or infrequently an IgG that has”held on” through this phase

How does reactivity differ at different stages of an antibody detection test

• same reaction strength at same phase of reactivity

  • single antibody

• reactions at different phases of reactivity → multiple Ab or one antibody with IgM and IgG components

• different strength of reaction at same phase of reactivity → multiple Ab or dosage

What does it mean if there is a reaction at ANY phase of an antibody screen test?

• POSITIVE

What does it mean if no agglutination occur at any phases of an antibody screen test?

• NEGATIVE

  • positive check cell confirm negative AHG reaction

The patient does not currently possess detectable common antibodies in their plasma directed against RBC antigens. Does this exclude the presence of all antibodies?

• NO

  • negative ab screen results predict with 95% certainty that ABO/Rh compatible blood transfused to the patent should survive / function normally

What causes false negatives in Ab detection and ID?

• neutralizing AHG

• inadequate washing

• contaminating reagent cells

• improper saline used

What causes false positives in Ab detection and ID?

• debris in testing tubes

• over centrifugation

• rouleaux

What are antigrams?

• charts showing antigen profiles of the screening / panel cells

• Ab screens MUST have homozygous examples of antigen expression within the 3-cell set

• except high/low pairs and big K antigen only

What occurs in Step 1 of using an antigram

• Highlight

  • look for any cells (rows) that have reactivity at IS, 37 C, and/or ahg, IAT/ IgG phase

  • highlight these rows

What occurs in Step 2 of using an antigram

• Rule Out

  • use homozygous + cells that are non-reactive with patient plasma to rule out antibody possibilities

  • one rule-out needed

  • eliminate all possibilities that you can

    • do NOT need to rule out low frequency:

      • Cw, Kpa, Jsa, Lua

What occurs in Step 3 of using an antigram

• Match the pattern

  • look to see if anything that you have NOT ruled out matches all highlighted rules

What occurs in Step 4 of using an antigram

• Rule in

  • 3+3 rule

    • 3 antigen pos cells reactive (pos) with patient plasma

    • 3 antigen-neg cells non-reatcive with patient plasma

  • if many - cannot use same cell as “antigen positive” for both antibodies

What does a Negative result mean when evaluating Autocontrol?

• no reactivity

  • alloantibody causing reactions in panel

What does a Positive result mean when evaluating Autocontrol and there has been no recent transfusions (past 3 months)?

• reactivity is likely an autoantibody

What does a Positive result mean when evaluating Autocontrol AND there has been a recent transfusion or other sensitization?

• could be autoantibody

• could be alloantibody

• could be both

What is the hallmark feature of allo-ab coating Rbc?

• mixed field agglutination

  • some donor cells, some patients own cells

What antibodies may show dosage

• - C, c, E,e

• - Fya, Fyb

• -Jka, Jkb

• -M,N,S,s

• -Lua, Lub

What does the phase of reactivity determine in antibody identification testing?

• may indicate possible clinical significance

• tells you about possible class of antibody

What does the strength of a reaction determine in antibody identification testing?

• all same strength / same phase → single Ab

• varying strength → many aB, or single with dosage

What does the varying phase determine in antibody identification testing?

• single Ab with multiple classes of immunoglobulin (transition from initial - anamnestic response )

• multiple antibodies

  • Igm like anti-P1 and IgG like anti-Fya

What should you do when doing an antigran if not everything is ruled out or in?

• run selected cells

  • cells from different panels/ chose based on suspected to help ruling in/out

• Pick and chose

  • only run cells that are helpful

• Advanced techniques

  • enzyme treatment panel of RBC

  • use of different enhancement media

  • inhibit / neutralization techniques

What does it mean if an antibody is clinically significant?

• it can cause hemolytic transfusion reactions or HDFN

• usually IgG (except ABO)

• react at 37 C and/or AHG

What is needed for someone Antibody history?

• once identified, it does not need to be confirmed on every sample

• may investigate again for changes to suspected clinical significance

• transfusion requirements persist for like once established

• must honor antigen requirements established by other institutions

What is the Standard Operating Procedure (SOP)

• will tell you what your allowed to do for rule out/ins

• will also have procedures to cover ABO/Rb typing, Ab screens, phenotyping, cross match, ect

How does antigen phenotyping occur?

• Use of known antibody from reagent manufacturer (i.e. Anti-E) to test patient cells for presence / absence of antigen

• ex: identify anti-E in patient plasma → must phenotype for E antigen

  • can ONLY perform if patient has NOT recently been transfused

    • within last 90 days cause donor cells can contaminate results

What is a positive control for antigen phenotyping?

• heterozygous positive for antigen (E+e+)

  • ensures reagent is detecting any weaker expression of antigen

  • based on premise that heterozygous people have less of antigen

What is a negative control for antigen phenotyping?

• homozygous negative for antigen (E-e+)

What does this mean

• negative, no agglutination

What is this

• grading scale for reading agglutination reactions

What is the Fisher-Race theory?

• 3 closely liked sets of allelic genes

  • D9d), Cc, Ee

• move as unit

• ex: Dce/dce

  • parent will pass Dce or dce

Wiener Theory

• 1 gene results in production of agglutinogen which is composed of blood factors

What is the current inheritance theory

• two loci theory

  • locus 1: codes for presence/ absence of D

  • locu 2: codes for presence of Cs and Es