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Immunohematology Review. Deborah Baudler MS, MT(ASCP)SBB Assistant Professor University of Illinois Springfield Patchwork Conference April 2014. University of Illinois-Springfield. UIS CLS Students. Objectives. Quick Review of most blood group systems

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Immunohematology review

Immunohematology Review

Deborah Baudler MS, MT(ASCP)SBB

Assistant Professor

University of Illinois Springfield

Patchwork Conference April 2014


  • Quick Review of most blood group systems

  • Highlight Antibody Identification and Special procedures

  • Review donor screening

  • Discuss component collection, testing and clinical indication

  • Discuss HDFN and Prenatal testing

  • Review transfusion reactions

  • End with Review Questions

Abo inheritance and genetics
ABO Inheritance and Genetics

  • The ABO gene is autosomal (the gene is not on either sex chromosomes)

  • The ABO gene locusis located on the chromosome 9.

  • A and B blood groups are dominant over the O blood group

  • A and B group genesareco-dominant

H substance
H Substance


  • The H gene codes for the enzyme fucosyltransferase that adds the sugar fucoseto the terminal sugar of a precursor substance (PS)


H antigen





Group a
Group A


  • The A gene codes for the enzyme N-acetyl galactosaminyl-transferase that adds the sugar N-acetyl galactosamineto the terminal sugar of H antigen







Group b
Group B


  • The B gene codes for the enzyme D-galactosyltransferase

  • that adds the sugar D-galactoseto the terminal sugar of H antigen

  • Glucose






    Abo blood g roup
    ABO Blood Group

    • Group A: you have A antigens on the surface of your RBCs and B antibodies in your plasma.

    • Group B, you have B antigens on the surface of your RBCs and A antibodies in your plasma.

    • Group AB, you have both A and B antigens on the surface of your RBCs and no A or B antibodies in your plasma.

    • Group O, you have neither A nor B antigens on the surface of your RBCs but you have both A and B antibodies in your plasma.

    Abo frequencies
    ABO Frequencies

    Subgroups of a and b
    Subgroups of A and B

    • Differ in the amount of antigen present on the red blood cell membrane

    • Subgroups of B are very rare

    • B3 is the most common, shows 2+mf with Anti-B and Anti-A,B


    • Doesn’t produce


    • No A, B or H antigens on rbcs

    • Non-secretor

    • Phenotypes as Group O

    • This is the ABO “Null” group

    Rh antigens
    Rh Antigens

    • Rh antigens are proteins that are highly immunogenic

    • the D antigen is most antigenic

    • Exposure to less than 1 ml of Rh positive red cells can stimulate Ab production

      D  c  E  C  e

      Highly Rarely Immunogenic

    • f [ce]: compound antigen, Dce or dce on same haplotype

    • Rhi [Ce]: compound antigen, DCe or dCe on same haplotype

    • D - - has strongest expression of D

      • Make Anti-Rh17 [anti-Hro]

    • D · · variation of D - -

    • Weak D: quantitative, position or mosaic

      • MUST PERFORM ON Rh neg Units initial testing and Rh Neg newborns

    Rh null
    Rh Null

    • Rh null = NORh antigens expressed

    • 2 types:

    • Regulator type: RhAG gene is mutated but has normal RhD and RhCE genes so can pass Rh antigens onto off spring

    • Amorphtype: RhAG is normal, but lacks RhD gene and RhCE is mutated ,

    • Affects other antigens: S, s, U depressed

    • Negative for Fy5 [Duffy 5] antigen

    • Lacks LW antigen

    • Lack Rh29 antigen [total Rh]

    Fisher race weiner nomenclature
    Fisher-Race & Weiner Nomenclature

    • Fisher-Race: CDE terminology

      • They theorized that the Rh antigens are controlled by a complex of 3 sets of genes with closely linked loci

    • Weiner: Rh-Hrterminology

      • A single gene at Rh locus leads to the expression of the Rh antigens

    • WeinerFisher-Race

    • R0 =Dce

    • R1 = DCe

    • R2 = DcE

    • r =dce

    • r’ = dCe

    • r” = dcE

    • Rz = DCE [rare]

    • ry = dCE[rare]

    Rh antibodies
    Rh Antibodies

    • 50- 70% of Rh negative patients will make anti-D

    • Most are IgG

    • React optimally at 37° C or after Antiglobulin testing

    • Clinically significant

    • Don’t bind Complement

    • Anti-D is most potent, followed by Anti-c, Anti-E, anti-C and last Anti-e

    • Anti-LW reacts stronger with D positive cells than D negative cells [mimics Anti-D]

      • Can differentiate from Anti-D: Anti-LW reacts with ALL cord cells, regardless of Rh type


    • The Le gene codes for enzyme L-fucosyltransferase to attach fucose to the subterminal to the GLcNAc on the ABH precursor substance to form Leª Ag

    • The Se gene codes for enzyme, α-2-L-fucosyltransferase to attach 2ndfucose to the terminal Gal on the ABH precursor substance to form Le b Ag

    • Genes: Le and le. The are co-dominant.

    • Solubleantigens produced by tissues and found in body fluids (plasma)

    • Adsorbed on the RBC

    • Examples:

      • Lese Le(a+b-)

      • LeSe Le(a-b+)

      • leSe Le(a-b-)

      • lese  Le(a-b-)

    Lewis antibodies
    Lewis Antibodies

    • Primarily IgM

    • Frequently detected at RT

    • Can activate complement and cause hemolysis in invivo and invitro testing

    • Enzymes enhance reactivity

    • Lewis antibodies can be neutralized by Lewis positive plasma or saliva

    • Often times a pregnant Mom will develop Lewis antibodies and phenotype will appear Le(a-b-)

    • Doesn’t cause HDN [doesn’t cross placenta] and cord cells Le(a-b-)

    • Can cause HTR

    P antigens
    P Antigens

    • 3 antigens: P, P1 and P(k) give rise to 5 phenotypes

      • P1 phenotype: P1, P and P(k) antigens on red cells

      • P2 phenotype: P and P(k) antigens on red cells

      • p phenotype: Null phenotype[no P antigens] (rare)

  • Common Precursor Substance is Lactosylceramide

  • Deteriorates in storage

  • Variable antigen expression on red cells

  • Found in secretions like plasma and Hydatid cyst fluid

  • Binding site for Parvovirus B19

  • P antibodies
    P Antibodies

    • P1 produces no antibody

      • Has all 3 antigens on red cells [P1, P, P(k)]

  • P2 produces Anti-P1

    • P and P(k) antigens

    • IgM, naturally occurring

    • Reacts best at RT or 4°C

    • Rarely activates Complement

    • Enhanced by enzymes

    • Can be neutralized (inhibited) by P1 substance

    • p produces Anti-PP1P(k) [antibody used to be called Anti-Tjª]

      • Reacts with all red cells except null p type

      • IgM and IgG antibody

      • Wide thermal range

      • Activates Complement

      • Causes severe HTR and HDN

      • Associated with spontaneous abortions in early pregnancy

    P antibodies1
    P Antibodies

    • Autoanti-P

      • IgG [autoantibody]

      • Causes Paroxysmal Cold Hemoglobinuria [PCH]

      • Biphasic hemolysin

      • Found in children <14 yr following a viral infection

  • PerformDonath–Landsteinertest

  • M and n antigens
    M and N Antigens

    • Glycophorin A: carries M and N antigens

    • Rich in sialicacid

    • Enª: found on all cells that are M+ or N+

    • If cells lack Glycophorin A, they will have no: Enª, M or N antigens

    • M and N are easily Destroyed by enzymes and by ZZAP

    • M and N antigens are well developed at birth

    • M(k) NULL gp

      • Silent (amorph) gene

      • RBCs lack M, N, S, s, and U antigens

      • Normal rbc survival

    S s u antigens
    S,s,U Antigens

    • Glycophorin B: carries the S, s and Uantigens

    • The U antigen is ALWAYSpresent when S & s are inherited [less than 1% of S=s= are U=

    • Less sensitive to destruction by enzymes

      • If cells lack Glycophorin B, they will have no: S, s, and U antigens and are only found in theBlack population

      • S, s and U antigens are well developed at birth

      • Glycophorin B associates with the Rh protein [explains why S, s expression is reduced on Rh null cells]

    M n s s u antibodies
    M, N, S, s, U Antibodies

    S,s and U

    IgG and react at 37°C / AHG

    Shows minimal dosage effect

    **May bind Complement

    May not react with enzyme-treated cells

    Can cause HTR and HDN

    Anti-U: Found mostly in African Blacks [35% are U=]

    M and N

    • Usually reacts at room temperature [saline reactivity] or below

    • IgM and IgG component

    • Do not bind Complement

    • **Shows Dosage: antibodies react better with homozygous cells [M/M and N/N] rather than heterozygous [M/N]

    Kell antigens
    Kell Antigens

    • K is rated second in Immunogenicity after D antigen

    • K antigen is detected early in fetal life, at 10 weeks

    • are well developed at birth

    • 3 antithetical partners: Co-dominant

      • K [Kell] and k [Cellano]

      • Kpa [Penny] and Kpb[Rautenberg]

      • Jsa [Sutter] and Jsb [Matthews]

        • K, Kpa and Jsa are low incidence

        • k, Kpb and Jsb are high incidence

    Kell antigens1
    Kell Antigens

    • sensitive to sulfhydryl reagents

    • Not denatured by enzymes

    • Kothe Null phenotype [no Kell antigens expressed]

    • K0 individuals can develop anti-Ku

      • Kx antigens are increased in those who are K0

    • Kxis produced by a gene XK1on the X chromosome [not part of the Kell system]

  • McLeod Phenotype

  • When the XK1 gene is not inherited, Kx is absent

  • Associated with Chronic Granulomatous Disease

  • Kell antibodies
    Kell Antibodies

    • Anti-K is most common

    • IgG(react well at AHG)

    • 20% bind Complement

    • Clinically significant

      • Can cause HDN and HTR

      • Fetus should be carefully monitored

    • Reactivity may be increased with PeG

    Duffy antigens
    Duffy Antigens

    • 3 main alleles: Fya and Fyb and Fy [silent allele]

    • 4 high incidence antigens: Fy3, Fy4, Fy5 and Fy6

      • Fy3 is a precursor common to Fya and Fyb

      • All normal red cells positive for Fy3, Fy4, Fy5 and Fy6

    • Fygene is Null phenotype [lack all Duffy antigens]

    • If Rh null, you lack Fy5 antigen

    • Fya and Fybantigens do not store well

    • Fya, Fyb, and Fy6 are destroyed by enzymes and ZZAP

    • Fy3, Fy4 and Fy5 are not destroyed by enzymes

    Duffy antibodies
    Duffy Antibodies

    • IgGin nature

      • React best at AHG phase

    • Enhanced by LiSS

    • Do not bind complement

    • showdosage

    • Clinically significant

      • Do cause acute and delayed HTR

      • Do cause mild to severe HDN

    • To distinguish Anti-Fy3 from Anti-Fy5: Rh null cells

    Kidd antigens
    Kidd Antigens

    • Jk3 is a high incidence antigen found on all Jka+ or Jkb+ cells

    • Kidd antigens are involved in urea transport in red cell

    • Kidd Antigens are well developed at birth

    • NULL phenotype: JKJK Jk(a-b-)

      • Found in Polynesians: Filipinos, Japanese and Chinese

      • Resistant to lysis in 2M urea [normal red cells lyse < 1 min]

      • Jk(a-b-) lyse in 30 minutes

      • Unable to concentrate urine

    Kidd antibodies
    Kidd Antibodies

    • IgG

    • Clinically significant: can cause HDN

    • The Kidd antibodies are notorious for causing delayed hemolytic transfusion reactions

    • Show dosage

    • Titers rise quickly and fall just as fast [DHTR]

    • Enhanced by enzymes, LiSS and PeG

    • Anti-Jk3: Found in some individuals who are Jk(a-b-)

    Pretransfusion testing
    Pretransfusion Testing

    • ID patient with name and date of birth

    • Complete label with 2 patient identifiers such as First and Last name and Medical ID number, date and time of collection, Phleb ID

    • Remember Type and Screens are good for 72 hrs

    • ABO and Rh testing

    • Antibody Screen

    • Antibody Identification [if necessary]

    • Antigen testing [if necessary]

    • Crossmatch

      • Immediate spin detects ABO compatibility

      • AHG detects clinically significant alloantibodies and autoantibodies

    Antibody identification1
    Antibody Identification

    • “Ruling out” means crossing out antigens that did not react on HOMOZYGOUS cells

    • Is everything else ruled out?

    • Circle the antigens that are not crossed out

    • Look for a matching pattern

    • The rule of three must be met to confirm the presence of the antibody. A p-value ≤ 0.05 must be observed

    • Phenotype patient if the patient has NOT been recently transfused


    • Useful for detecting alloantibodies masked by an autoantibody

    • Differential Adsorption

      • Used in patients who have been recently transfused or positive DAT

      • 3 cells: R1R1, R2R2 and rr

      • Among the 3 cells: 1 cell must be negative for K, another negative Jka and third one negative for Jkb

    • Autologous adsorption

      • Uses washed patient cells

      • Incubate patient’s plasma with patient’s cells for up to 1 hr to remove autoantibody

    Antigen frequency
    Antigen Frequency

    • Calculation used to estimate number of units to screen vs. ordering from reference lab

    • Uses the antigen negative frequency [100-frequency]

      Y = x number of units needed

      # combined antigen neg frequencies


    • Categories

    • ABO Antibodies

    • Mild HDN due to Anti-A,B

    • DAT: 1-2+

    • Can affect every pregnancy

    • Mom is Group O, child is A or B

    • Bilirubin peaks in 1-3 days

    • IgG Antibodies

    • Anti-D: most severe

      • Anti-K: 2nd most common

      • DAT: 2-4+

      • Affects 2nd and future babies

      • Bilirubin levels higher and longer

    • Mother has an antibody capable of crossing the placental barrier that is specific to an antigen present on the red blood cells of the fetus.

    • Fetal red cells become coated with the IgGalloantibody and undergo accelerated destruction both before and after birth.

    • Kernicterus: Bilirubinemiadue to increased red cell destruction


    • Mom

    • Type and Screen

    • Antibody ID [if necessary]

    • Antibody Titers

    • Amniocentesis [if necessary]

    • RhIG Evaluation on Rh Neg moms

      • Fetal Screen [Rosette test]

      • KleihauerBetke

      • PCR

    • Infant

    • Cord Blood: Type and DAT

    • Elution [if necessary]

    • Bilirubin levels [if necessary]

    Neonatal transfusions
    Neonatal Transfusions

    • Intrauterine and Exchange

    Mosby 2009

    Donor deferrals
    Donor Deferrals

    Quality control
    Quality Control

    • RBC ≤ 80% HCT

    • Leukoreduced:

      • RBC or SDP: 95% ≤ 5.0 x 106wbcs

      • Random plt: 95% ≤ 8.3 x 105wbcs

      • Maintains 85% of rbcs

  • Platelets:

    • Single: ≥ 5.5 x 1010 platelets

    • Pheresis: ≥ 3.0 x 1011 platelets

    • pH ≥ 6.2

  • Cryo:

    • 1 unit: ≥ 80 IU of F8 and ≥ 150 mg fibrinogen

  • Granulocytes:

    • ≥ 1.0 x 1010wbcs

  • Transfusion reactions
    Transfusion Reactions

    Acute Intravascular

    • Tends to present immediately or within 24 hours after transfusion


    • Fever, chills, chest pain or hypotension.

    • Transfused patients develop oliguria, hemoglobinuriaand hemoglobinaemia.


    • clerical errors (ABO antibodies)

    Delayed Extravascular

    • A type of transfusion reaction occurs 3 to 10 days later


      • unexplained fever

      • Anemia [no evidence of bleeding]

      • The patient may also have jaundice, high bilirubin, positive DAT.


      • patient develops an IgG antibody to a red cell antigen that they lack.

    Transfusion reactions cont
    Transfusion Reactions cont..


    • Adverse reaction with hypotension and pulmonary edema.


    • Occur when human leucocyte antigen (HLA) or human neutrophil antigen (HNA)

    • Symptoms:

    • Acute onset of fever, chills, dyspnea,respiratory failure

    • hypotension.


    • Adverse reaction with hypertension and pulmonary edema.


    • This is usually due to rapid or massive transfusion of blood- cardiac problems


    • Dyspnea, hypertension and pulmonary edema

    It’s in the Bank! April 2014

    Transfusion reactions cont1
    Transfusion Reactions cont..


    • Urticarial

      Allergic reaction


    • Antibody to plasma protein


    • hives


      • Anaphylaxis is a life-threatening allergic reaction that can occur after only a few milliliters of blood have been transfused


    • Because they lack IgA, their immune systems develops anti-IgA


    • mild upper respiratory symptoms.

    Bacterial Contamination

    • Severe reaction to component


  • Due to gram neg endotoxin


  • Shock, fever, low BP, chills


  • Adverse reaction 1° after plasma products, within 6 hrs

  • Cause:

  • HLA or WBC antibodies

  • Symptoms:

  • Acute onset of fever, chills, dyspnea, respiratory failure

  • hypotension.

  • Transfusion reaction workup
    Transfusion Reaction Workup

    • Stop transfusion

    • Keep saline lines open

    • Return product to blood bank for inspection

    • Check for clerical errors

    • Test the post-transfusion sample:

    • Check for visual hemolysis

    • Perform DAT if positive, compare to pre-transfusion DAT

    • Repeat ABO and RH if necessary

    • Perform Elution and Antibody ID if necessary

    • Additional testing per pathologist

    Let the g ames begin
    Let the Games Begin…..

    • An example of a technical error that can result in an ABO discrepancy is:

      • Acquired B phenomenon

      • Missing antibodies or antigens

      • Leaving out the plasma in the reverse cells

      • Washing the cells 4 times instead of 3 after the 37 C incubation

    • Excluding ABO, this antibody is rated second only to D in immunogenicity:

      • Anti-Fya

      • Anti-K

      • Anti-Jka

      • Anti-S

    Review questions
    Review Questions

    • A red cell unit that has AS1 preservative added to it, has an expiration date of:

      • 5 days

      • 21 days

      • 42 days

      • 1 year

    • Symptoms of dyspnea, cough, hypoxemia, and pulmonary edema within 6 hrs of transfusion is most likely which type of reaction?

      • Febrile Non-hemolytic

      • TRALI

      • Hemolytic

      • Anaphylactic

    Review questions1
    Review Questions

    • A patient has an Anti-E antibody. What kind of crossmatch, if any, should the technologist perform?

      • An immediate spin crossmatch only

      • An immediate spin and an AHG crossmatch

      • No crossmatch is necessary, just give out E negative units

      • No crossmatch is required, the antibody is not clinically significant

    •  Which of the following tests can distinguish a patient who has had Hepatitis B from one that has received the Heptavax [Hep B] immunization series?

      • Hbs Ag

      • Hbs Ab

      • Hbcore Ab

      • HCV Ab

    Review questions2
    Review Questions

    • Extravascular destruction of the red cells in a delayed transfusion reaction is characterized by:

      • Increased bilirubin, decreased haptoglobin, increased hemoglobin

      • Increased bilirubin, falling hemoglobin, positive DAT

      • Decreased bilirubin, falling hemoglobin, negative DAT

      • Decreased bilirubin, increased haptoglobin, falling hemoglobin

    • The H antigen is found in highest concentration on what blood group type?

      • A

      • B

      • O

      • AB

    Review questions3
    Review Questions

    • With Warm Autoimmune Hemolytic Anemia, the DAT is results are usually:

      • Positive for IgG only

      • Positive for C3 only

      • Positive for either IgG and/or C3

      • Negative most of the time

    •  The most serious hemolytic transfusion reactions are due to antibodies from which blood group system?

      • Rh

      • Kell

      • Duffy

      • ABO

    Review questions4
    Review Questions

    • Which of the following constitutes permanent rejection for a blood donor?

      • Had a tattoo 5 months ago

      • Received 2 units of blood 3 years ago

      • Had a confirmed positive test for Hepatitis B 13 months ago

      • Received an immunization for Rubella 2 months ago

    •  A moderate type of post-donation reaction that is characterized by a decreased pulse, decreased blood pressure and loss of consciousness is called:

      • Hyperventilation

      • Shock

      • Vasovagel

      • Hematoma

    Review questions5
    Review Questions

    • Fresh Frozen Plasma is the product of choice for treating all the following disorders except:

      • Bleeding caused by DIC

      • Bleeding due to deficiency of coagulation factors

      • Bleeding due to decreased platelets

      • Bleeding due to Coumadin overdose

    • An individual that is identified as Rh null lacks which of the following antigens?

      • M,N,S,s

      • ABO

      • Rh and Fy5

      • Kidd

    Review questions6
    Review Questions

    • What is the minimal interval between 2 1-unit red cell donations?

      • 2 days

      • 2 weeks

      • 4 weeks

      • 8 weeks

    • What is the main purpose of performing an antibody screen?

      • To detect mostly IgM antibodies

      • To detect clinically significant IgG antibodies

      • To detect ABO antibodies

      • To detect all antibodies

    Review questions7
    Review Questions

    • What is the terminal sugar on the B antigen?

      • L-fucose

      • N-acetylglucosamine

      • N-acetylgalactosamine

      • D-galactose

    • In an emergency situation and you have no blood type for the patient, which type of red cells should you give?

      • AB Negative

      • A Positive

      • O Negative

      • O Positive

    Review questions8
    Review Questions

    • Which of the following statements about Rh antibodies is true?

      • They are predominately IgG

      • They activate Complement

      • They best react at RT

      • They are predominately are IgM

    • What is the terminal sugar on the H antigen?

      • L-fucose

      • N-acetylglucosamine

      • N-acetylgalactosamine

      • D-galactose

    Review questions9
    Review Questions

    • Which of the following is the correct Wiener notation for DCe/DcE?

      • R1R1

      • R2R2

      • R1R2

      • rr

    • What is the purpose of the immediate spin crossmatch?

      • To detect clinically significant antibodies

      • To detect a low frequency antibody

      • To detect ABO incompatibility

      • To detect an ABO discrepancy