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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

objectives
Objectives
  • 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

RBC

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

Glucose

H antigen

Galactose

N-acetylglucosamine

Galactose

Fucose

group a
Group A

RBC

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

Glucose

Galactose

N-acetylglucosamine

Galactose

N-acetylgalactosamine

Fucose

group b
Group B

RBC

    • The B gene codes for the enzyme D-galactosyltransferase
  • that adds the sugar D-galactoseto the terminal sugar of H antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

Fucose

Galactose

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

http://liengdieng.tumblr.com

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
bombay
Bombay
  • Doesn’t produce

L-fucosyltransferase

  • 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
lewis
Lewis
  • 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
adsorptions
Adsorptions
  • 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

slide38
HDFN
  • 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
testing
Testing
  • 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

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

Symptoms:

  • Fever, chills, chest pain or hypotension.
  • Transfused patients develop oliguria, hemoglobinuriaand hemoglobinaemia.

Causes:

  • clerical errors (ABO antibodies)

Delayed Extravascular

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

Symptoms:

    • unexplained fever
    • Anemia [no evidence of bleeding]
    • The patient may also have jaundice, high bilirubin, positive DAT.

Causes:

    • patient develops an IgG antibody to a red cell antigen that they lack.
transfusion reactions cont
Transfusion Reactions cont..

TRALI

  • Adverse reaction with hypotension and pulmonary edema.

Cause:

  • Occur when human leucocyte antigen (HLA) or human neutrophil antigen (HNA)
  • Symptoms:
  • Acute onset of fever, chills, dyspnea,respiratory failure
  • hypotension.

TACO

  • Adverse reaction with hypertension and pulmonary edema.

Cause:

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

Symptom:

  • Dyspnea, hypertension and pulmonary edema

It’s in the Bank! April 2014

transfusion reactions cont1
Transfusion Reactions cont..

Allergic

  • Urticarial

Allergic reaction

Cause:

  • Antibody to plasma protein

Symptoms:

  • hives

Anaphylaxis

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

Cause:

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

Symptoms:

  • mild upper respiratory symptoms.

Bacterial Contamination

    • Severe reaction to component

Cause:

  • Due to gram neg endotoxin

Symptoms:

  • Shock, fever, low BP, chills

TRALI

  • 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
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