BLOOD BANK
M.G.
◆ Has agg. → test valid
TYPES OF AHG REAGENTS ◆ No agg → test invalid (AHG neutralized) → REPEAT
1. POLYSPEFIC AHG d) DAT Panel
➔ Contains anti-IgG + anti-C3d ➔ Uses monospecific anti-IgG and anti-C3d
➔ Determines if sensitization is due to IgG or complement
2. MONOSPECIFIC AHG ➔ Includes saline control → detects spontaneous agg.
➔ Contains either anti IgG or anti-C3d
FACTORS AFFECTING AHG TEST
3. POLYCLONAL AHG
➔ From multiple plasma cell clones (usually rabbits) 1. Serum to cell ratio
➔ Detects various IgG antibodies. ➔ Optimal:
◆ 2 drops serum : 1 drop 5% RBC (40:1)
4. MONOCLONAL AHG 2. Reaction medium
➔ From a single plasma cell clone (hybridoma technology) ➔ Albumin
➔ Highly specific, identical antibodies ◆ Reduces zeta potential
➔ LISS
TYPES OF ANTIGLOBULIN TESTS ◆ Enhances antibody uptake (shorter incubation)
➔ PEG
a) Direct Antiglobulin Test (DAT) ◆ Concentrates antibodies
➔ Detects: 3. Temperature
◆ antibodies/complement already bound IN VIVO to ➔ IgG & complement optimal at 37 C
patient’s RBCs. 4. Incubation time
➔ Specimen: ➔ Albumin: 15-60 mins
◆ Patient’s washed RBC (whole blood EDTA) ➔ LISS: 10-15 mins
➔ 37 C incubation: NO 5. Washing (CRUCIAL)
➔ PROCEDURE: TUBE METHOD ➔ Wash at least 3x
1. 1 drop 2-5% RBCs ➔ Removes free globulins
2. Wash 3x with NSS → decant ➔ Inadequate washing → FALSE NEGATIVE
3. Add 2 drops of AHG 6. Saline pH
4. Centrifuge → check agg. ➔ 7.2 - 7.4
7. Add AHG immediately after washing
➔ Clinical uses: 8. Centrifugation
◆ HEMOLYTIC TRANSFUSION REACTION ➔ 1000 RCF for 20 sec.
◆ HEMOLYTIC DISEASE OF FETUS & NEWBORN
◆ AUTOIMMUNE HEMOLYTIC ANEMIA FALSE RESULTS IN AHG
◆ DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA
FALSE POSITIVE FALSE NEGATIVE
b) Indirect Antiglobulin test (IAT)
➔ Detects:
◆ Free serum antibodies (IN VIVO) Clotted sample Inadequate washing
➔ Specimen:
◆ Patient serum + Patient RBCs Bacterial contamination Improper incubation
➔ 37 C incubation: YES
➔ PROCEDURE: Dirty glassware Old serum
1. 2 drops serum + 1 drop RBCs
2. Incubate at 37 C overcentrifugation AHG not added
3. Wash 3X → decant
4. Add 2 drops of AHG overreading undercentrifugation
5. Centrifuge → check for agg.
Contaminated AHG Incorrect cell suspension
➔ Clinical uses:
◆ CROSSMATCHING Inactive AHG
◆ ANTIBODY SCREENING
◆ ANTIBODY IDENTIFICATION
◆ ANTIBODY TITRATION ABO BLOOG GROUP SYSTEM
◆ WEAK D TESTING ➔ Discovered by:
◆ Karl Landsteiner (1901)
c) Check cells (COOMB’s control cels) ➔ Genes Involved
➔ Type O RBCs coated with IgG (usually anti-D) a) ABO Gene
➔ Added to negative AHG tests only ➔ Chromosome 9
➔ Interpretation: ➔ Encodes glycosyltransferase enzyme
BLOOD BANK
M.G.
➔ Adds specific sugar to H antigen ◆ SMALL AMOUNT IgG and IgA
➔ Reaction properties:
b) H gene ◆ STRONG AGGLUTINATORS
➔ Chromosome 19 ◆ EFFICIENT COMPLEMENT ACTIVATION
➔ Produces H antigen (precursor substance) ◆ CAN CAUSE INTRAVASCULAR HEMOLYSIS
➔ HDFN
c) Se gene ◆ Usually mild
➔ Chromosome 19 ◆ Because mostly IgM (does not cross placenta)
➔ controlS ABH expression in body fluids
➔ Secretor (Se/ se)
➔ Non secretor (se / se)
LAB DETECION OF ABO
IMMUNODOMINAT SUGARS
1. FORWARD TYPING (CELL TYPING)
➔ Tests patient RBCs
GENE ENZYME SUGAR ADDED ANTIGEN
➔ Reagents:
FORMED
◆ anti-A (blue dye)
◆ anti-B (yellow dye)
H a-2-L-fucosyltr L-fucose H antigen 2. REVERSE TYPING (SERUM TYPING)
ansferase ➔ Test patient serum
➔ Regent cells:
A a-3-N-acetylgal N-acetyl-D-gal A antigen ◆ A1 cells
actosaminyltra actosamine ◆ B cells
nsferase
LECTINS
B a-3-D-galactos D-galactose B antigen
yltransferase
LECTIN DETECTS SOURCE
anti-A1 A1 cells Dolichos biflorus
NOTES:
anti-H H antigen Ulex europaeus
➔ AHG detects IgG only, not IgM
➔ DAT = in vivo, IAT = in vitro
➔ Washing step is the most common cause of false negatives anti-M M antigen Vicia graminea
➔ Check cells validate negative AHG tests
➔ H antigen is required before A or B antigen can be formed BOMBAY PHENOTYPE
➔ LISS shortens incubation time
➔ PEG increases sensitivity but may increase false positives ➔ Genotype: hh
◆ No H gene → no H antigen
ABO ANTIGENS ◆ Cannot form A or B antigen
➔ RBCS:
➔ Formation: ◆ No A, B, or H antigens
◆ Present on 5-6 weeks fetal life ➔ Serum:
◆ Fully developed at birth ◆ anti-A
◆ Inherited (codominant: A & B; O is silent) ◆ anti-B
➔ Composition: ◆ anti-A,B
◆ On RBCs ◆ anti-H (Strong IgM)
● Glycolipids ➔ Clinical significance:
● Built on type 2 chain paragloboside ◆ Can receive blood only from bombay
◆ In secretions ◆ Severe hemolytic reaction of given O blood
● Glycoproteins
DIFFERENTIATING O vs BOMBAY
● Built on type 1 chain
● Controlled by Secretor (Se) gene
TEST TYPE O BOMBAY
ABO ANTIBODIES
anti-A 0 0
➔ Characteristics:
◆ NATURALLY OCCURING anti-B 0 0
◆ DETECTABLE AT 3-4 MONTHS OLD
◆ PEAK LEVEL: 5-10 YRS OLD anti-H Lectin 4+ 0
◆ PREDOMINANTLY IgM
BLOOD BANK
M.G.
Serum anti-H weak/absent strong
NOTES:
➔ ABO antibodies =naturally occurring IgM
➔ H antigen must be present before A or B formation
NOTES: ➔ O has highest H antigen conc,
➔ H antigen strength: ➔ Bombay = no H antigen
O > A2 > B > A2B > A1 > A1B ➔ Most ABO discrepancies occur in reverse typing
➔ O = highest antigen ➔ Rouleaux mimics agglutination but disperses in saline
➔ A1B = lowest ➔ anti-A1 is detected using dolichos biflorus lection
➔ anti-H lectin source = ulex europaeus
ABO TYPING DISCREPANCIES
Rh BLOOD GROUP SYSTEM
1. Rouleaux formation
➔ Increase plasma proteins ➔ Most clinically significant system after ABO
➔ Seen in: ➔ D antigen = most immunogenic RBC antigen
◆ MULTIPLE MYELOMA ➔ immunogenicity : D > c > E > C > e
◆ HYPERFIBRINOGENEMIA ➔ Antigens are non glycosylated proteins
➔ Resolution: ➔ Antibodies:
◆ SALINE REPLACEMENT ◆ Usually IgG
◆ WASH RBCs ◆ React at 37 C / AHG phase
◆ Cause extravascular hemolysis
2. Weak antibodies (reverse typing problem) ◆ Major cause of HDFN
➔ Seen in: ➔ Weak D
◆ NEWBORNS ◆ Reduced expression
◆ ELDERLY ◆ Detected at AHG phase
◆ HYPOGAMMAGLOBULINEMIA ◆ Test blood donors
◆ LEUKEMIA ◆ Partial D can form anti-D
◆ HODGKIN’S DISEASE ➔ Rare
◆ BRUTON’S AGAMMAGLOBULINEMIA ◆ Rh null → no Rh antigens → mild hemolytic anemia
➔ Resolution:
◆ Extend incubation 15-30 min at RT LEWIS BLOOD GROUP SYSTEM (ISBT 007)
3. Acquired B phenomenon ➔ Lewis antigens are adsorbed from plasma
➔ Cause: ➔ Poorly expressed at birth
◆ Bacterial deacetylation of A antigen ➔ Antibodies:
◆ Seen in GI disorders ◆ Usually IgM
➔ Resolution: ◆ Cold reacting
◆ Acidify anti-B reagent ◆ Rarely clinically significant
◆ Secretor studies
MNS BLOOD GROUP SYSTEM (ISBT 002)
4. Wharton’s Jelly (Cord blood)
➔ Cause: ➔ Key antigens
◆ Mucopolysaccharide contamination ◆ M, N → usually insignificant antibodies
➔ Resolution: ◆ S, s → clinically significant
◆ Wash cord cells 6-8x with NSS ➔ Important
◆ anti-S and anti-s = IgG, 37 C, AHG reactive
5. Weak antigens (forward typing problem) ◆ Slow dosage
➔ Cause: ◆ Destroyed by enzymes
◆ A/B subgroups
◆ Leukemia P BLOOD GROUP SYSTEM
◆ Excess soluble substances
➔ Resolution: ➔ Important antibodies
◆ Extend incubation 15-30 min ◆ ANTI-P1 → usually IgM, cold
◆ ANTI-PP1PK → seen in p phenotype, associated with
6. Cold reactive autoantibodies recurrent abortion
➔ React at 22 C ◆ AUTO ANTI-P → biphasic IgG seen in paroxysmal cold
➔ Resolution hemoglobinuria (donath landseiner antibody)
◆ Prewarm technique (37 C)
◆ Use DTT or 2-ME (Denatures IgM) I BLOOD GROUP SYSTEM (ISBT 027)
◆ Adsorption method
➔ i antigen → fetal RBCs
BLOOD BANK
M.G.
➔ I antigen → adult RBCs ➔ Antibodies:
➔ Antibodies: ◆ IgG
◆ anti-I → cold agglutinin, ass with mycoplasma ◆ 37 C / AHG reactive
pneumoniae infection ◆ weak, hard to detect
◆ Anti-i → ass with infectious mononucleosis ◆ Titer drops rapidly
NOTES: NOTES:
➔ D antigen = strongest immunogen ➔ Causes delayed hemolytic transfusion
➔ Rh antibodies = IgG, warm, HDFN reaction
➔ Lewis = IgM, cold, usually insignificant ➔ RBCs resist lysis in 2M urea
➔ MNS (S/s) = clinically significant ➔ Jk(a-b-) more common in polynesians and
➔ anti-P (DL antibody) = biphasic hemolysin asians
➔ anti-I = cold agglutinin ➔ Notorious blood bank antibody
LUTHERAN BLOOD GROUP SYSTEM (ISBT 005)
KELL BLOOD GROUP SYSTEM (ISBT 006)
➔ Gene: LU (chr 19)
➔ Second most immunogenic after D ➔ Poorly developed at birth
➔ Gene: KEL (chr 7) ➔ Most people are Lu (b+)
➔ K antigen → 9% (low incidence) ➔ Antibodies:
➔ k cellano → 99.8% (high incidence) ◆ anti-Lua → usually IgM
➔ Antibodies: ◆ anti-Lub → IgG, may shorten RBC survival
◆ anti-K = most common after ABO/Rh ◆ Often mixed field agg
◆ Usually IgG DIEGO BLOOD GROUP SYSTEM (ISBT 010)
◆ React at 37 C / AHG
◆ Causes HTR & HDN ➔ Located on band 3 (anion exchanger protein)
➔ Important in anthropologic studies (asian ancestry)
➔ Antibodies:
NOTES: ◆ IgG
➔ Kell antigens destroyed by DTT, AET, 2-ME, ◆ Clinically significant
ZZAP
➔ anti -Ku → seen in K0 indiv DOMBROCK (ISBT 014)
➔ McLeod phenotype → X-linked, weak Kell,
acanthocytosis, muscular dystrophy ➔ Weak, hard to detect IgG
➔ Clinically significant
➔ Located on GPI linked glycoporotein
DUFFY BLOOD GROUP SYSTEM (ISBT 008) COLTON (ISBT 015)
➔ Gene: FY (chr 1) ➔ Located on aquaporin-1
➔ antigens : Fya , Fyb ➔ IgG
➔ Destroyed by enzymes ➔ Clinically sig
➔ Moderately immunogenic
➔ Antibodies: LANDSTEINER-WIENER (ISBT 016)
◆ IgG
◆ 37 C / AHG reactive ➔ Located on ICAM-4
◆ Causes HTR & HDFN ➔ anti-LW reacts stronger with D+ cells
➔ IgG
NOTES: CHIDO RODGERS (ISBT 017)
➔ Fy (a-b-) common in african ancestry
➔ Confers resistance to plasmodium vivax ➔ Antigens adsorbed from plasma (C4 component)
malaria ➔ Classified as HTLA antibodies
➔ Weak IgG
➔ Rarely clinically sig
KIDD BLOOD GROUP SYSTEM (ISBT 009)
➔ No HTR/HDF
➔ Gene: JK (chr 18)
KNOPS (ISBT 022)
➔ Antigens: Jka, Jkb
➔ Enhanced by enzymes
➔ Located on complement receptor 1 (CR1)
➔ Show dosage
➔ HTLA ab
BLOOD BANK
M.G.
➔ Not clin sig
JR (ISBT 032)
➔ Antigen on ABCG2 transporter
➔ Anti Jr = IgG
➔ Rare but clinically significant
LAN (ISBT 033)
➔ Located on ABCB6
➔ anti-Lan = IgG
➔ Clin sig
VEL (ISBT 034)
➔ Antigen: SMM1
➔ anti-Vel = IgG
➔ Fixes complement
➔ Can cause severe hemolysis
NOTES:
➔ Kell = 2nd most immunogenic (after D)
➔ Duffy = malaria resistance (Fy a-b-)
➔ Kidd = delayed HTR, weak Ab
➔ Kell destroyed by DTT
➔ Chido/rodgers & knops = HTLA (not sig)
➔ Vel & Lan = clinically significant