Blood Banking & Immunohematology
Master blood group systems, compatibility testing, and transfusion medicine for the MedTech board exam.
Table of Contents
1. ABO Blood Group System
Discovery and Genetics
- • Karl Landsteiner (1901): Discovered ABO system, Nobel Prize 1930
- • Chromosome location: 9q34
- • Inheritance: Codominant (A and B), O is recessive
- • H gene: Located on chromosome 19, produces H antigen (precursor)
- • Secretor gene (FUT2): Determines ABH antigens in body fluids
ABO Antigens and Antibodies
| Blood Type | Antigens on RBCs | Antibodies in Plasma | Genotypes | Frequency (Filipino) |
|---|---|---|---|---|
| A | A, H | Anti-B | AA, AO | ~27% |
| B | B, H | Anti-A | BB, BO | ~25% |
| AB | A, B, H | None | AB | ~7% |
| O | H only | Anti-A, Anti-B | OO | ~41% |
ABO Subgroups
A Subgroups
- • A1: 80% of type A, strong reactions
- • A2: 20% of type A, weaker reactions
- • A2 cells may have anti-A1 in serum
- • A1 lectin (Dolichos biflorus) differentiates
- • Other weak A: A3, Ax, Aend, Am, Ael
B Subgroups
- • Less common than A subgroups
- • B3, Bx, Bm, Bel variants exist
- • Weak B may be missed in routine testing
- • Need extended incubation for detection
Bombay Phenotype (Oh)
Definition: Individuals lack H antigen due to homozygous hh genotype
- • Cannot produce A, B, or H antigens
- • Have anti-A, anti-B, and anti-H antibodies
- • Can only receive blood from other Bombay individuals
- • Forward typing: Appears as O
- • Reverse typing: Reacts with all cells including O cells
- • Discovered in Bombay (Mumbai), India
ABO Typing Methods
Forward (Cell) Typing
- • Patient RBCs + known antisera
- • Tests for antigens on RBCs
- • Use anti-A and anti-B reagents
Reverse (Serum) Typing
- • Patient serum + known cells
- • Tests for antibodies in serum
- • Use A1 cells, B cells, (O cells optional)
2. Rh Blood Group System
Discovery and Genetics
- • Landsteiner & Wiener (1940): Discovered using Rhesus monkey RBCs
- • Chromosome location: 1p36 (RHD and RHCE genes)
- • Most immunogenic antigen: D antigen (after ABO)
- • D-positive: ~85% Caucasians, ~95% Filipinos
- • D-negative: Lack D antigen, can develop anti-D
Rh Antigens (Fisher-Race Nomenclature)
| Fisher-Race | Wiener | Immunogenicity Rank | Notes |
|---|---|---|---|
| D | Rho | 1st | Most clinically significant |
| c | hr' | 2nd | Common cause of HDFN |
| E | rh" | 3rd | Often develops with anti-c |
| e | hr" | 4th | Less common antibody |
| C | rh' | 5th | Least immunogenic of five |
Weak D and Partial D
Weak D (Du)
- • Reduced expression of complete D antigen
- • Negative with direct typing, positive with IAT
- • Usually cannot make anti-D
- • Can donate as D-positive
- • Can receive D-positive blood
Partial D
- • Missing some D epitopes
- • CAN make anti-D (against missing epitopes)
- • Should receive D-negative blood
- • Categories: DII, DIII, DIV, DV, DVI, DVII
- • DVI most clinically significant
Rh Typing Methods
- • Slide/Tube method: Direct agglutination with anti-D
- • Weak D testing: Indirect antiglobulin test (IAT) required
- • Gel technology: Sensitive, standardized method
- • Monoclonal anti-D: IgM (immediate spin), IgG (IAT phase)
3. Other Blood Group Systems
Kell Blood Group System
- • K antigen: Third most immunogenic (after D and c)
- • Antibodies: IgG, clinically significant, cause HDFN
- • K-positive: ~9% Caucasians, rare in Asians
- • Kell null (Ko): McLeod syndrome association
- • Important antigens: K, k (cellano), Kpa, Kpb, Jsa, Jsb
Duffy Blood Group System
- • Antigens: Fya and Fyb
- • Fy(a-b-) phenotype: Common in African populations
- • Malaria resistance: Duffy antigens are receptors for Plasmodium vivax
- • Antibodies: IgG, can cause HDFN and HTR
- • Dosage effect: Stronger reactions with homozygous cells
Kidd Blood Group System
- • Antigens: Jka and Jkb
- • Antibodies: IgG, notorious for causing delayed HTR
- • Characteristics: "Disappearing antibody" - drops below detectable levels
- • Show dosage: Stronger with homozygous cells
- • Enhance detection: Use enzyme-treated cells, PEG
MNS Blood Group System
- • Antigens: M, N, S, s, U
- • Anti-M: Usually IgM, cold-reacting, clinically insignificant
- • Anti-N: Rare, usually from dialysis patients
- • Anti-S and anti-s: IgG, clinically significant
- • Anti-U: Can cause severe HDFN, found in S-s- individuals
Lewis Blood Group System
- • Antigens: Lea and Leb (adsorbed from plasma)
- • Not truly RBC antigens: Produced in plasma, adsorbed onto RBCs
- • Antibodies: IgM, NOT clinically significant
- • Do not cause HDFN: Antigens not developed on fetal cells
- • May change during pregnancy: Le(a-b-) phenotype common
P1PK and I Blood Group Systems
P1PK System
- • Anti-P1: IgM, cold-reacting
- • Usually clinically insignificant
- • p phenotype: Very rare, anti-PP1Pk
I System
- • I antigen: Adults; i antigen: Newborns
- • Anti-I: Cold autoantibody (Mycoplasma)
- • Anti-i: Associated with EBV infection
4. Antibody Screening & Identification
Antibody Screening Purpose
- • Detect unexpected antibodies in patient serum/plasma
- • Use 2-3 reagent red cell panel (screening cells)
- • Screen cells must express common clinically significant antigens
- • Perform at 37°C and AHG phase for IgG antibodies
- • Required before transfusion and during pregnancy
Antibody Identification
Panel Cell Method
- • Use 10-16 cell panel with known antigen profiles
- • Test patient serum against each cell
- • Match reaction pattern with antigram
- • Rule out antigens: Cells negative for antigen, reaction negative
- • Confirm antibody: At least 3 positive and 3 negative reactions
Enhancement Techniques
LISS (Low Ionic Strength Saline)
- • Reduces zeta potential
- • Speeds up antibody uptake
- • Shorter incubation time
- • May enhance non-specific reactions
PEG (Polyethylene Glycol)
- • Removes water, concentrates antibodies
- • Very sensitive method
- • Cannot read immediate spin phase
- • Must wash cells well before AHG
Enzyme Treatment
- • Ficin, papain, bromelin
- • Enhances: Rh, Kidd, Lewis, P, I
- • Destroys: Fya, Fyb, M, N, S, s
- • Useful for antibody identification
Gel Technology
- • Standardized, stable endpoint
- • Requires specialized cards/equipment
- • No washing required
- • Can store results for review
Antibody Characteristics
| Characteristic | IgM | IgG |
|---|---|---|
| Optimal temperature | 4°C - Room temp | 37°C |
| Detection phase | Immediate spin | AHG phase |
| Complement activation | Strong | Variable |
| Crosses placenta | No | Yes |
| Causes HDFN | No | Yes |
5. Compatibility Testing & Crossmatching
Pre-Transfusion Testing Steps
- Verify patient identity and sample labeling
- ABO and Rh typing (forward and reverse)
- Antibody screening
- Antibody identification (if screen positive)
- Crossmatch (major crossmatch)
- Selection of compatible units
- Issue and transfuse
Types of Crossmatches
Immediate Spin (IS)
- • Patient serum + donor cells
- • Centrifuge immediately
- • Detects ABO incompatibility
- • Used when antibody screen negative
- • Fastest method
Full Crossmatch (AHG)
- • Incubate at 37°C
- • Add AHG reagent
- • Detects IgG antibodies
- • Required if antibody screen positive
- • Most sensitive method
Electronic/Computer
- • No serologic testing
- • Computer verifies ABO compatibility
- • Requires validated computer system
- • Two ABO typings on record
- • Negative antibody screen required
Major vs Minor Crossmatch
Major Crossmatch
- • Patient SERUM + Donor CELLS
- • Detects patient antibodies vs donor cells
- • REQUIRED before transfusion
- • Most important test
Minor Crossmatch
- • Donor SERUM + Patient CELLS
- • Detects donor antibodies vs patient cells
- • NOT routinely performed
- • Donor screening replaces this
Blood Selection Rules
| Patient Type | Compatible RBC Units | Compatible Plasma |
|---|---|---|
| O | O only | O, A, B, AB |
| A | A, O | A, AB |
| B | B, O | B, AB |
| AB | AB, A, B, O | AB only |
* Universal donor (RBC): O negative | Universal recipient (RBC): AB positive
6. Blood Component Therapy
Blood Component Preparation
From 1 unit whole blood (450 mL ± 45 mL):
- • Packed RBCs: Centrifuge, remove plasma (Hct 65-80%)
- • Fresh Frozen Plasma: Freeze within 8 hours of collection
- • Platelets: From platelet-rich plasma or buffy coat
- • Cryoprecipitate: Thaw FFP at 4°C, collect precipitate
Blood Components Summary
| Component | Storage | Shelf Life | Indications |
|---|---|---|---|
| Packed RBCs | 1-6°C | 35-42 days (additive) | Anemia, blood loss |
| Whole Blood | 1-6°C | 21-35 days | Massive transfusion |
| FFP | ≤-18°C | 1 year | Clotting factor deficiency |
| Platelets | 20-24°C, agitation | 5 days | Thrombocytopenia, bleeding |
| Cryoprecipitate | ≤-18°C | 1 year | Fibrinogen deficiency, vWD |
| Granulocytes | 20-24°C | 24 hours | Severe neutropenia with sepsis |
Special Products
Leukocyte-Reduced
- • Filter removes WBCs (<5 × 10⁶)
- • Prevents febrile reactions
- • Reduces CMV transmission
- • Prevents HLA alloimmunization
Irradiated Products
- • 25 Gy gamma irradiation
- • Prevents TA-GVHD
- • For immunocompromised patients
- • Directed donations from relatives
Washed RBCs
- • Removes plasma proteins
- • For IgA-deficient patients
- • Severe allergic reactions
- • Short shelf life (24 hours)
CMV-Negative
- • Donor tests negative for CMV
- • For CMV-negative transplant patients
- • Leukoreduction is alternative
- • Neonates, pregnant women
Cryoprecipitate Contents
- • Fibrinogen: 150-250 mg per unit
- • Factor VIII: 80-120 IU per unit
- • von Willebrand Factor (vWF)
- • Factor XIII
- • Fibronectin
7. Transfusion Reactions
Acute Hemolytic Transfusion Reaction (AHTR)
Most Serious Reaction!
- • Cause: ABO incompatibility (usually clerical error)
- • Mechanism: Intravascular hemolysis, complement activation
- • Symptoms: Fever, chills, flank pain, hypotension, hemoglobinuria, DIC
- • Lab findings: Positive DAT, hemoglobinemia, elevated bilirubin
- • Treatment: STOP transfusion immediately, maintain renal perfusion, supportive care
Delayed Hemolytic Transfusion Reaction (DHTR)
- • Onset: 3-14 days post-transfusion
- • Cause: Anamnestic response to foreign RBC antigens (Kidd, Rh, Kell)
- • Mechanism: Extravascular hemolysis
- • Symptoms: Mild fever, unexplained drop in hemoglobin, jaundice
- • Lab findings: Positive DAT, new antibody detected, elevated bilirubin
- • Treatment: Usually mild, supportive care
Non-Hemolytic Transfusion Reactions
Febrile Non-Hemolytic (FNHTR)
- • Most common reaction
- • Cytokines from stored WBCs
- • Temperature rise ≥1°C
- • Prevention: Leukoreduction
- • Treatment: Antipyretics
Allergic Reaction
- • Antibodies to plasma proteins
- • Urticaria, hives, itching
- • Usually mild
- • Treatment: Antihistamines
- • May resume transfusion if mild
Anaphylaxis
- • IgA deficiency with anti-IgA
- • Hypotension, bronchospasm
- • Occurs within minutes
- • Treatment: Epinephrine
- • Future: Washed products or IgA-deficient donors
TRALI
- • Transfusion-Related Acute Lung Injury
- • Donor anti-HLA antibodies
- • Within 6 hours of transfusion
- • Non-cardiogenic pulmonary edema
- • Treatment: Supportive, oxygen
Other Transfusion Complications
- • TACO: Transfusion-Associated Circulatory Overload - volume overload, CHF
- • TA-GVHD: Donor T-cells attack recipient tissues - prevented by irradiation
- • Bacterial contamination: Most common in platelets (room temp storage)
- • Iron overload: Chronic transfusion therapy, >20 units
- • Post-transfusion purpura: Severe thrombocytopenia 5-10 days post-transfusion
Transfusion Reaction Workup
- Stop transfusion, keep IV line open
- Clerical check - verify patient and unit identification
- Visual inspection of plasma (hemolysis?)
- Direct Antiglobulin Test (DAT)
- Repeat ABO/Rh on pre and post samples
- Antibody screen
- Urinalysis (hemoglobinuria)
- Bilirubin, LDH, haptoglobin
- Blood culture (if bacterial contamination suspected)
8. Special Topics in Blood Banking
Hemolytic Disease of the Fetus and Newborn (HDFN)
Pathophysiology
- • Maternal IgG antibodies cross placenta
- • Attack fetal RBCs if antigen positive
- • ABO HDFN: Most common, usually mild (Group O mother, A or B baby)
- • Rh HDFN: D-negative mother, D-positive fetus - more severe
- • Other antibodies: c, K, Fy, Jk can cause HDFN
Rh Immune Globulin (RhIg)
- • Purpose: Prevent sensitization of D-negative mothers
- • Dosing: 300 mcg covers 30 mL whole blood (15 mL RBCs)
- • Antepartum: At 28 weeks gestation
- • Postpartum: Within 72 hours if baby is D-positive
- • Other indications: Abortion, amniocentesis, ectopic pregnancy, trauma
- • Rosette test: Screens for large fetomaternal hemorrhage
- • Kleihauer-Betke: Quantifies fetal cells in maternal blood
Direct and Indirect Antiglobulin Test
DAT (Direct Coombs)
- • Tests for in-vivo sensitization
- • Patient RBCs + AHG reagent
- • Detects antibody/complement on RBCs
- • Uses: AIHA, HDFN, HTR, drug-induced
IAT (Indirect Coombs)
- • Tests for in-vitro sensitization
- • Patient serum + reagent RBCs, incubate, add AHG
- • Detects antibody in serum
- • Uses: Antibody screen, crossmatch, weak D
Autoimmune Hemolytic Anemia (AIHA)
| Type | Warm AIHA | Cold Agglutinin Disease | PCH |
|---|---|---|---|
| Antibody | IgG (37°C) | IgM (4°C) | IgG (biphasic) |
| DAT | IgG positive | C3d positive | C3d positive |
| Specificity | Rh-like | Anti-I | Anti-P |
| Hemolysis | Extravascular | Intravascular | Intravascular |
Donor Screening and Testing
Required Testing (Philippines - NVBSP)
- • ABO and Rh typing
- • Antibody screening
- • Hepatitis B surface antigen (HBsAg)
- • Hepatitis C antibody (anti-HCV)
- • HIV-1/2 antibody
- • Syphilis (VDRL/RPR or TPHA)
- • Malaria (in endemic areas)
- • NAT testing (where available)
Quality Control in Blood Banking
- • Reagent antisera: Test with known positive and negative cells daily
- • AHG reagent: Add IgG-coated check cells if negative test
- • Temperature monitoring: Refrigerators, freezers, platelet incubators
- • Centrifuge calibration: Timer, speed verification
- • Component QC: Hematocrit, platelet count, factor assays
- • Sterility testing: Culture of components
- • Proficiency testing: External quality assessment
Key Takeaways
- ✓ABO: Most important blood group system for transfusion safety
- ✓D antigen: Most immunogenic Rh antigen after ABO
- ✓Kidd antibodies notorious for delayed HTR (disappearing antibody)
- ✓Major crossmatch: Patient serum + donor cells (most important)
- ✓AHTR: Usually due to ABO incompatibility (clerical error)
- ✓RhIg prevents D sensitization in D-negative mothers
- ✓Platelets stored at room temp - highest bacterial contamination risk
- ✓DAT detects in-vivo sensitization; IAT detects serum antibodies