Immunohematology Review Notes

Criteria for Whole Blood Donors (AABB)

• Allogeneic:

  • Age: ≥ 16 or as allowed by state law

  • HGB: ≥ 12.5 g/dL

  • HCT: ≥ 38%

  • Temperature: ≤ 37.5°C (99.5°F)

  • Venipuncture site: No infectious skin disease or scars indicative of drug use

• Autologous:

  • Age: As determined by medical director.

  • HGB: ≥ 11.0 g/dL or HCT ≥ 33%. As determined by medical director.

  • Bacteremia: Bacteremia is cause for deferral. As determined by medical director.

  • Autologous Donations: No donations within 72 hr of surgery.

Donor Deferrals (AABB)

• 2 days: Aspirin, if donor is sole source of platelets

• 2 weeks: Measles (rubeola), mumps, polio, typhoid, yellow fever vaccines

• 4 weeks: Rubella, chicken pox (varicella-zoster) vaccine, Pregnancy

• 6 weeks: Whole blood donation

• 8 weeks: Syphilis, Gonorrhea

• 12 months:

  • Mucous membrane exposure to blood

  • Skin penetration with sharp contaminated with blood or body fluids

  • Household or sexual contact with individual with hepatitis

  • Sexual contact with individual with HIV or at high risk

  • Incarceration in correctional facility for >72 consecutive hr

  • Travel to Iraq or area endemic for malaria

  • Recipient of blood, blood components, plasma-derived clotting factor concentrates, or transplant

• 3 years: Malaria, or from an area endemic for malaria

• Permanent:

  • Parenteral drug use

  • Family history of Creutzfeldt-Jakob disease

  • Treatment with pituitary growth hormone of human origin

  • Viral hepatitis after 11th birthday

  • Confirmed positive HBsAg

  • Repeatedly reactive anti-HBc on >1 occasion

  • Repeatedly reactive HTLV on >1 occasion

  • Present or past clinical or laboratory evidence of infection with HIV, HCV, HTLV

  • History of babesiosis or Chagas’ disease

Collection of Whole Blood

• Skin preparation: Aseptic method, e.g., povidone-iodine scrub & prep solution.

• Volume of blood routinely collected: 450 mL ± 10% or 500 mL ± 10%, depending on collection bag.

• Maximum volume: 10.5 mL of blood per kg of donor’s weight, including samples for testing (e.g., 473 mL from 100 lb [45 kg] donor).

• Low-volume collections:

  • 300–404 mL in 450-mL bag or 333–449 mL in 500-mL bag.

  • Labeled “low-volume.”

  • RBCs may be transfused, but other components shouldn’t be prepared because of abnormal anticoagulant-to-plasma ratio.

• Volumes of anticoagulant: 63 mL anticoagulant for 450-mL collection, 70 mL for 500-mL collection.

• Time of collection: Usually <10 min. If >15–20 min, unit may not be suitable for preparation of platelets or plasma.

• Samples for testing: From diversion pouch or by 2nd phlebotomy.

• Storage temperature of unit between collection & processing: 20° –24°C if platelets are to be prepared; otherwise 1° –6°C.

Apheresis

• Explanation: Automated blood collection system that allows removal of 1 or more components from blood & return of remainder to donor.

• Advantages:

  • Allows collection of larger volume of specific components.

  • Can reduce # of donors to which pt is exposed.

• Donor requirements: Vary with procedure.

• Components collected:

  • RBCs (2 units can be collected at same time from donors who are larger & have higher HCT. 16 wk between donations).

  • Platelets: Plateletpheresis. Can collect HLA matched for pts who are refractory to random plts. Can be leukoreduced during collection. Contain ≥ 3 x 10^11 plt.

  • Plasma: Plasmapheresis.

  • Granulocytes: Leukapheresis. Not widely used to date.

  • Stem cells: For bone marrow reconstitution in pt with cancer, leukemia, lymphoma. Autologous or HLA matched.

• Therapeutic uses:

  • Therapeutic plasmapheresis (plasma exchange) used to remove abnormal plasma proteins & replace with crystalloid, albumin, or FFP.

  • Therapeutic cytapheresis used to remove cellular elements, e.g., abnormal # platetets (to ↓ risk of hemorrhage or thrombosis), leukemic WBCs, lymphocytes (to induce immunosuppression).

Donor Testing Required by AABB and/or FDA

• Typing:

  • ABO

  • Rh (including weak D)

• Antibody screen

• Syphilis testing: Antibodies to Treponema pallidum or nontreponemal serological test for syphilis, e.g., RPR

• Hepatitis testing:

  • HBsAg

  • Anti-HBc

  • Anti-HCV

  • HCV RNA (NAT)

• HIV testing:

  • Anti-HIV-1/2

  • HIV-1 RNA (NAT)

• Other infectious disease testing:

  • Anti-HTLV-I/II

  • West Nile virus RNA (NAT)

  • Anti–Tryanosoma cruzi (FDA recommends 1-time donor screening)

• Test to detect bacterial contamination of platelets: Culture of platelets or FDA-approved rapid test (e.g., Pan Genera Detection [PGD] test)

• For autologous donations: complete donor testing required if tf will occur outside collection facility; otherwise only ABO & Rh required.

Anticoagulant/Preservative Solutions

• ACD-A (Acid citrate-dextrose Formula A)

  • RBC Shelf Life: 21 days

  • Comments: Citrate prevents coagulation by chelating $Ca^{2+}$. Dextrose (glucose) supports ATP generation. Used for apheresis.

• CPD (Citrate-phosphate-dextrose)

  • RBC Shelf Life: 21 days

  • Comments: Higher pH preserves 2,3-DPG better, better O2 delivery.

• CP2D (Citrate-phosphate-double-dextrose)

  • RBC Shelf Life: 21 days

  • Comments: Contains 100% more glucose than CPD.

• CPDA-1 (Citrate-phosphate-dextrose with adenine)

  • RBC Shelf Life: 35 days

  • Comments: Adenine increases ADP, which increases synthesis of ATP. Contains more glucose to sustain cells during longer storage.

Additive Solutions

• Purpose: Extend shelf life of RBCs to 42 days.

• Constituents: Glucose for energy, adenine to support ATP levels.

• Procedure: Plasma expressed from WB. 100–110 mL additive transferred from attached satellite bag to RBCs within 72 hr of collection or per manufacturer’s instructions.

• Final hematocrit: 55%–65% (HCT of RBCs without additive 65%–80%). Lower viscosity facilitates tf.

• Examples: Adsol (AS-1), Nutricel (AS-3), Optisol (AS-5)

Open and Closed Systems

• Open system:

  • Explanation: Seal on unit is broken to attach external transfer bag. Exposure to air poses threat of bacterial contamination.

  • Effect on expiration date of component: Components stored at 1° –6°C must be used within 24 hr after system opened; components stored at 20° –24°C within 4 hr.

• Closed system:

  • Explanation: Sterility maintained through use of attached satellite bags or sterile connecting device that welds tubing from 1 bag to another. No exposure to air.

  • Effect on expiration date of component: No change.

Blood Components—RBCs

• RBCs

  • Preparation: Separated from WB by centrifugation or sedimentation any time before expiration date of WB, or collected by apheresis.

  • Storage Temperature: 1° –6°C

  • Shelf Life: 35 days in CPDA-1, 42 days in additive solution

  • Indications: Inadequate tissue oxygenation

  • Other: Should have HCT ≤ 80%; otherwise not enough preservative to support RBCs. 1 unit should ↑ HGB 1 g/dL or HCT 3%. Most commonly used RBC product.

• RBCs adenine, saline added

  • Preparation: Additive solution added to RBCs following removal of most plasma.

  • Storage Temperature: 1° –6°C

  • Shelf Life: 42 days

  • Indications: Same as RBCs

  • Other:

• RBCs frozen

  • Preparation: Frozen in glycerol within 6 days of collection. High glycerol (40%) method most commonly used.

  • Storage Temperature: Frozen in high glycerol: ≤ –65°C. After deglyc (washing in ↓ concentration of saline): 1° –6°C

  • Shelf Life: Frozen: 10 yr. After degylc: 24 hr (unless closed system used)

  • Indications: Same as RBCs

  • Other: Osmolality to monitor glycerol removal. Virtually all plasma, anticoagulant, WBCs, & platelets removed. Safe for IgA- deficient pt. Used to store rare cells.

• Washed RBCs

  • Preparation: RBCs washed with saline.

  • Storage Temperature: 1° –6°C

  • Shelf Life: 24 hr after washing

  • Indications: History of severe allergic rxn (e.g., IgA, other plasma proteins)

  • Other: Not a substitute for leuko- reduced RBCs. About 20% of RBCs lost in process. Must retain 85% of original RBCs.

• RBCs leukocytes reduced

  • Preparation: Filtration or apheresis processing.

  • Storage Temperature: 1° –6°C

  • Shelf Life: Closed system: same as RBCs. Open system: 24 hr

  • Indications: History of febrile rxn

  • Other:

• RBCs irradiated

  • Preparation: Irradiation at 2,500 cGy.

  • Storage Temperature: 1° –6°C

  • Shelf Life: Original outdate or 28 days from irradiation, whichever comes 1st

  • Indications: Immunodeficiency, malignancy, bone marrow transplant, tf with blood from blood relative, intrauterine & neonatal tf

  • Other: For prevention of graft-vs.-host disease. Kills donor T cells.

Blood Components—Plasma and Derivatives

• Fresh frozen plasma (FFP)

  • Preparation: Plasma separated from WB & frozen within 8 hr of collection

  • Storage Temperature: Frozen: ≤ –18°C. After thawing: 1° –6°C

  • Shelf Life: Frozen: 12 mo. After thawing: 24 hr

  • Indications: Deficiency of coag factors

  • Other: Contains all coag factors. Check for evidence of thawing & refreezing.

• Cryoprecipitate

  • Preparation: Prepared by thawing FFP at 1° –6°C, removing plasma, & refreezing within 1 hr

  • Storage Temperature: Frozen: ≤ –18°C. After thawing: RT

  • Shelf Life: Frozen: 12 mo. After thawing: single units 6 hr, pools 6 hr if sterile connecting device used, otherwise 4 hr

  • Indications: Fibrinogen & factor XIII deficiencies

  • Other: Thawed at 30° –37°C or by FDA-approved microwave. Used for hemophilia A & von Willebrand disease only if factor VIII concentrate or recombinant factor preparations not available. Should contain ≥ 80 IU of factor VIII & ≥ 150 mg of fibrinogen.

Blood Components—Platelets

• Platelets

  • Preparation: Centrifugation of WB at RT within 8 hr of collection. 1st soft spin yields platelet- rich plasma. 2nd hard spin separates plt from plasma.

  • Storage Temperature: 20° –24°C

  • Shelf Life: 5 days from collection, with agitation. After pooling, 4 hr

  • Indications: Severe thrombocytopenia or abnormal platelet function

  • Other: 40–70 mL plasma. ≥ 5.5 x 10^10 plt. pH ≥ 6.2. 1 unit should ↑ plt by 5,000–10,000/μL in 75-kg recipient. Shouldn’t be used if visible aggregates present. May contain residual RBCs. Usually pooled (4–6 units).

• Apheresis platelets

  • Preparation: Apheresis.

  • Storage Temperature: 20° –24°C

  • Shelf Life: 5 days with agitation Open system—4 hr. Apheresis—5 days

  • Indications: Same

  • Other: ≥ 3.0 x 10^11 plt. Equivalent to 4–6 units. Exposes recipient to fewer donors.

• Leukocyte-reduced platelets

  • Preparation: WBCs removed by filtration or during apheresis processing.

  • Storage Temperature: 20° –24°C

  • Shelf Life: 5 days from collection

  • Indications: Recurrent febrile rxn & to ↓ risk of CMV transmission or HLA alloimmunization

  • Other:

• Prestorage pooled platelets

  • Preparation: 4–6 ABO- identical plt pooled using closed system.

  • Storage Temperature: 20° –24°C

  • Shelf Life: 5 days from collection

  • Indications: Same

  • Other: Extended outdate overcomes need for tf service to pool just before administration.

Labeling Requirements for Blood and Components

• ISBT 128 bar-code symbology

• Name of product

• Method of preparation (whole blood, apheresis)

• Storage temperature

• Preservatives/anticoagulant

• Number of units in pooled components

• Name, address, registration number, & license number of collection & processing facility

• Expiration date (& time if applicable). When expiration time not indicated, expiration is at midnight.

• Identification number for unit or pool

• Donor category (volunteer, autologous)

• ABO group & Rh type, if applicable.

• Special handling information

• Statements regarding recipient identification, Circular of Information, infectious disease risk, & prescription requirement

• Autologous units: “For Autologous Use Only.” Biohazard label if any infectious disease markers are pos or “Donor Untested” if testing not performed.

Leukocyte Reduction (Leukoreduction)

• Purpose: To ↓WBCs to ↓ febrile nonhemolytic transfusion reactions, transmission of CMV, & HLA alloimmunization

• Methods:

  • <5 × 10^6

  • By apheresis processing

  • By filtration during manufacture of components or after storage. Prestorage leukocyte reduction is most effective. WBCs removed before they release cytokines

  • Use of filter during infusion

RBC Storage Lesion

• Increased:

  • Lactic acid

  • Plasma K+

  • Plasma hemoglobin

  • Microaggregates

• Decreased:

  • ATP

  • 2,3-DPG

  • pH

  • Glucose

  • Viable cells

  • Labile coagulation factors

• Other Changes:

  • Shift to left of O2dissociation curve (↑ HGB/O2 affinity, ↓ O2 delivery to tissues)

Factors That Affect Agglutination in Tube Testing

• Sensitization stage: Attachment of ab to ag

  • Temperature: Clinically significant abs react best at 37°C.

  • pH: Most abs react at pH 5.5–8.5.

  • Ionic strength: Reducing ionic strength of medium facilitates interaction of ab with ag (e.g., low ionic strength solution [LISS]).

  • Ag/ab ratio: Too much ab can cause prozone (false neg). Optimum serum-to-cell ratio is 80:1. Usually 2 drops serum to 1 drop of 2%–5% RBCs.

  • Incubation time: Follow manufacturer’s directions. Depends on medium. Usually 10–30 min. Follow manufacturer’s directions.

• Agglutination stage: Formation of ag-ab bridges between RBCs

  • Type of ab molecule: IgM is larger, can span distance between RBCs more easily.

  • Density of ags & location on RBC surface: Affects ease of attachment of abs.

  • Zeta potential: Difference in charge between neg-charged RBC surface & cloud of pos ions that surround RBCs. Reducing zeta potential allows RBCs to move closer together (e.g., enzyme treatment of test cells).

IgG Versus IgM

• IgG:

  • Structure: Monomer

  • Number of ag-binding sites: 2

  • Type of ab: Immune

  • Optimum temperature of reactivity: 37°C

  • Reacts in saline? No

  • Reacts best by IAT? Yes

  • Complement fixation: Moderate

  • Causes transfusion reactions? Yes

  • Crosses placenta? Yes

  • Causes hemolytic disease of the newborn/fetus? Yes

  • Destroyed by sulfhydryl compounds (dithiothreitol [DTT], 2-mercaptoethanol [2-ME])? No

• IgM:

  • Structure: Pentamer

  • Number of ag-binding sites: 10

  • Type of ab: Naturally occurring

  • Optimum temperature of reactivity: 25°C or lower

  • Reacts in saline? Yes

  • Reacts best by IAT? No

  • Complement fixation: Strong

  • Causes transfusion reactions? Not usually, except ABO

  • Crosses placenta? No

  • Causes hemolytic disease of the newborn/fetus? No

  • Destroyed by sulfhydryl compounds (dithiothreitol [DTT], 2-mercaptoethanol [2-ME])? Yes

Primary Versus Secondary Response

• Primary:

  • Stimulus: 1st exposure to ag

  • Lag phase: Days to months

  • Type of antibody: IgM at first. May switch to IgG after 2–3 weeks

  • Titer: Rises slowly, peaks, then declines

• Secondary (Anamnestic):

  • Stimulus: Subsequent exposure to ag

  • Lag phase: Hours

  • Type of antibody: IgG (isotype switching)

  • Titer: Rises faster & higher, stays elevated longer

Comparison of Tube, Gel, and Solid-Phase Testing

• Tube Testing:

  • Reaction container: Glass test tubes

  • Principle: Abs attach to corresponding ags on RBCs, forming bridges between cells. RBCs agglutinate.

  • Pos rxn: Agglutinated RBCs or hemolysis

  • Neg rxn: No agglutinated RBCs or hemolysis

  • Adaptable to automation? No

  • Advantages: Low cost
    *Note: A cell button in tube testing is a solid agglutinate—4+ rxn. A cell button in solid phase is unagglutinated cells—neg rxn.

• Gel Testing:

  • Reaction container: Plastic microtube containing dextran- acrylamide gel.

  • Principle: Ag-ab rxn results in agglutinated RBCs. Gel acts as sieve. Large agglutinates can’t pass through, remain at top. Small agglutinates pass into gel. Unagglutinated cells go to bottom.

  • Pos rxn: Agglutinated RBCs suspended in gel. Position indicates strength of rxn. Larger agglutinates at top.

  • Neg rxn: Button of unagglutinated RBCs in bottom of microtube.

  • Adaptable to automation? Yes

  • Advantages: Standardized. More sensitive than tube testing. Rxn stable 2–3 days; can be captured electronically. AHG tests don’t require washing or control cells.

• Solid-Phase Testing:

  • Reaction container: Microplate with RBC membranes bound to surface of wells.

  • Principle: Abs in sample attach to RBC ags on surface of wells. After incubation, unbound ab removed by washing. Anti-IgG-labeled indicator RBCs added. Attach to abs bound to reagent RBC ags during centrifugation.

  • Pos rxn: Indicator RBCs adhere diffusely to surface of well.

  • Neg rxn: No adherence of RBCs. Button of RBCs in bottom of well.

  • Adaptable to automation? Yes

  • Advantages: Standardized. More sensitive than tube testing. Rxn stable 2 days.

Grading Reactions—Tube Versus Gel

• Tube

  • 4+: One solid agglutinate

  • 3+: Several large agglutinates

  • 2+: Medium-sized agglutinates, clear background

  • 1+: Small agglutinates, turbid background

  • Mixed field: Some agglutinated RBCs in sea of free RBCs

  • Neg: No agglutinates
    *In tube testing, hemolysis is also a pos rxn.

• Gel

  • 4+: Solid band of agglutinated RBCs at top

  • 3+: Band of agglutinated RBCs near top with a few staggered below

  • 2+: Agglutinates throughout

  • 1+: Agglutinates predominantly in lower half of column with some RBCs at bottom

  • Mixed field: Layer of agglutinated RBCs at top & pellet of unagglutinated RBCs at bottom

  • Neg: Well-defined pellet of unagglutinated RBCs at bottom

ABO System

• O

  • Antigen(s) on RBC: Neither

  • Antibody(s) in Serum: Anti-A, Anti-B

• A

  • Antigen(s) on RBC: A

  • Antibody(s) in Serum: Anti-B

• B

  • Antigen(s) on RBC: B

  • Antibody(s) in Serum: Anti-A

• AB

  • Antigen(s) on RBC: A & B

  • Antibody(s) in Serum: Neither anti-A nor anti-B

ABO Genotypes and Phenotypes

• A

  • Genotype(s): AA, AO

• B

  • Genotype(s): BB, BO

• AB

  • Genotype(s): AB

• O

  • Genotype(s): OO
    *Example Punnett Square for predicting ABO Type:

• Mother’s genotype: A

• Father’s genotype: B, AB, BO or O

Frequency of ABO Types

• O

  • Whites (%): 45

  • Blacks (%): 49

  • Hispanics (%): 57

  • Asians (%): 40

• A

  • Whites (%): 40

  • Blacks (%): 27

  • Hispanics (%): 31

  • Asians (%): 27

• B

  • Whites (%): 11

  • Blacks (%): 19

  • Hispanics (%): 10

  • Asians (%): 25

• AB

  • Whites (%): 4

  • Blacks (%): 4

  • Hispanics (%): 2

  • Asians (%): 7

ABO Typing

• Forward Grouping: Uses known antisera (Anti-A, Anti-B) to identify antigens on patient RBCs.

• Reverse Grouping: Uses known A and B cells to identify antibodies in patient serum.

• Use of anti-A,B & A2 cells optional.

ABO Discrepancies

• Discrepancies between forward and reverse grouping results must be resolved before transfusion.

*Other explanations may be possible.

Rh Antigens

• Fisher-Race: D, C, E, c, e

• Weiner: Rh0, rh’, rh”, hr’, hr”

• Rosenfield: Rh1, Rh2, Rh3, Rh4, Rh5

Breaking the Rh Code

• r before h: “big” (C)

• h before r: “little” (c)

• R: presence of D

• r: absence of D

• 1 or ’: C (if no 1 or ’, then c)

• 2 or ”: E (if no 2 or ”, then e)

• 0: c + e

• Z or y: C + E

• Notes:

  • Italics & superscripts denote genes, e.g., Rh’.

  • Standard type is used for agglutinogens (antigens).

  • Subscripts are used with R, e.g., Rho.

  • Superscripts are used with r, e.g., rh’.

  • The h’s are left out in shorthand notation, e.g., Rho = Ro.

Frequency of Rh Antigens

• D

  • Whites (%): 85

  • Blacks (%): 92

• C

  • Whites (%): 68

  • Blacks (%): 27

• E

  • Whites (%): 29

  • Blacks (%): 22

• c

  • Whites (%): 80

  • Blacks (%): 96

• e

  • Whites (%): 98

  • Blacks (%): 98
    *Note which antigens are the most and least common.

Frequency of Rh Genes

• Note which genes are the most and least common and which show marked racial differences.

Rh Typing Sera

• High-Protein Anti-D

  • Source: Prepared from pools of human sera (immunized Rh-neg individuals).

  • Control: Same ingredients as reagent, except no anti-D. Should be purchased from same manufacturer as anti-D.

  • Comments: More false-pos than low-protein reagents, e.g., RBCs with pos DAT.

• Low-Protein Anti-D

  • Source: Mixture of monoclonal IgM & monoclonal or polyclonal IgG.

  • Control: Any neg typing rxn serves as control, e.g., neg rxn with anti-A or anti-B. When RBCs react with all antisera (i.e., AB pos), run control recommended by manufacturer. (Usually pt RBCs with autologous serum or 6% or 8% albumin.)

  • Comments: Most widely used. Lower rate of false pos with Ig-coated RBCs.

Interpretation of Rh Typing

*Test for weak D if donor or infant of mother being evaluated for RhIG.

Causes of False Rh Typing Results

Weak D Testing and Interpretation

• When performed: When anti-D & Rh control are neg in Rh typing of donor or infant of mother being evaluated for RhIG.

• Reagent: Not all anti-D reagents are appropriate for use. Refer to manufacturer’s package insert.

• Method: Incubate Rh typing tubes at 37°C for 15–60 min & carry through AHG.

*RBCs with pos DAT will react in any IAT test.

Selection of Rh Type for Transfusion