RBC Preservation, Antigen, Antibody

RBC Preservation:

• Goal:

• Maintain RBC viability during storage

→ >75% of transfused RBCs shouyld remain viable in bloodstream for 24 hrs

→ free hemoglobin should be less than 1% of total hemoglobin

Temperature requirement:

• _ → storage temperature of liquid whole blood/RBC units

• _ → shipping/transport temeperature of liquid whole blood / RBC units

• 1 to 6 degrees celcius → storage temperature of liquid whole blood/RBC units

• 1 to 10 degrees celcius → shipping/transport temeperature of liquid whole blood / RBC units

RBC Storage Lesion

• HIGH (3)

• LOW (3)

• HIGH (3)

→plasma potassium

→plasma hemoglobin

→lactic acid

• LOW (3)

→ATP and glucose

→2,3-DPG

→pH

Approved Preservative Solutions

• has lower pH; loss of 2,3-DPG @ 1st week

• 21 days

Acid Citrate Dextrose

Approved Preservative Solutions

• phosphates preserves 2,3-DPG

• 21 days

Citrate Phosphate Dextrose

Approved Preservative Solutions

• Adenine maintains ATP levels

• 35 days

Citrate-phosphate-dextrose-adenine

Approved Preservative Solutions

• 100% more glucose than CPD

• 21 days

Citrate phosphate double dextrose

Additive Solution

• Shelf-life:

• Process:

• Content (4):

• Example (4):

• Shelf-life: extension up to 42 days

• Process: added to RBCs within the first 72 hours of storage

• Content (4):

→saline

→adenine

→glucose

→mannitol

• Example (4):

→Adsol / AS-1

→Nutricel / AS-3

→Optisol / AS-5

→SOLX / AS-7

Additive Solution

• Shelf-life:

• Process:

• Content (4):

• Example (1):

• Shelf-life: extension upto 24 hours

• Process: can be used up to 3 days after expiration of red cells

• Content (4):

→phosphate

→inosine

→pyruvate

→adenine

• Example (1):

→rejuvesol (FDA-approved rejuvenation solution in US)

Red Cell Freezing

• Shelf-life:

• Process:

• Example:

• Deglycerolization is done prior transfusion: ( Washing: _)

• Shelf-life: 10 years (-65 degrees celcius)

• Process: includes addition of a cryoprotective agent to RBCs that are <6 days old

• Example: glycerol (40% w/v or 20% w/v)

• Deglycerolization is done prior transfusion: ( Washing: 12% NaCl, 1.6% NaCl, 0.9% NaCl with 0.2% dextrose)

RBC Substitutes

• carry and transfer oxygen in the absence of intact RBCs

• Example: _

• Advantage: abundant supply, long shelf life ( 1 to 3 years), no blood typing / crossmatching required

• perfluorochemicals and hemoglobin-based oxygen carriers

Genetics in Bloodbank

• Humans have a total of 23 pairs of chromosomes

→22 pairs are autosomes, 1 pair of sex chromosomes

• Most blood group genes are inherited as _ and are on _ chromosomes

• _ - is the complement of DNA that is inherited (actual genes)

→homozygous - presence of two identical chromosomes

→heterozygous - different alleles present at a particular locus

• _ - observable expression of the genotype, description of which antigens are present

• Humans have a total of 23 pairs of chromosomes

→22 pairs are autosomes, 1 pair of sex chromosomes

• Most blood group genes are inherited as CODOMINANT and are on AUTOSOMAL chromosomes

• GENOTYPE - is the complement of DNA that is inherited (actual genes)

→homozygous - presence of two identical chromosomes

→heterozygous - different alleles present at a particular locus

• PHENOTYPE - observable expression of the genotype, description of which antigens are present

All blood group antigens are assigned to:

_ - inheritance is controlled by a gene or two closely-linked genes

_- demonstrates a serologic relationship but no independent inheritance

_ - observed in >90% of random population

_ - observed in <1% of random population

SYSTEM - inheritance is controlled by a gene or two closely-linked genes

COLLECTION (200) - demonstrates a serologic relationship but no independent inheritance

HIGH-INCIDENCE (901) - observed in >90% of random population

LOW-INCIDENCE (700) - observed in <1% of random population

ISBT No. and Blood Group System

• 001

• 002

• 003

• 004

• 005

• 001 - ABO

• 002 - MNS

• 003 - P

• 004 - Rh

• 005 - Lutheran

ISBT No. and Blood Group System

• 006

• 007

• 008

• 009

• 010

• 006 - Kell

• 007 - Lewis

• 008 - Duffu

• 009 - Kidd

• 010 - Diego

ISBT No. and Blood Group System

• 011

• 012

• 013

• 014

• 015

• 011 - Yt

• 012 - Xg

• 013 - Scianna

• 014 - Dombrock

• 015 - Colton

ISBT No. and Blood Group System

• 016

• 017

• 018

• 019

• 020

• 016 - Landsteiner-Wiener

• 017 - Chido/Rodgers

• 018 - H

• 019 - Kx

• 020 - Gerbich

ISBT No. and Blood Group System

• 021

• 022

• 023

• 024

• 025

• 021 - Cromer

• 022 - Knops

• 023 - Indian

• 024 - Ok

• 025 - Raph

ISBT No. and Blood Group System

• 026

• 027

• 028

• 029

• 030

• 026 - John Milton Hagen

• 027 - I

• 028 - Globoside

• 029 - Gill

• 030 - Rh-associated glycoprotein

ISBT No. and Blood Group System

• 031

• 032

• 033

• 034

• 035

• 031 - FORS

• 032 - JR

• 033 - LAN

• 034 - VEL

• 035 - CD59

ISBT No. and Blood Group System

• 036

• 037

• 038

• 039

• 040

• 041

• 042

• 043

• 036 - Augustine

• 037 - Kanno

• 038 - Sid

• 039 - CTL2

• 040 - PEL

• 041 - MAM

• 042 - EMM

• 043 - ABCC1

ISBT No. and Blood Group System

• 044

• 045

• 046

• 047

• 048

• 044 - ER

• 045 - CD36

• 046 - ATPIIC

• 047 - MAL

• 048 - PIG2

Naturally Occuring

• Antibody production occurs:

• Antibody type:

• Optimal tempt. reactivity:

• Examples:

• Antibody production occurs: even without exposure to RBC antigen

• Antibody type: IgM

• Optimal tempt. reactivity: <22 degrees celcius

• Examples: ABO, H, MN, Ii, Lewis, P antibodies

Immune Antibodies

• Antibody production occurs:

• Antibody type:

• Optimal tempt. reactivity:

• Examples:

• Antibody production occurs: after exposure to RBC antigen

• Antibody type: IgG

• Optimal tempt. reactivity: 37 degrees celcius

• Examples: Rh, Kell, Kidd, Duffy, Ss antibodies

• Any antibody that reacts at or near body temperature (30 degrees to 37 degrees) regardless pf specificity

• Potentially cause HTR or HDN

Clinically-Significant Antibodies

_ - produced after exposure to non-self antigens (RBC antigens after transfusion)

_ - produced in response to self antigens (require special techniques to be identified)

ALLOANTIBODIES - produced after exposure to non-self antigens (RBC antigens after transfusion)

AUTOANTIBODIES - produced in response to self antigens (require special techniques to be identified)

Positive sign of antigen-antibody reaction (2)

• hemagglutination

• hemolysis

Blood Bank Methods: TRADITIONAL

• Reaction chamber:

• Reaction pattern:

• Reaction matrix:

• Special equipment:

• Washing required:

• Stable reactions:

• Reaction readings:

• Reaction chamber: Tube

• Reaction pattern: Hemagglutination

• Reaction matrix: None

• Special equipment: No

• Washing required: Yes

• Stable reactions: No

• Reaction readings: 0 to 4+ mf

Blood Bank Methods: GEL

• Reaction chamber:

• Reaction pattern:

• Reaction matrix:

• Special equipment:

• Washing required:

• Stable reactions:

• Reaction readings:

• Reaction chamber: Microtube card

• Reaction pattern: Hemmaglutination

• Reaction matrix: Dextran acrylamide gel

• Special equipment: Yes

• Washing required: No

• Stable reactions:Yea ( 2-3 days)

• Reaction readings: 0 to 4+, mf

Blood Bank Methods: SOLID PHASE

• Reaction chamber:

• Reaction pattern:

• Reaction matrix:

• Special equipment:

• Washing required:

• Stable reactions:

• Reaction readings:

• Reaction chamber: Microplate wells

• Reaction pattern: Immune adherence

• Reaction matrix: Modified polystyrene wells

• Special equipment: Yes

• Washing required: Yes

• Stable reactions: Yes (2 days)

• Reaction readings: Strong pos, Weal pos, Neg

Grading of Agglutination Reactions

• Tube Method

→4+

→3+

→2+

→1+

→0

→MF

→4+ - one solid agglutinate, clear supernatant

→3+ - several large agglutinates, clear supernatant

→2+ - medium-sized agglutinates, clear background

→1+ - small agglutinates, turbid background

→0 - No agglutinantion

→MF - presence of minor population of agglutinated cells superimposed on a negative bg

Grading of Agglutination Reactions

• Gel Method

→4+

→3+

→2+

→1+

→0+

→MF

→4+ - solid band of RBC agglutinates at the top of gel column, no RBCs are visible at the bottom

→3+ - predominant amount of RBC agglutinates near the top / upper half of gel column, few agglutinates below

→2+ - RBC agglutinates that are dispersed throughout gel column, few aggutinates at bottom

→1+ - RBC agglutinates that are predominantly in the lower half of gel column, some RBCs at bottom

→ 0+ = RBCs form a well-delineated pellet at the bottom of microtibe, gel above RBC pellet is clear

→MF - presence of layer of agglutinated RBCs at the top of the gel accompanied by a pellet of unaaglutinated cells at the bottoms of microtubes

Other Grading Observed on Tube Method:

• H

• + or w

• +m

• R

• H - hemolysis

• + or w - few small aggregates with many unagglutinated cells

• +m - aggregates visible only under microscope

• R - rouleaux

Column Agglutination Technology / Gel Test Method

• developed by _

• uses _ that contains pre-dispensed reagents

• a gel card measures approximatel;y 5 × 7 cm and has 6 - 8 columns

• developed by Dr. Yves Lapierre

• uses dextran-acrylamide gel that contains pre-dispensed reagents

• a gel card measures approximatel;y 5 × 7 cm and has 6 - 8 columns

Column Agglutination Technology / Gel Test Method

• Advantages:

• standardization

• stability of results

• decreased sample volume needed

• no need for washing of RBCs before AHG phase

• no need to use check cells to validate AHG results

• enhanced sensitivity and specificity

Factors that Influence Agglutination Reactions

• _ - simplest and most common technique to enhance agglutination

High Speed Centrifugation

Factors that Influence Agglutination Reactions

• Effect of antigen-antibody ratio

→_ should be obtained

→prozone (_ excess) and postzone (_ excess) will lead to _

→remedies:

POSTZONE: _

PROZONE: _

→_ should be obtained

→prozone (antibody excess) and postzone (antigen excess) will lead to FALSE NEGATIVE REACTIONS

→remedies:

POSTZONE: serum dilution technique

PROZONE: increase serum to cell ratio

Factors that Influence Agglutination Reactions

• Dosage

→_ alleles - presence of less antigen on red cells = weak agglutination upon testing

→ _ alleles - presence of more antigen on red cells = stroger agglutination upon testing

→Example of blood groups with dosage effect: _

→HETEROZYGOUS alleles - presence of less antigen on red cells = weak agglutination upon testing

→ HOMOZYGOUS alleles - presence of more antigen on red cells = stroger agglutination upon testing

→Example of blood groups with dosage effect: Rh, MNS, Kidd, Duffy, Lutheran

Factors that Influence Agglutination Reactions

• ph

→optimal ph: _

6.5 to 7.5

Factors that Influence Agglutination Reactions

• Effect of temperature

→ _ reacts best at cold tempt

→ reacts best at warm tempt

→ IgM reacts best at cold tempt

→ IgG reacts best at warm tempt

Factors that Influence Agglutination Reactions

• Effect of Enhancement Media and Potentiators

→primarily used to detect _ antibodies

→aimed at reducing _ of cell membrane

→primarily used to detect IgG antibodies

→aimed at reducing zeta potential of cell membrane

expression of the difference in electrostatic potential at the surface of red cells and the ionic cloud of positive cations that are attracted to the negative charges on the surface

zeta potential

Enhancement Media / Potentiators

1) _

• incrases dielectric constant which reduces the zeta potential

• examples:

→_ - causes agglutination by adjusting zeta potential of red cells; incubation period: 37 degrees celcius for 10 to 15 minutes

→_ - removes water from test system thereby concentrating any antigens present

-causes non-specific aggregation of cells, incubation: 37 for 10 to 30 mins

-centrifugation after incubation not performed

-more effective than albumin, polybrene, LISS

→Others: _

1) PROTEIN MEDIA

• incrases dielectric constant which reduces the zeta potential

• examples:

→ 22% albumin - causes agglutination by adjusting zeta potential of red cells; incubation period: 37 degrees celcius for 10 to 15 minutes

→Polyethylene glycol (PEG) - removes water from test system thereby concentrating any antigens present

-causes non-specific aggregation of cells, incubation: 37 for 10 to 30 mins

-centrifugation after incubation not performed

-more effective than albumin, polybrene, LISS

→Others: polybrene, polyvinyllpyrrolidone, protamine

Enhancement Media / Potentiators

2) _

• decresed the ionic strength of a reaction medium and so reduces the zeta potential

• increase the attraction between positive charge antibody and negatively charged red cells

• contains 0.2% NaCl and often used because of increased rate of antibody uptake during sensitization

• incubation at 37 degrees for _

2) Low Ionic Strength Solution (LISS)

• decresed the ionic strength of a reaction medium and so reduces the zeta potential

• increase the attraction between positive charge antibody and negatively charged red cells

• contains 0.2% NaCl and often used because of increased rate of antibody uptake during sensitization

• incubation at 37 degrees for 10 - 15 minutes

Enhancement Media / Potentiators

3) ENZYMES

• Ex: _

• Effects:

→enhances reactivity to _

→destroys reactivity to _

3) ENZYMES

• Ex: Ficin (fig plant), Papain (papaya), trypsin (pig stomach), bromelin (pineapple)

• Effects:

→enhances reactivity to ABO, RH, Kidd, Lewis, I, P

→destroys reactivity to MNS, Duffy, Chido-Rodgers, Xga

Enhancement Media / Potentiators

4) _

• crosslinks sensitized cells, resulting in visible agglutination

• designed to detect cellscoated with antibody or complement

• dyes present: _

4) ANTI-HUMAN GLOBULIN

• crosslinks sensitized cells, resulting in visible agglutination

• designed to detect cellscoated with antibody or complement

• dyes present: patent blue and tartrazine

REACTION PHASE

1) Immediate Spin

• detects:

• process:

• visible result:

• detects: IgM

• process: test mixture is centrifuged at room tempt

• visible result: hemagglutination

REACTION PHASE

1) 37 degrees incubation

• detects:

• process:

• visible result:

• detects: IgG

• process: sensitization of red cells occur, complement may be bound during reactivity

• visible result: hemolysis

REACTION PHASE

1) Antiglobulin test

• detects:

• process:

• visible result:

• detects: IgG

• process: AHG has specificity for the Fc portion of IgG acting as a bridge and cross linking red cells

• visible result: hemagglutination

ANTIGLOBULIN TEST

• also known as _

• based on the principle that _

• primarily detect _

• also known as COOMB's TEST

• based on the principle that AHG obtained from immunized non-human species can bind to human globulins in free state or attached to RBCs

• primarily detect IgG or complement-sensitized RBCs

Types of AHG reagents

• _ - contains both anti-IgG and anti-C3d

• _ - contains either anti-IgG or anti-C3d only

• _ - pool of heterogenous anti-IgG from many immunized rabbits

• _ - capable of detecting many different IgG antibodies

• POLYSPECIFIC - contains both anti-IgG and anti-C3d

• MONOSPECIFIC - contains either anti-IgG or anti-C3d only

• POLYCLONAL - pool of heterogenous anti-IgG from many immunized rabbits

• MONOCLONAL - capable of detecting many different IgG antibodies

Types of Antiglobulin Test

→Direct Antiglobulin Test

• Purpose:

• Specimen:

• Procedure:

• 37C incubation:

• Validation:

• Clinical conditions/applications:

• Purpose: detects bound antibodies to RBCs, in vivo sensitization of RBCs

• Specimen: patient's washed RBCs

• Procedure: 1 drop of 2 - 5% RCS, wash 3x with NSS, 2 drops of AHG, centrifuge and observe for agglutination

• 37C incubation: No

• Validation: check cells, coomb's control

• Clinical conditions/applications: HTR, HDFN, AIHA, drug-induced immune hemolytic anemia

Types of Antiglobulin Test

→Indirect Antiglobulin Test

• Purpose:

• Specimen:

• Procedure:

• 37C incubation:

• Validation:

• Clinical conditions/applications:

• Purpose: detects serum antibodies / in vitro sensitization of RBCs

• Specimen: patient's serum + reagent red cells

• Procedure: 2 drops serum + 1 drop red cells and centrifuge, incubate at 37 and centrifuge, wash 3x + 2 drops of AHG and centrifuge

• 37C incubation: Yes

• Validation: Check cells / Coomb's control

• Clinical conditions/applications: crossmatching, antibody screening, antibody identification, antibody titration, red cell phenotyping

Check Cells / Coomb's Control

• composed of type _ RBCs coated with IgG antibodies

• only added to _ tubes for validation

• _ - test is valid

• _ - invalid, repeat test from the start

• composed of type O RBCs coated with IgG antibodies

• only added to NEGATIVE tubes for validation

• AGGLUTINATION - test is valid

• NO AGGLUTINATION - invalid, repeat test from the start

DAT Panel Reactions

• positive results are monitored by DAT panel using _ to detct specific type of antibody sensitizing the cell

• _ control is included and serves to detect spontaneous agglutination

• positive results are monitored by DAT panel using MONOSPECIFIC anti-IgG and anti-C3d to detct specific type of antibody sensitizing the cell

• SALINE control is included and serves to detect spontaneous agglutination

Factors that affect Antiglobulin Test

1) Ratio of Serum to Cells

• ratio should be _

40:1 or 2 drops of serum and 1 drop of 5% RCS

Factors that affect Antiglobulin Test

2) Reaction Medium

• _ - allow antibody-coated cells to come into closer contact with each other

• _ - enhance antibody uptake and decrease incubation time

• _ - remove water and concentrates antibody

• ALBUMIN- allow antibody-coated cells to come into closer contact with each other

• LISS- enhance antibody uptake and decrease incubation time

• POLYETHYLENE GLYCOL - remove water and concentrates antibody

Factors that affect Antiglobulin Test

3) Temperature

• IgG activity and complement activation optimum at _

37C

Factors that affect Antiglobulin Test

4) Incubation Time

• using 22% albumin: _

• using LISS: _

• extended incubation = decreased sensitivity

• using 22% albumin: 15 to 60 minutes

• using LISS: 10 to 15 minutes

Factors that affect Antiglobulin Test

5) Washing of Cell

• washed RBC minimum of 3 times before addition of AHG

• purpose is to removed _

• inadequate washing will result to false _ because of neutralization

• should be performed in as short time possible to minimize elution of low-affinity Abs

• washed RBC minimum of 3 times before addition of AHG

• purpose is to removed UNBOUND/FREE ANTIBODIES

• inadequate washing will result to false NEGATIVE because of neutralization

• should be performed in as short time possible to minimize elution of low-affinity Abs

Factors that affect Antiglobulin Test

6) Saline for Washing

• should have a pH of _

• saline stored for long periods in plastic container = _

• should have a pH of 7.2 - 7.4

• saline stored for long periods in plastic container = DECREASE PH

Factors that affect Antiglobulin Test

7) Addition of AHG

• add AHG immediately after washing

8)Centrifugation for reading

• recommended for AHG:_

1000 RCF for 20 sec

Causes of False Positive Results in AHG

• clotted specimen, presence of fibrin

• bacterial contamination of cells

• metal contamination of saline

• dirty glassware

• overcentrifugation

• overreading

• contaminated AHG

Causes of False Negative Results in AHG

• Inadequate washing of cells

• Inadequate incubation conditions

• Old serum samples

• Serum/AHG not added

• Undercentrifugation

• Cell suspension roo weak/too heavy

• Non-reactive AHG