E. Ann Steiner, MT(ASCP)SBB Blood Bank Technical ... · Blood Bank Technical Specialist Ortho Clinical Diagnostics. ... Positive crossmatch Positive inert control, IAT control, or

E. Ann Steiner, MT(ASCP)SBB

Blood Bank Technical Specialist

Ortho Clinical Diagnostics

Why Do We Care?� Considering change? Considering automation?

� To better understand current methodology, for

use in troubleshooting, problem prevention

� Evaluation of current alternative method(s)

� Facilitate antibody identification

Serological Results That Trigger

an ABID study

�Certain parts of routine pretransfusion testing

�Certain parts of optional steps in

pretransfusion testing

�The results of diagnostic, or ‘special’, testing

Serological Results That Trigger

an ABID study

�Apparently ‘extra’ reaction in ABO reverse

�Positive antibody screen

�Positive crossmatch

�Positive inert control, IAT control, or DAT

�The results of diagnostic, or ‘special’, testing

Pretransfusion Testing�Test suspension of patient’s red blood cells

(RBC’s) with anti-A, -B, and -D

aka, the ABO/Rh forward group

�Test plasma/serum against known A1 and B

reagent RBC’s

aka, the ABO reverse group

�Test plasma (or serum) against Group O

reagent RBC’s of known phenotype

aka, the antibody screen or detection test

Pretransfusion Testing�Test suspension of patient’s red blood cells

(RBC’s) with anti-A, -B, and -D

aka, the ABO/Rh forward group

�Test plasma/serum against known A1 and B

reagent RBC’s

aka, the ABO reverse group

�Test plasma (or serum) against Group O

reagent RBC’s of known phenotype

aka, the antibody screen or detection test

Optional Serological Steps �Test suspension of patient’s RBC’s with:

�Anti-A1, -A,B, second –D

� Inert control (reagent, autologous plasma)

�Test plasma/serum against known A2 RBC’s

�Run a 3 or 4-cell screen (rather than 2-cell)

� IAT autocontrol or Direct Antiglobulin Test (DAT)

�Perform a crossmatch

�For major ABO confirmation only

�For the detection of IAT reactive antibodies

Optional Serological Steps �Test suspension of patient’s RBC’s with:

�Anti-A1, -A,B, second –D

� Inert control (reagent, autologous plasma)

�Test plasma/serum against known A2 RBC’s

�Run a 3 or 4-cell screen (rather than 2-cell)

� IAT autocontrol or Direct Antiglobulin Test (DAT)

�Perform a crossmatch

�For major ABO confirmation only

�For the detection of IAT reactive antibodies

‘Special’ Testing

� Studies to aid in diagnosis such as:

�Direct Antiglogulin Tests (DATs), with

Polyspecific and/or Monospecific AHG

�Preparation and testing of an eluate, with or

without the presence of a positive DAT

�Use of Polyspecific AHG & fresh serum in IAT

�Other special methods and modified testing

parameters

Diagnostic Studies

�Differential diagnostic investigations such as:

�Transfusion (Tx) Reaction

�Hemolytic Disease of the Fetus/Newborn

�Autoimmune Hemolytic Anemia

�Drug-related Hemolysis

�Graft vs host, other complications of solid

organ or stem cell/marrow transplantation

History of Antibody Detection�Review of antibodies:

� Identified subsequent to the detection of an

incompatible crossmatch (not attributed to

ABO incompatibility)

�Associated with a hemolytic tx reaction

� It was determined that the corresponding

antigens for >95% of these antibodies could be

located on RBC’s from 2-3 select individuals

What Really Is This Test Called

ANTIBODY DETECTION?�Historically – developed in the 1950’s to

lessen the incidence of antibodies detected at

time of crossmatching (XM) and therefore too

close to time of need for transfusion

�Recent past – simultaneous to time of XM,

intended to be, in some instances, more

sensitive and more predictable in antigenic

make-up, e.g., double dose expression of

certain antigens

What Really Is This Test Called

ANTIBODY DETECTION?�Current definition and use

�Detect the majority of antibodies likely to

cause significantly shortened RBC survival

�Done as replacement to crossmatch (with

certain defined exceptions)

�Minimum antigen make up defined by CFR

�Common practice to select RBCs whose

antigenic make up enhance sensitivity

Variables in Antibody Screens �Number of reagent RBC’s (for pretransfusion

patients, can NOT be pooled cells)

�Antigenic make up of these reagent RBC’s

�Methodology/enhancement

�Variables between and within a method: � Low Ionic Strength saline (LISS), Polyethylene

Glycol (PEG), Bovine Albumin, other

� Time, temperature

� Tube, solid-phase, column

FDA 21 CFR Requirements� For Reagent RBC’s for antibody detection:

Antigens that must be present

� For compatibility testing

�Time of sample collection relative to tx

� <3 days if recipient has been transfused or

pregnant in preceding 3 months

�Type of testing

� Capable of demonstrating incompatibility

AABB Standards�Method to detect clinically significant

antibodies:

�Defined as capable of producing a

significant adverse reaction

�Must include 37°C incubation and include

an antiglobulin test

�Reagent RBC’s must not be pooled

Antigens Required�D, C, c, E, and e

�K, k

� Fya, Fyb

� Jka, Jkb

� Lea, Leb

�M, N, S, s

�P1

Dichotomy Abounds�Want to find “clinically significant” and yet:

�Antigens are missing whose antibodies are

known to cause significant RBC destruction

� Doa, Kpa, Lan, (and dozens more)

�Antigens are present whose antibodies are

not generally associated with significant

RBC destruction

� M, N, Lea, Leb, P1 (and a few dozen more)

Dichotomy Abounds�Want to find “clinically significant” and yet:

�Antigens are present whose antibodies are

not generally associated with significant

RBC destruction

� M, N*, Lea, Leb, P1 (and a few dozen more)

There’s More…�Autoantibodies

�Generally a panagglutinin, but not always

�May be clinically significant

�May affect tx and donor selection

�May be warm/cold; IgM/IgG; combinations

�Passively-acquired antibodies

�May be only temporarily present, but

clinically significant during that time

�Sources include transplantation, donor

components, infusions (IVIG, RhIG, etc.)

Goals for Antibody Detection�Detect the necessary clinically significant

antibodies

�Avoid false positives or unreadable tests, i.e.,

have a high first pass yield

�Quick enough to meet our customers’ needs

�Reasonably low amount of plasma needed

�Cheap and easy to perform

Causes of False Positive or

Unreadable Results� False positives

�Unwanted antibodies, i.e., not considered

likely to be clinically significant

�Rouleaux

�Anomolous reactions such as antibodies to

preservatives and additives

�Unreadable results

�Contamination, fibrin, AHG neutralization, etc.

Maximizing Predictive Value:

Method Selection

C Generally Unwanted Considered Significant W

O Allo Anti-M, -N, -Lea, -Leb, -P1 Allo Anti-D, -C, -c, -E, -e, -K A

L Auto Anti-I, -i, -H, -HI -Jka, Jkb, Fya, Fyb, S, s R

D False or Unreadable Results AHG neutralized, etc. M

(IS, RT) Direct agglutination IAT (≈37°C)


Method Performance

�Minimize the unwanted

�Maintain sample quality, avoid fibrin, cryo,

contamination, hemolysis, etc.

�Adhere to procedure guidelines, have a

quality staff training program

�Quality check reagents and equipment

�Avoid inadvertent cold exposure


Method Selection

� Select what’s best for your setting

�Staff capable of proper performance

�Meets budget and time constraints

� Increase sensitivity?� Reagent RBC make-up

� Additional antigens

� Double-dose expression of selected antigens

� Length of 37°C incubation

Routine Alternative Methods

�Tube testing

� Low Ionic Strength Saline (LISS)

�Polyethylene Glycol (PEG)

�Bovine albumin, 22% or 30%

� Solid-phase (in micro titer plates [MTPs])

�RBC stroma adherence

�Protein A adherence

�Column technology

�Gel columns

Tube Methods

�Procedure steps in common

�Can employ 2 or 3 reagent RBC’s

� Incubation at 37°C for first stage of reactivity

– antibody uptake

�Enhancement reagent present during

incubation

� Include Indirect Antiglobulin Testing (IAT)

� IAT needs coated cells as quality check for

each negative result

Tube Methods�Variable steps in common

�Time at 37°C� Minimum required: manufacturer, regulations

� Minimum for maximum sensitivity

� Maximum to avoid risk of antibody elution

�RBC make up� Double-dose requirements, e.g., R1R1, R2R2,

Jk(a+b-), Fy(a+b-), etc.

� Presence of low incidence antigens, e.g., Kp(a+)

�Phases of testing: Imm. Spin, Room Temp.

Tube Methods�Variable steps in common

�Phases of reading: 37°C

�Optical aid for AHG reading, e.g.,� Microscope

� Viewlight

�Type of AHG employed� Monoclonal monospecific anti-IgG

� Detects subclasses 1, 2, and 3; not 4

� Polyclonal monospecific anti-IgG

� Polyspecific anti-AHG

� Monoclonal, Polyclonal, Blended

Tube Method - LISS�Delivery of LISS

�As an additive, i.e., drops from reagent vial

�As an RBC suspension medium

�Reactants

�Critical control of proportions, generally

equal parts of LISS and plasma� Quality checked pipettes may be necessary

�Additives may contain additional agents

�Can use anti-IgG or Poly AHG

�37°C incubation as short at 10 minutes

Tube Method - LISS� “Wanted” ↑ SensiWvity

�Kidd system antibodies

� “Wanted” ↓ SensiWvity

�Certain anti-K, esp. with shortened 37°C inc.� Personal experience: some anti-K ↑

� “Unwanted” ↑ SensiWvity

�Anti-M

�Certain cold autoantibodies� Esp. if serum and Poly AHG is used


�Can omit readings other than IgG

Tube Method - PEG�Delivered as an additive, i.e., drops from a

reagent vial

�Use of anti-IgG strongly recommended

�Do not read at phases other than IAT

�Non-specific aggregates may mimic

agglutination

�Precipitins may form in high protein samples

�Mimic agglutination

�Neutralize AHG (not washed away)

�37°C incubation as short at 10 minutes

Tube Method - PeG� “Wanted” ↑ SensiWvity



�Some Knops-system antibodies

�Perhaps some Chido-Rodgers antibodies


�Anti-P1

�Warm autoantibodies


�No readings other than IgG

Tube Method – Bovine Albumin�Delivered as an additive, i.e., drops from a

reagent vial

�Affects second-stage of agglutination

�No increase in antibody uptake

�Creates low-ionic environment

�Facilitates direct agglutination of IgM or IgG

� Lack of innate sensitivity prompts use of other

enhancing alternatives, e.g.,

�Serum + poly AHG

� Longer 37°C incubation: at least 20’, better 30’

Tube Method – Bovine Albumin� “Wanted” ↑ SensiWvity

�Rh, at least as direct agglutination


� Less AHG carry-over of cold IgM vs LISS/PeG



�Complement-binding cold autoantibodies

� “Unwanted” ↓ SensiWvity

�Pretty much everything

Other Methods�Performed in MTPs

�RBC adherence� Available with automation or manual testing

�Protein A adherence� Available with automation

�Performed by column agglutination technology

(CAT)� In the U.S., available as gel column, with

automation or manual testing

�All use smaller reactant volumes than tube

Solid Phase – RBC Stroma� Stroma of reagent RBC’s is bound to bottom

of U-shaped MTP

�Plasma and LISS are added

�37°C incubation (15-60’ followed by x4 wash)

� Indicator cells with affixed anti-IgG added

�Centrifugation

� Indicator cells adhere if stroma has

antibody affixed

�No adherence with negative test

Solid Phase – RBC Stroma� “Wanted” ↑ SensiWvity


�However may not be in vivo significant


�Anti-K due to LISS



�Non-specific adherence



�Manual test may be difficult to read

Solid Phase – Protein A�Protein A, from cell wall of S. aureus, is bound

to bottom of U-shaped MTP

�Protein A has high affinity for Fc of most Ig

�Reagent RBC’s are suspended in LISS

�Plasma is added; 37°C incubation for 20’

�Washing followed by addition of anti-IgG

�Centrifugation

�RBC’s adhere if antibody affixed

�No adherence with negative test

Solid Phase – Protein A� Sensitivity/Specificity

�Newer method, data incomplete

�AnWcipate ↓ AnW-K due to LISS

�Some reports indicate “non-specific”

reactions comparable to Stroma method

�AnWcipate ↑ warm autoanWbodies



�Manual test not available

Column Technology - Gel�Performed in molded plastic columns

�Contain small particles of gel + anti-IgG

�Particles serve as sieve to filter RBCs by size

� LISS suspended Reagent RBC’s added

�Plasma added, followed by 37°C inc. for 15’

�Controlled centrifugation

�Sensitized RBC’s react with IgG and trap

�Negative RBC’s centrifuge to the bottom

� “Wanted” ↑ SensiWvity

�Some Rh system antibodies


�Anti-K due to LISS

�Historically other such as anti-E, ? currently



�Non-specific reactions� Sample prep and reactant storage critical



Column Technology - Gel

Comparing & ContrastingWithin “same” method, e.g.,

� Source of LISS or PeG

�Other additives in LISS or PeG

�Variables of solid phase, e.g.,

�Parameters of buffer, indicator cells, etc.

�Variables of gel, e.g.,

�Manufacturer’s reagents cells vs. in-house

preparation

Comparing & Contrasting�No method is perfect or ideal

�All meet minimum requirements

�Many exceed minimum, but in different ways

�Gain of increased sensitivity (weak clinically

significant antibodies) may be at the cost of

decreased specificity (increased false positives)

�Antibody ID work is geared to characteristics of

antibody detection method

Documents

E. Ann Steiner, MT(ASCP)SBB Blood Bank Technical ... · Blood Bank Technical Specialist Ortho Clinical Diagnostics. ... Positive crossmatch Positive inert control, IAT control, or