e. ann steiner, mt(ascp)sbb blood bank technical ... · blood bank technical specialist ortho...

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

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

e. ann steiner, mt(ascp)sbb blood bank technical ... · blood bank technical specialist ortho...

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