Ghislain Noumsi MD,SBB(ASCP)CM

Molecular ImmunoHematologist

Scientific Support Services

LifeShare Blood Centers

Shreveport, LA

The Monocyte Monolayer Assay (MMA):

An Adjunct to Compatibility Testing

Objectives

Review the mechanism of RBC destruction

by alloantibody

Describe the MMA technique

Analyze the MMA impact as secondary

crossmatch method for patients with RBC

alloantibody(ies)

Blood bank response

to blood request

Request and specimen

received

Initial

investigation

Interpretation and

additional test

Crossmatch

Blood delivered

Patient transfused

No reaction (hemolysis)

Sometimes

things are more

complicated!!!

Any decision can have a direct impact on

the patient prognosis

Meet the transfusion request Requested

Phenotypes

Total

number of

requests

(n=141)

Requests

completely

filled

Requests

partially filled

Requests

unfilled

Number % Number % Number %

All patients 1070 912 85.2 90 8.4 68 6.4

Patients with

SCD

351 303 86.3 30 8.6 18 5.1

Total 1421 1215 85.5 120 8.4 86 6.1

14.5% of requests for rare RBCs unfilled or partially filled (1 out of 7

patients) over a 18 months period. Flickinger C. Immunohematology

2006,22(3):136-42

Central Europe (Germany, Austria, Switzerland): transfusion support

for one-third (1/3) of patient with RBC antibody to high incidence

antigen was unsatisfactory. Seltsam et al. Transfusion 2003,43:1563-1566

Meet the transfusion request Requested

Phenotypes

Total number of

requests (n=141)

Requests

completely filled

Requests partially

filled

Requests unfilled

Number % Number % Number %

U-,D+ 62 47 75.8 8 12.9 7 11.3

Js(b-) 42 40 95.2 1 2.4 1 2.4

U-,D- 8 5 62.5 2 25.0 1 12.5

r”r” 7 1 14.3 4 57.1 2 28.6

Hy- 6 1 16.7 3 50.0 2 33.3

Jo(a-) 5 2 40.0 1 20.0 2 40.0

E-,hrB- 4 3 75.0 1 25.0 0 -

Lu(b-) 2 2 100 0 - 0 -

K- 2 2 100 0 - 0 -

I- 1 0 - 1 100 0 -

Ge:-2 1 0 - 0 - 1 100

E-,hrS- 1 0 - 0 - 1 100

Total 141 103 73.0 21 14.9 17 12.1

Flickinger C. Immunohematology. 2006;22(3):136-42.

What is the clinical significance of my

antibody(ies)?

“A clinically significant RBC antibody is defined as an

antibody that is frequently associated with HDFN,

with hemolytic transfusion reactions, or with a

notable decrease in the survival of transfused

RBCs”. AABB Technical Manual. 16th Ed. P466

In vivo: RBCs are considered incompatible if their

survival is curtailed by the presence of clinically

significant alloantibody

Factors related to

the antibody

• Binding constant

• Ig class and IgG subclass

• Ability to bind to macrophage Fc receptors

• Ability to activate complement

• Thermal reactivity range

• Plasma concentration

Factors related to the

antigen

• Antigenic determinant “epitope”

• Distribution in the body

• Abudance of sites on the red cell

• Appearance on the fetus RBC and placenta (in

case of evaluation of risk of HDFN)

• Association with complement activation

• Other: antigen expression and modification

during storage; number of RBC transfused…etc

Factors affecting the antigen-antibody

bond formation

Spatial complementarity between antigen

and antibody: “Lock and Key” concept

Weak non-specific intermolecular forces

including: electrostatic charges (ionic

groups), hydrogen bonds, hydrophobic

(non-polar) bonds, Van der Waals forces

The equilibrium (association) constant of

the Ag-Ab formation

Factors related to the mononuclear

phagocytic system (RES) • The Fc receptor polymorphism:

• The phagocytic activity of the mononuclear

phagocytic system (RES)

Where do we go

from here ?

Do we just STOP ?

One element missing in

the above pathogenesis

process can result in normal

survival of antigen positive RBCs !!!

The Monocyte Monolayer Assay (MMA)

In vitro assay

Predict the outcome of transfused

antigen positive RBCs to patients

with corresponding antibody

Predict the risk of HDFN in

maternal alloimmunization with

feto-maternal incompatibility

Early development

• Stevens JO, Braley JF, Schanfield MS. Detection

of clinically significant IgG antibodies by an in vitro

human peritoneal macrophage phagocytosis assay. Transfusion

1976; 16:523

• Arndt PA, Garratty G. A retrospective analysis of

the value of monocyte monolayer assay results for

predicting the clinical significance of blood group

alloantibodies. Transfusion 2004;44:1273-81

MMA

MMA: Monocyte Index (MI%)

MI = number of monocytes with one or more RBCs

adhered and/or ingested divided by the total number

of monocytes x 100

ROBERT TEMPKIN

Interpretation

“…MI values of ≤5% have indicated that incompatible blood can be given

without the risk of an overt hemolytic transfusion reaction but it does not

guarantee normal long-term survival of those RBCs…”

Arndt PA, Garratty G. Transfusion 2004;44:1273-81

Can MMA be used as a secondary

crossmatch technique for patients with

unusual antibodies presentation ?

RBC units tested and MI distribution RBC antibodies Number of RBC

units tested

Monocyte index (MI)

0-5 5.1-20 > 20

hrB 54 51 2 1

Fy3 15 11 4 0

AnWj 15 7 6 2

Yta 11 11 0 0

Jsb 9 8 1 0

LW 9 9 0 0

Rg1 8 8 0 0

Lub 7 7 0 0

Jra 4 0 1 3

Hy 3 1 1 1

Coa 3 3 0 0

Tca 2 2 0 0

Lan 1 0 0 1

U 1 1 0 0

hrS 1 0 0 1

Multiple antibodies 26 24 2 0

Unidentified high 52 48 3 1

Total 221 192 16 13

Number of RBC units transfused Antibodies

Specificities (Anti-)

Number of RBC

units tested

Number of RBC

with

MI < 5%

Number of RBC units

transfused

Number %

hrB 54 51 39 76.5

Fy3 15 11 2 18.2

AnWj 15 7 7 100

Yta 11 11 10 90.9

Jsb 9 8 0 0

LW 9 9 9 100

Rg1 8 8 0 0

Lub 7 7 7 100

Jra 4 0 0 0

Hy 3 1 0 0

Coa 3 3 1 33.3

Tca 2 2 2 100

Lan 1 0 0 0

U 1 1 0 0

hrS 1 1 0 0

Multiple antibodies 26 24 11 45.8

Unidentified high 52 48 28 58.3

Total 221 192 118 61.6

> 20

0

5

10

15

20

25

0 1 2 3 4

MI

Tube-Saline

(60’-37C/AHG)

Serological crossmatch reactivity vs MMA

Antibody Specificity and MI distribution Antibody

Specificity (Anti-)

Number of

source

Monocyte index distribution (MI)

0-5 5.1-20 > 20

hrB 10 9 1 0

Fy3 4 3 1 0

AnWj 2 1 1 0

Yta 3 3 0 0

Jsb 2 1 1 0

LW 1 1 0 0

Rg1 2 2 0 0

Lub 1 1 0 0

Jra 1 0 1 0

Hy 1 0 1 0

Coa 1 1 0 0

Tca 1 1 0 0

Lan 1 0 0 1

U 1 1 0 0

hrS 1 0 0 1

PREDICTING THE CLINICAL SIGNIFICANCE OF 51 RBCs ALLOANTIBODIES USING MONOCYTE

MONOLAYER ASSAY (MMA): A 5 YEAR REVIEW NOUMSI GT, BILLINGSLEY K, MOULDS JM, MOULDS JJ

Conclusion Positive crossmatch:

In vivo, the capacity of an antibody to curtail RBCs

survival involves other pathways and….

THIS IS A COMPLETELY DIFFERENT STORY !!!

When all RBCs

seems to be

crossmatch

incompatible

think MMA

you may be

SURPRISED !!!

Thank you !!!