Why We Do What We Do: Serologic Testing 10/20/2007 2:00PM – … · 2009. 4. 30. · EVENT FACULTY...

Why We Do What We Do: Serologic Testing 10/20/2007

2:00PM – 5:30PM

Event Outline

Event Title: 5123-TC Why We Do What We Do: Serologic Testing Event Directors: Lieta Maffei

Lieta Maffei Event Date: Saturday, October 20, 2007, 2:00 PM to 5:30 PM Presenters: W. John Judd Susan Johnson

Theresa Downs Debra Bailey

Time Speaker Presentation

2:00 PM to 2:30 PM Susan Johnson

Why Are We Performing the ABO Test and What Are We Detecting

3:30 PM to 4:00 PM Debra Bailey

Why Do We Perform an Antibody Detection Test and How it Can Help with the Antibody Identification

4:00 PM to 4:30 PM Theresa Downs Which Crossmatch Do I Do Now?

6629 W. John Judd What to do about Weak D?

EVENT FACULTY LIST

Event Title: 5123-TC Why We Do What We Do: Serologic TestingEvent Date: Saturday, October 20, 2007 Event Time: 2:00 PM to 5:30 PM

Director Lieta Maffei CHE, MT(ASCP)SBB 3143 Chauncey Drive San Diego, CA, 92123-2909 (619) 400-8254 [email protected]

Disclosures: No

Director Lieta Maffei CHE, MT(ASCP)SBB 3143 Chauncey Drive San Diego, CA, 92123-2909 (619) 400-8254 [email protected]

Disclosures: NoSpeaker Debra Bailey MT(ASCP)SBB 100 Red Cross Circle Pomona, CA, 91768, USA 909-859-7414 [email protected]

Disclosures: No

Speaker Susan Johnson MSTM, MT(ASCP)SBB 414-937-6274 [email protected]

Disclosures: No

Speaker W. John Judd FIBMS, MIBiol 414 Lockwood Lane SW Supply, NC, 28462, USA 910.842.9893 [email protected]

Disclosures: No

1

Why do we do what we do?

Antibody Detection

Debra Bailey MT(ASCP)SBBIRL Assistant DirectorAmerican Red Cross Blood ServicesSouthern California Region

Antibody detection

Context of presentation– U.S. practices – Pretransfusion testing

Why do antibody detection?

Detect potentially clinically significant irregular RBC antibodies in a potential transfusion recipient– 0.5-2% of the general patient population

have irregular RBC antibodies– 6-35% of various multiply transfused

populations have irregular antibodies

Issitt PD, Anstee DJ. Applied Blood Group Serology, 4th edition 1998

Why do antibody detection?

Avoid – Transfusion of incompatible donor RBCs– Decreased survival of donor RBCs– Clinical effects of Hemolytic Transfusion

Reactions (HTRs)Causing morbidity

Promote– Effective RBC transfusion therapy– Timely RBC transfusion

Historical perspective Antibody detection evolution

1950’s– Major (patient plasma/donor RBCs) and

minor (patient RBCs/donor plasma) crossmatch the standard

reading for direct agglutination with and without albumin

– Some performed testing with selected RBCs to “screen” for antibodies

2

Antibody detection evolution

1960’S– IAT crossmatch requirement introduced

(3rd edition AABB Standards, 1962)– Brief mention that a screen of patient

serum for antibodies against selected RBCs could be an alternative to IAT crossmatch (3rd and 4th edition AABB Standards only; then removed)


1970’s– Requirement for the detection of

unexpected antibodies using reagent RBCs introduced (5th edition AABB Standards, 1970)

– Mid 70’s, reports support use of antibody detection instead of IAT crossmatch for pre-surgical patients (cost/benefit), but standards still required IAT crossmatch

Antibody detection vs. IAT crossmatch

Antibody detection– Can be performed in

advance– Carefully selected

RBCs to include all common antigens

– Is not inventory dependent

– Does not sequester units for improbable transfusion

IAT crossmatch– Direct indication of

compatibility– Capable of detecting

antibodies to low incidence antigens


1980’s– IAT crossmatch no longer required if

antibody screen is negative (11th edition AABB Standards, 1984)

Antibody detection current requirements

AABB Standards for Blood Banks and Transfusion Services 24th edition 2006– A test for unexpected antibodies to red

cell antigens is required– “methods of testing shall be those that

demonstrate clinically significant antibodies….”

Antibody detection current requirements

AABB Standards for Blood Banks and Transfusion Services 24th edition 2006– “…They shall include incubation at 37C

preceding an antiglobulin test using reagent red cells that are not pooled.”

3

Antibody detection-current

Antibody detection has become primary means to ensure a safe & effective transfusion for general patient population– Much emphasis has been placed on

appropriate detection of irregular red cell antibodies through analysis of the factors that contribute to a successful antibody detection test

Factors that contribute to the antibody detection test

No standard requirement for selection– Method – Enhancement media

(applicable to tube method only) – Configuration of antibody detection RBC sets– Reading phases of agglutination

(applicable to tube method only)– Autocontrol with antibody detection

What are we doing?

Source data for charts on subsequent slides– 1995-1996 U.S. data

Maffei LM, et al. Transfusion 1998;38:343-349– 2001-2004 North American data

Shulman IA, et al. Archives of Pathology and Laboratory Medicine 2005;129:984-989

– 2007 (First Trimester) North American dataPersonal communication with Shulman IA. Data from first trimester 2007 CAP survey. Complete 2005-2007 data pending publication by Downes KA, Shulman IA

– 2007 Regional Southern California dataInformal hospital survey; unpublished80 hospitals queried about antibody detection practices





Antibody detection-method

Currently available and acceptable options– Tube– Gel– Solid Phase Red Cell Adherence– Liquid Microwell

Antibody detection-method

20071st trimester

only

Manual Testing- National Data

100

70 67

61

56

43

26 29

36

41

54

4 4 3 3 3

0102030405060708090

100

1995-1996

1997 -2000

2001 2002 2003 2004 2005 -2006

2007

Year

App

rox.

% o

f tot

al p

artic

ipan

ts

TUBE

GEL

OTHER

4



(applicable to tube method only)– Configuration of antibody detection RBC sets– Reading phases of agglutination


Antibody detection-enhancement media

If antibody detection is performed by tube test, the following enhancement media options are available– None (“saline”)– Albumin– LISS– PEG


69 69 71 71 72 70

24

13 12 11 11 96

13 12 13 14

18

4 5 5 4 4 4

0102030405060708090

100

1995-1996

1997 -2000

2001 2002 2003 2004 2005 -2006

2007

Year

App

rox.

% o

f tub

e m

etho

d us

ers

LISS

ALB

PEG

NONE

Antibody detection-enhancement media

20071st trimester

only

Method and enhancement considerations

Balance of sensitivity and specificityStrive to detect clinically significant antibodies while avoiding clinically insignificant antibodiesStill no one perfect method or enhancement for the detection of clinically significant RBC antibodiesTransfusion Services must choose





Antibody detection-reagent RBC sets

FDA require the following antigens be represented on at least one of the antibody detection RBC suspensions in the set– D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k,

Fya, Fyb, Jka and Jkb

The AABB standards require and FDA recommends the use of RBCs that are not pooled for pretransfusion testing

5


Reagent RBC set options for pretransfusion testing– 2 RBC suspension set– 3 RBC suspension set– Other

Pooled- does not meet AABB requirements for pretransfusion testing4 RBC suspension set- expresses a low incidence antigen of interest


20071st trimester

only


39 38 40 40

61 60 59 58

0 2 1 2

0102030405060708090

100

1995-1996

2001 2004 2007Year

App

rox.

Per

cent TWO

THREE

OTHER

2 RBC suspension set exampleDifficult to consistently achieve double dose

3 RBC suspension set exampleUsually able to provide a double dose


D C E c e P1 M N S s Lea Leb K k Fya Fyb Jka Jkb

1 + + 0 0 + 0 + + 0 + + 0 0 + 0 + + 02 + 0 + + 0 + 0 + + + 0 + + + + 0 + +


1 + + 0 0 + 0 + + 0 + + 0 0 + 0 + + 02 + 0 + + 0 + 0 + + + 0 + + + + 0 + +3 0 0 0 + + + + 0 + 0 0 + 0 + + + 0 +

Antigen dose

Individuals that inherit two copies of the same gene at a particular locus, will be homozygous for that gene and their RBCs will express a double dose of the corresponding antigenIndividuals that inherit two dissimilar genesat a particular locus, will be heterozygousfor those genes and their RBCs will express a single dose of each antigen

Dose example

Genotype: Fya/Fya

Phenotype: Fy(a+b-)Result: 100% Fy sites = Fya antigen (double dose)

Genotype: Fya/Fyb

Phenotype: Fy(a+b+)Result: 50% of Fy sites = Fya antigen (single dose)

Antibodies that sometimes exhibit dosage

Rh: C, E, c, eMNS: M, N, S, s

Example of anti-Jka exhibiting dosage

Duffy: Fya, Fyb

Kidd: Jka, Jkb

D C E c e Lea Leb M N S s K k Fya Fyb Jka JkbgelIgG

1 + + 0 0 + + 0 + + 0 + + + 0 + + 0 1+2 + 0 + + 0 0 + 0 + 0 + 0 + 0 + 0 + 03 + 0 0 + + 0 + + 0 + + 0 + 0 0 0 + 04 0 + 0 + + 0 0 + + + 0 0 + + + + 0 1+5 0 0 + + + 0 + + 0 + 0 0 + 0 + 0 + 06 0 0 0 + + + 0 0 + 0 + + 0 + + + + 07 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 1+8 0 0 0 + + 0 0 + 0 + + 0 + 0 + + + 09 0 0 0 + + + 0 + + 0 + 0 + + 0 0 + 0

10 0 0 0 + + 0 + + + + 0 0 + + 0 + 0 1+11 + + 0 0 + 0 + 0 + 0 + 0 + + + 0 + 0PT 0 0

6

Dosage studies

Shulman IA, et al. Laboratory Medicine. 1985;16:602-604– 12 of 26 examples of anti-Jka did not react

with some or all Jk(a+b+) by albumin IATShulman IA, et al. Laboratory Medicine 1989;20:37-39– 4 of 25 examples of anti-Fya did not react

with some Fy(a+b+) but reacted with all Fy(a+b-)

Antibody detection RBCs considerations

DoseGoalCost





Antibody detection-reading for agglutination

If antibody detection is performed by tube test, reading for agglutination at the following phases is not required– Immediate spin (IS)– Room temperature (RT)– 37C

Indirect Antiglobulin Test (IAT) phase is required


1995-1996 National Data

70

5

92 96

0

20

40

60

80

100

IS RT 37C IAT

Perc

ent

2007 Regional Data

36

8

72

100

0

20

40

60

80

100

IS RT 37C IAT

Perc

ent


According to the 1995-96 US data, approximately 70 % of those testing by tube test were performing an IS reading, approximately 5% a RT reading during pre-transfusion antibody detectionWhy?

7

Antibody detection-IS? RT?

Anti-MAnti-NAnti-P1

Anti-Lea

Anti-Leb

Anti-IAnti-iAnti-IHAnti-HAnti-Lua

Antibody specificities typically detected a temperatures below 37C


Common antibodies not reacting at body temperature (37C) are typically rarely considered capable of causing in vivo red cell destruction of antigen positive transfused RBCs


1 + + 0 0 + 0 + + 0 + + 0 0 + 0 + + 02 + 0 + + 0 + 0 + + + 0 + + + + 0 + +3 0 0 0 + + + + 0 + 0 0 + 0 + + + 0 +


Detection of such antibodies in pretransfusion testing can cost time and moneyIdentification of such antibodies have little relevance in transfusion safetyIS/RT reading when performing antibody detection is not recommended

Antibody detection- 37C reading for agglutination?

Newer studies have questioned the necessity of the 37C reading for tube testingThere is no requirement to perform a reading for agglutination at 37CAntibody detection tests performed using gel test, SPRCA, or PEG IAT by nature have no 37C reading for agglutination



70

5

92 96

0

20

40

60

80

100

IS RT 37C IAT

Perc

ent

2007 Regional Data

36

8

72

100

0

20

40

60

80

100

IS RT 37C IAT

Perc

ent


Proponents of the 37C reading cite examples of antibody specificities commonly considered clinically significant that have agglutinated at 37C and not reacted by IAT (e.g., anti-E)They are correct but the more important question is, are these antibodies clinically significant?

8


Proponents for omitting the 37C reading cite fewer clinically insignificant reactions detected that would have required time, effort and resources to investigateA few studies have shown no increase in HTRs in the labs that have dropped the 37C reading





Antibody detection-routine autocontrol

2007 Regional Data

6

94

YES NO


46

41

13

YES NO Other

Antibody detection-routine autocontrol

Was thought to be needed to detect– Antibodies adsorbed onto transfused RBCs– Auto or drug induced antibodies

Studies have shown that the predictive value of routinely including an autocontrol during antibody detection for detecting clinically significant alloantibodies to be lower than routinely performing an antiglobulin crossmatch for a patient with a negative antibody screen

One last thought

Information obtained from a positive antibody screen is valuable information to be used as we move into antibody identification– Antibody detection RBCs that are non-reactive

with the patient’s plasma can be used just like panel RBCs to exclude red cell antibodies

– Strength and phase of reactivity (if applicable) of reactive RBCs can be clues to streamline the identification process

Finally

Why do you do what you do????It is a good idea from time to time to evaluate our testing practices to determine if the reasons why we are doing what we are doing are still applicable

9

Literature

Boral LI, et al. The type and screen: a safe alternative and supplement in selected surgical procedures. 1977;17:163-168.Treacy M. Pretransfusion testing for the ‘80s: a technical workshop. AABB 1980.Garratty, G. The role of compatibility tests. Transfusion 1982;22:169-172.Shulman IA, et al. The dependence of anti-Jkadetection on screening cell zygosity. Laboratory Medicine 1985;16:602-604.

Literature

Judd WJ, et al. The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited. Transfusion 1986;26:220-224.Stec N, et al. The efficacy of performing red cell elution studies in the pretransfusion testing of patients with positive direct antiglobulin tests. Transfusion 1986;26:225-226. Shulman IA, et al. Influence of reagent red cell zygosity on anti-Fya detection. Laboratory Medicine 1989;20:37-39.

Literature

Pestaner JP, et al. Is it safe to omit the 37C reading from pretransfusion red blood cell antibody detection testing? American Journal of Clinical Pathology 1994;101:361-364Beck, ML, et al. Red cell compatibility testing: a perspective for the future. Transfusion Medicine Reviews. 1996;X:118-130.Judd WJ. Commentary: testing for unexpected red cell antibodies-two or three reagent red cell samples. Immunohematology 1997;13:90-92.Issit PD, Anstee DJ. Applied Blood Group Serology, 4th edition. Durham, NC: Montgomery Scientific Publications 1998.

Literature

Maffei LM, et al. Survey on pretransfusion testing. Transfusion 1998;38:343-349.Issitt PD. Antibody screening: elimination of another piece of the test. Transfusion 1999;39:229-230. Judd WJ, et al. Revisiting the issue: can the reading for serologic reactivity following 37C incubation be omitted? Transfusion 1999;39:295-299. Judd WJ. Modern approaches to pretransfusion testing. Immunohematology 1999;15:41-52.

Literature

Issitt PD. From kill to overkill: 100 years of (perhaps too much) progress. Immunohematology 2000;16:18-25.Shulman IA, et al. Pretransfusion compatibility testing for red blood cell administration. Current Opinion in Hematology 2001;8:397-404.Garratty G. Screening for RBC antibodies-what should we expect from antibody detection RBCs. Immunohematology 2002;18:71-77Duran J, et al. Antibody screening in 37C saline. Is it safe to omit it using the indirect antiglobulin (gel) test? Immunohematology 2002;18:13-15

Literature

Shulman IA, et al. North American pretransfusion testing practices, 2001-2004 Archives of Pathology and Laboratory Medicine 2005;129:984-989.Brecher ME (ed). Technical Manual, 15th edition. AABB. Bethesda, MD 2005.Standards for Blood Banks and Transfusion Services, 24th edition. AABB Bethesda, MD 2006Casina TS. In search of the Holy Grail: comparison of antibody screening methods. Immunohematology 2006;22:196-202

1

Why We Do What We Do:Crossmatch

Terry Downs MT(ASCP)SBBSupervisorUniversity of Michigan Health [email protected]

What is a Crossmatch?

A test performed to determine the compatibility of a donated unit of blood for its intended recipient

Why do we Crossmatch?

Determine whether the recipient serum contains an antibody

capability of reacting with and destroying the donor red cells.This includes ABO antibodies.

Crossmatch Methods

TubeGel columnMicrotiterComputer

What We Used to Do…

We performed an antiglobulin crossmatch on everyone-IS, 37 C and IAT

If we didn’t, we made the physician come to the Blood Bank to sign the consent to transfuse uncrossmatched blood

Currently…

ComputerImmediate SpinAntiglobulinNo crossmatch at all!

2

What do the Standards Say?

Before issue, a sample of the recipient’s serum shall be crossmatched against a sample of donor cells from an integrally attached red blood cell segmentThe crossmatch shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens and shall include an antiglobulin test

5.15.1 AABB Standards, 24th Edition

Standards, continued…

If no clinically significant antibodies were detected in tests and there is no record of previous detection of such antibodies, detection of ABO incompatibility shall be performed

5.15.1.1 AABB Standards, 24th Edition


Computer CrossmatchValidated on site2 determinations of ABO are made:

Current sampleHistorical or 2nd on current sample

Computer contains unit number, component name, ABO, Rh, confirmed ABO


Computer crossmatch:Method exists to verify correct entry before release of componentsSystem contains logic to alert user to ABO/Rhdiscrepancies between the donor and the recipient

5.15.2 AABB Standards, 24th Edition

What About the FDA?

21 CFR 606.151:Method of collecting and identifying the blood samples of recipients to ensure positive IDUse of fresh recipient serum or plasma less than 3 days old for all pretransfusion testing if the recipient has been pregnant or transfusedProcedures to demonstrate incompatibility between the donor’s cell type and the recipient’s serum or plasma type

FDA

A provision that, if the unit of donor’s blood has not been screened by a method that will demonstrate agglutinating, coating and hemolytic antibodies, the recipient’s cells shall be tested with the donor’s serum (minor crossmatch)Procedures to expedite transfusion in life-threatening emergencies (signed by a physician)

3

Antiglobulin Crossmatch

Use when patient has antibodies to clinically significant antigensUse when immediate spin crossmatch is positive

Patient has clinically insignificant room temperature antibody

Other Methods

No clinically significant antibodies in sample No history of clinically significant antibodiesMethod to detect ABO incompatibility

Immediate spinComputer

Immediate Spin Crossmatch

Works well for Blood Banks that do not have the computer system needed for computer crossmatchesComfort level when performing a serological test

Advantages of IS XM

Decreased turn around time versus antiglobulin crossmatchDecreased workloadReduced reagent costsEffective use of blood inventory

Computer Crossmatch

Even faster than immediate spin, but…Validate on siteSystem contains logic to alert the user to discrepancies2 ABO determinations

Current sampleHistorical sample or 2nd on current sample

Advantages of Computer XM

Decreased turnaround timeDecreased workloadReduced sample volume for pre-transfusion testingReduced exposure to personnelBetter use of blood inventory

4

What Kind of Crossmatch to Perform?

Depends on SOPsDepends on comfort level of Medical Director and staffNeed guidelines in place when various crossmatches are in SOPDepends on the patient’s results and urgency of the blood

History of Clinically Significant Antibodies?

Immediate Spinor Computer

Current Antibody Screen Negative?

No

Yes

What About Patients with Antibodies?

Depends…on the clinical significance of the antibodyIs the antibody known to cause destruction of red cells?

What Determines Clinical Significance?

Antibodies reactive at 37 C and/or IAT are generally significantReactivity at room temp and below are generally not significantThere are always exceptions!Medical Director to set policy

Is the antibodyclinically significant?

Yes No

Perform AntiglobulinCrossmatch

Yes No

Antibody Screen Positive?

Perform AntiglobulinCrossmatch

Perform Immediate Spinor Computer Crossmatch

(Immediate Spin maybe positive)

When NOT to perform the antiglobulin crossmatch

Massive transfusions-replace blood volume in 24 hoursPretransfusion serum does not represent the blood currently in the patientOnly required to confirm ABO compatibilityCan use immediate spin or computer

5

Massive Transfusion in Patients with Antibodies

Antibody usually not present to cause problems at that pointAfter stabilization, resume antiglobulincrossmatchNote to physician of potential for delayed reaction

When NOT to Crossmatch?

InfantsEmergencies

Infants

Difficult to obtain blood samplesNeonates do not typically form antibodies during the first 4 months of lifeUse Group O reds cells for transfusionCheck for passive antibodies if type specific red cells are used

Infants

If infant has passive antibody from mother, issue antigen negative red cells Continue for as long as maternal antibody persists in infantCrossmatch not necessary when using Group O red cells

Emergencies

Patient bleeding-any blood may better than no blood at allIssue Group O red cells for emergenciesNot the time for staff to consider optionsImportant to have procedures in place to define what blood to giveCrossmatch after the fact when calmer

Resources

AABB Technical Manual, 15th EditionAABB Standards for Blood Banks and Transfusion Services, 24th EditionCode of Federal Regulations

1

Why Do We Do What We Do?ABO TYPING

AABB Annual Meeting 2007

Susan T. Johnson, MSTM, MT(ASCP)SBBBloodCenter of Wisconsin

Milwaukee, WI

ABO TYPING• Why do we ABO type?• What are we detecting?• Why do anti-A & anti-B cause hemolysis?

• Should we worry about anti-A1?

Why is ABO important?The plasma of an individual contains naturally occurring antibodies to A and B antigens, if these antigens are absent from the individual’s red cells.

LANDSTEINER’S RULE

ACUTE IMMUNE HEMOLYSIS

• ABO antibodies cause the most serious form of acute immune hemolysis• Intravascular

• Fatality Rate of 20%

2

WHY DO ANTI-A & ANTI-B

CAUSE HEMOLYSIS?

PATHOGENECITY OF ABO ANTIBODIES

• Antigen Characteristics

• Antibody Characteristics

ANTIGEN CHARACTERISTICS

• Antigenic Make-up

• Quantity

• Distribution

ABO ANTIGEN QUANTITY

• Carbohydrate antigen

Up to 1.5 million antigens per RBC!

N

HO

HO

The Sugars

HOHO

HO

HO

HO

HOHO

O

D-Galactose N-Acetylglucosamine

HO

HO

HO

Numbering the Carbons

HOHO

N

HO

HO

HOHO

O2

34

5

6

1 1

243

65

D-Galactose N-Acetylglucosamine

3

GlcNACGal

i ANTIGEN

GlcNACGal

Type II Unbranched Chain

R

I ANTIGENI gene

β-1,6-acetylglucosaminyltransferase“Branching Enzyme”

Gal

GlcNAC

GlcNAC

GlcNAC

Gal

Gal

β-1,6-acetylglucosaminyltransferase

Gl cN

AC

Gal

Gal GlcN

AC

ABH GLYCOSYLTRANSFERASES

GENE GENE PRODUCTH (FUT 1) α -2-L-fucosyltransferaseA1 α -3-N-acetylgalactosaminyltransferaseB α -3-D-galactosyltransferaseO None

Fuc

H ANTIGEN

α -2-L-fucosyltransferaseGal

GlcNACGal

H gene

4

GalGlcNACGal

Fuc

A ANTIGEN

GalNAC

α -3-N-acetylgalactosaminyltransferase

A gene

GalGlcNACGal

Fuc

Gal

B ANTIGEN

α -3-D-galactosyltransferase

B gene

H1 to H3 BRANCHING

Gal - GlcNAc - Gal – R H1 (i)

Gal - GlcNAc - Gal - GlcNAc - Gal - R H2 (I)

Gal - GlcNAc - Gal - GlcNAc - Gal R H3

Gal - GlcNAcFuc

Fuc

Fuc

FucFuc

H4

Gal - GlcNAc – (Gal – GlcNAc)n - Gal - Glc - R H4

Gal - GlcNAc

Fuc

Gal - GlcNAc

Fuc Gal - GlcNAc

Fuc

5

ABH ANTIGENS AT BIRTH

• Weaker expression • Occasionally mixed field

• Branching enzyme not active • Lack branched H3 & H4 glycolipids(I-i+)

• As i converts to I; H, A, B

i I H A or BABH ANTIGEN DISTRIBUTION

• Carried on protein and lipid structures• Most are glycoproteins carried on….

• Anion Transport Protein (Band 3)•1,000,000 copies

• Glucose Transport Protein (Band 4.5)•500,000 copies

• Rh Associated Glycoprotein (RhAG)• 5-10% associated with glycolipids

C/c

GLYCOLIPID

HISTO-BLOOD GROUP ANTIGENS• Skin• Vascular endothelium• Digestive epithelia

• Colon• Small intestine

• Respiratory mucosae - Lung• Most Epithelial Cells• Platelets

• Low levels on most

ABO ANTIGENS

• Environment• Bacteria• Animals

6

ANTIBODY CHARACTERISTICS

Factors Influencing Antibody Pathogenicity

• Antibody characteristics:• Class (IgM vs IgG)• Subclass• Specificity• Thermal Range• Efficiency of complement activation• Antigen affinity

Adapted from Petz and Garratty Immune Hemolytic Anemia, 2nd ed. 2004

Factors Influencing Antibody Pathogenicity

• Quantity of RBC-bound antibody and/or complement

• Type of complement on RBCs• Activity of RE system

Adapted from Petz and Garratty Immune Hemolytic Anemia, 2nd ed. 2004

ABO ANTIBODIES

• Non-red cell stimulated (“naturally occuring”)

• IgM / IgG – detected at IS, RT, 4C• Active at 37C• Present in high titer• Efficient activator of complement• Cause intravascular hemolysis

IgGantibody

C1Complement

recognition unit

C2aC3C4bComplementactivation unit

C5b6789Membrane

attack complex

RBC lysisRBC

Intravascular Hemolysis ABO TYPING

B

7

BARNEYI’ve got Bs in my blood!

IS ANTI-A1

SIGNIFICANT?

Anti-A1

• Found in A2, A2B and weaker subgroups of A

A1 AND A2 PHENOTYPES

• Qualitative difference• A2 can make anti-A1• Transferase activity differs

•A1 transferase adds GalNActo repetitive Type 3H and Type 4H

GalNAc - Gal - GlcNAc

Fuc

Type 2A

Type 2H

8


Fuc

Gal -

Fuc

Type 2A

Type 2H

Type 3H

Repetitive Type 3H Structures


Fuc

Gal -

Fuc

GalNAc -

Type 2A

Type 2H

Type 3H

Type 3A Repetitive Type 3A Structures

Present on glycolipids

SIGNIFICANCE OF ANTI-A1

• Rarely causes hemolysis• 4 reports in the literature of HTR

• 1946, 1959, 1975, 1978• Antibody must be reactive at 37C

ABO TYPING - SUMMARY

• Why do we ABO type?• What are we detecting?• Why do anti-A & anti-B cause hemolysis?

• Should we worry about anti-A1?

1

What to do about Weak D?W. John Judd, FIBMS, MIBiol

April, 2007University of Michigan, Ann Arbor

ObjectivesUnderstand the fundamental difference between Weak D and Partial D phenotypes

Develop a testing strategy to decrease RhD alloimmunization in Partial D patients

Assess the impact of such a strategy on the supply of Rh-negative blood when applied to transfusion candidates

What is D?

multi-pass membrane protein encoded by RHD at 1p36.13-p34its presence on RBCs defines the Rh-positive phenotypehighly immunogenic, second only in clinical importance to A and B antigens

What is D?first defined by an agglutinin (IgM) in the serum of Mary Seno, who delivered a still-born fetus (Levine and Stetson, 1939)

initially thought to be the same determinant as that defined by diluted antibody from rabbits/guinea pigs immunized with Rhesus monkey cells (Landsteiner and Wiener, 1940)

NH2 COOH

What is D?

5’ 3’ 3’ 5’

RHD1 2 3 54 6 7 9 108

RHCE1235 467910 8

upstreamRh box

downstreamRh box

SMP1

2

NH2 COOH

CE polypeptideCc Ee

1 2 3 54 6 7 9 108 1235 467910 8

Rh-positive

1235 467910 8

Rh-negative

hybridRh box

Molecular Backgrounds for D-

63% RHD deletion28% (all C+) intact RHD2% RHD-CE-D

Japanese

18% RHD deletion67% RHDψ (37-bp insert; TYR259stop)15% RHD-CE-DS (r’S; VS+, V-)

African

RHD deletionEnglish

Distribution of D Antigen

0.050.2Chinese

45.5Nigerian

15.117.5English

D-C-E-D-

SE Michigan Data

0.10.4r”r1.22.5R2R2

1.70.6r’r14.912.1R2r4.715.3rr3.513.0R1R2

-0.1R1RZ2.017.4R1R1

49.81.8RO22.136.8R1r%AA%C%AA%C

The Entity Known as Du

1st described by Stratton* in 1946mistakenly called the Du antigenresults from:– Cde haplotype in trans (Ceppellini effect)– weak D alleles– hybrid Rh genes

* Developed Stratton sandwich technique for Rh D typing.

3

The Ceppellini Effect

DCe/Ce ce/ce

Ce/ce DCe/ce DCe/CeCe/ce

Ce/ce DCe/ce

DCe/cece/ce

Du

Du

Weak D PhenotypesFlegel and Wagner: Clin Lab 2002:4853-9

arise from missense mutations to regions of RHD encoding transmembrane portion of Dless D protein inserted into RBC membraneusually do not make anti-D if exposed to normal Rh+ RBCsshould type as Rh+ with high-affinity IgM monoclonal anti-D

weak D types

partial D including D categories Partial D Phenotypesarise from hybrid genes and missense mutations to regions of RHD encoding parts of D external to RBC membranesome react weakly with anti-Dmay make antibody to missing part of D

if transfused, should receive Rh- negative RBCsif pregnant, are candidates for RhIG

account for FDA-reportable errors when hospitals and blood centers use different anti-D reagents

5’ 3’

3’5’

1 2 3 54 6 7 9 108

1 2 3 54 6 7 9 108

RHD

RHCE

V1.1

V11

IIIb

IVb

Va

IIIa

IVa

DFR

rG

r”G

D-V-VS+

ROHar

4

NH2 COOH

partial D novelsequence Reasons to Test for Weak D

conserve Rh-negative blood for true D-negative recipients*do early in pregnancy, to eliminate false-positive results due to large FMH at deliveryavoid giving RhIG to women who do not need it*

* Arguments viable only if weak D individualscannot make anti-D.

0.4%2004GermanyFlegel

0.4%2004USAJenkins

Frequency of Weak D

0.66%1974FranceGarretta

1990

1967

0.2%USABeck*

0.56%ScotlandHopkins

* Frequency of DVI = 0.02%.

If You Don’t Test for Weak D… if frequency of weak D is 0.4%, andif frequency of D-negatives is 16.0%∴frequency of weak D among apparent D-negatives is (0.4 ÷ 16) x 100 = 2.5%2.5% RhIG doses go to weak D women2.5% Rh-negative RBCs go to weak D recipients

Should We Test for Weak D?

required for donor bloodnot required for recipientsstandard-of-care for infants of Rh-women (to determine need for RhIG)not required for prenatal testing

mAb Anti-D’s

gel

tube

tube

tube

tube

n/aMS201ID-MTS

MS26Th28Immucor-5

MS26MS201Immucor-4

F8D8GAMA 401Gamma

humanMAD2Ortho

IgGIgM

5

Direct Tests (IS)

999

10mf10G-T

12129

12mf12

I4-T

10128

12mf11

I5-T

12128

12mf11

O-T

12121112mf12O-G

DIVbDIVaDIIIbDIIIa*DII

* transfused with Rh- RBCs

Direct Tests

00000DFR00

G-T

00

I4-T

00

I5-T

00

O-T

00

O-G

DVI.2DVI.1

tube tests by IS and after 37 C incubation

Indirect Tests (IAT)

11121212DFR1112G-T

1011

I4-T

1011

I5-T

88

O-T

DVI.2DVI.1

Conflicts in Direct Tests

110896DVa

110101110DBT

120(0)0(0)0(6)6(8)RoHar-1

120(0)6(10)6(10)6(6)RoHar-20(3)

I5-T

90(0)0(3)3(4)RoHar-3

O-GO-TI4-TG-T

( )= after incubation

Findings at IAT

11121210DVa

8///DBT

0999RoHar-1

0101010RoHar-2

8

I5-T

088RoHar-3

O-TI4-TG-T

We Switched to1 normal RHD1 corrupted intron 4/52 weak D Type 16 weak D Type 23 DAR (weak D Type 4.2)– DVI by exon mapping– DVa by serology (mAb panel)

6

Strategies for Partial DPretransfusion/Prenatal Testing

do not test for weak Dgive Rh-negative RBCs; give RhIG consider two anti-D’s; look for conflictsgrade reactions; interpret weak reactors as Rh-negative

Partial D in Pregnancy

partial D women “at risk”most mAb anti-D do not detect DVI (RhIG presumed effective)most mAb anti-D react strongly with ROHarRBCs in contrast to human anti-D; ∴ROHar women unlikely to receive RhIG if tested with modern-day reagents

evaluateby K-B

norosettetest

yes

no

give RhIG at 26-28 weeks’

no RhIGyes

direct testwith anti-D >3+

infantRh+

no

no RhIG

0

single doseof RhIG

yes

Strategies for Partial DDonor Testing*

select reagentstest for weak D (tube IAT)or manage the conflicts

* to avoid FDA-reportable errors

Terminology Issues

many partial D’s react weakly with anti-Dsome weak D’s make anti-Dconsider all as D-variants with clear indication of those that can produce anti-D

Food For Thought

Why is prevention of anti-D formation so sacrosanct?Should we not provide CcEeK-matched RBCs for all (female age <50) patients?

Why We Do What We Do: Serologic Testing 10/20/2007 2:00PM – … · 2009. 4. 30. · EVENT FACULTY...

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Transcript of Why We Do What We Do: Serologic Testing 10/20/2007 2:00PM – … · 2009. 4. 30. · EVENT FACULTY...