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Completing the CIBMTR Completing the CIBMTR Confirmation of HLA Typing Form Confirmation of HLA Typing Form

(Form 2005)(Form 2005)

Stephen SpellmanResearch ManagerNMDP Scientific Services

Maria BrownScientific Services Specialist

Data Management Conference 2007

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Introduction to Form 2005

Basic Biology of HLA

Overview of HLA nomenclature

Laboratory report interpretation and data transcription

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IntroductionIntroduction

The CIBMTR product information is collected on three separate forms:

• The IDM form (Form 2004)

• The Confirmation of HLA Typing form (Form 2005)

• Infusion form (Form 2006)

HLA form modeled on NMDP Form 22/117

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Confirmation of HLA Typing FormConfirmation of HLA Typing Form

The Confirmation of HLA Typing form is completed for:

• Non-NMDP allogeneic or syngeneic donors

• Non-NMDP recipients

• Non-NMDP cord blood units

Complete a separate copy of this form for each donor, recipient, or cord blood unit

Not required on NMDP-facilitated transplants!

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FormsNetFormsNet 2.02.0

All data fields identical to the paper form

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Basic Biology of HLABasic Biology of HLA

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What is HLA?What is HLA?

Human Leukocyte Antigen (HLA) First discovered through antigenic differences on white blood cells

Highest expressed antigens are the class I and class II HLA molecules (0.5 - 1 million per cell)

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HLA and the MHCHLA and the MHC

HLA is part of a larger system know as the Major Histocompatibility Complex (MHC)

MHC is a set of closely linked genes inherited as a unit (complex)

MHC was initially recognized by its major influence on transplant rejection

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Histocompatibility MoleculesHistocompatibility Molecules

MHC is divided into two main regions:

•HLA class I

•HLA class II

Encoded by genes on chromosome 6

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HLA InheritanceHLA Inheritance

HLA genes on the same haplotype are inherited together, along with the rest of the MHC ‘Complex’

Maternal Copy

Paternal Copy

HLA Class II HLA Class IC

C

CC

C

C DRB1 DQB1 B C A

DRB1 DQB1 B C A

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HLA InheritanceHLA Inheritance

d

O F

F S

P R

I N

G

c

db

b

b/a

25%

25%

Recombinant Sibling (1%)

c

a25%

25%a

dA B DR

a

b

c

d

FATHER

MOTHER

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HLA DiversityHLA Diversity

Highest degree of polymorphism in human genome

The diversity makes possible thousands of different combinations

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HLA Typing MethodsHLA Typing Methods

SerologyIdentifies HLA molecules on the cell surface using antigen specific anti-sera

DNAIdentifies HLA molecules by defining the DNA code in the cell nucleus

AntigenProteinDNA

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HLA NomenclatureHLA Nomenclature

DNA DNA SerologySerology

DNA SerologyA*0201 A2A*0202 A2A*0203 A2A*0204 A2A*0205 A2…..A*0273 A2

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Number of Antigens/Alleles

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Why So Diverse?Why So Diverse?

Present many different types of peptides

Protection from a myriad of pathogens

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The Function of HLA MoleculesThe Function of HLA Molecules

Present antigen (peptides) to T-lymphocytes

Facilitate immune surveillance

Detection of:

• Viral or bacterial infected cells

• Mutated genes or proteins

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Class I HLA MoleculesClass I HLA Molecules

Co-dominantly expressed on the surface of almost all nucleated cells (except RBCsand CNS)

Present intracellular peptides to CD8+ T cells

Individuals express up to 6 different class I molecules; two HLA-A, two HLA-B and two HLA-C

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Class II HLA MoleculesClass II HLA Molecules

Expressed on the surface of Antigen Presenting Cells (APCs)• B lymphocytes, dendritic cells and thymic

epithelial cells• Class II HLA-DRB1/3/4/5, DQA1, DQB1, DPA1,

DPB1

Present extracellular (and intracellular) peptides to CD4+ T cellsConsist of a and b chains

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HLA Class I and IIHLA Class I and II

Antigen Presenting Cell

Groove-Domain CytoplasmicTM

Class I

Class II

1 5 6 87Exon 4Exon 3Exon 2

1 4 65Exon 3Exon 2

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HLA Class I and IIHLA Class I and II

Antigen Presenting Cell

Peptide

Groove-Domain CytoplasmicTM

Class I

Class II

1 5 6 87Exon 4Exon 3Exon 2

1 4 65Exon 3Exon 2

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Groove-Domain CytoplasmicTM

Class I

Class II

1 5 6 87Exon 4Exon 3Exon 2

1 4 65Exon 3Exon 2

HLA Class I and IIHLA Class I and II

Antigen Presenting Cell

T Cell

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HLA and TransplantationHLA and Transplantation

Stem cell transplantation is transplanting of the immune system

Graft rejection occurs when the residual host immune system attacks the transplanted immune system

Graft vs. Host Disease (GvHD) occurs when the new immune system begins to attack the body of the recipient

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HLA Basic Biology HLA Basic Biology -- SummarySummary

HLA genes part of MHC complex

Inherited as maternal and paternal haplotypes

HLA genes are the most polymorphic in the human genome

Facilitate immune surveillancePresent self and non-self peptides (viral, bacterial, mutated proteins, etc…) to circulating T cells

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Overview of HLA NomenclatureOverview of HLA Nomenclature

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Examples of Serological TypingExamples of Serological TypingNomenclatureNomenclature

order ofantigen

discovery

gene

A10 B15 DR4

A2 B35 DR15

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Examples of DNA Typing Examples of DNA Typing NomenclatureNomenclature

gene

order ofallele discovery

A*2501 B*1503 DRB1*0402

A*0210 B*3502 DRB1*1504

“ * ” DNAmethodology allele group

•structure•serologic

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DNA Nomenclature:DNA Nomenclature:Silent SubstitutionsSilent Substitutions

DRB1*150201DRB1*150202DRB1*150203DRB1*150204

The fifth & sixth digits indicate a difference in DNA structure, but the HLA antigens specified do not differ

Example: DRB1*1502

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DNA Nomenclature OdditiesDNA Nomenclature Oddities

A*020101 = Silent mutationA*2409N = (Null) No antigen expressedA*2402L = Low antigen expressionB*4402S = HLA protein is secretedB*3565Q = Antigen level is questionable

Antigen

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NMDP Allele CodesNMDP Allele Codes

11AAF

01AG13ADG

07XX01PP

01AD

15AAA02GS

04FH

01TY

01FG

13ST

01DD

11AKA

02EF 04FH

13EG

01TY01DD01BAB

03HJ

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NMDP Allele Codes used for Multiple NMDP Allele Codes used for Multiple Allele DesignationsAllele Designations

Used to report multiple possible alleles (intermediate/high resolution)

Provides maximum information on the person’s typing

NMDP Bioinformatics Web site contains look-up Web tool

www.bioinformatics.nmdp.org

Used to report multiple possible alleles (intermediate/high resolution)

Provides maximum information on the person’s typing

NMDP Bioinformatics Web site contains look-up Web tool

www.bioinformatics.nmdp.org

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NMDP Codes for Multiple Allele NMDP Codes for Multiple Allele DesignationDesignation

AB = 01/02

A*0201 or *0202 = A*02AB

B*4001 or *4002 = B*40AB

DRB1*1501 or *1502 = DRB1*15AB

A*0201 or *0202 = A*02AB

B*4001 or *4002 = B*40AB

DRB1*1501 or *1502 = DRB1*15AB

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CIBMTR Confirmation of HLA CIBMTR Confirmation of HLA Typing Form (Form 2005)Typing Form (Form 2005)

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Key Fields BoxKey Fields Box

•The Key Fields Box contains information about the transplant –recipient, donor, cord, transplant date and type of transplant.

•The Key Fields link the donor forms to the patient forms.

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Key Fields BoxKey Fields Box

The section on the bottom of Page 5 is used if multiple donors or cords were infused into one recipient.

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Whose HLA typing data are you Whose HLA typing data are you reporting? reporting?

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Are you submitting a laboratory Are you submitting a laboratory report?report?

If you have any questions about how to interpret a lab report, please contact either:

Maria Brown 612/627-8186 mbrown2@nmdp.orgSteve Spellman 612/617-8334 sspellma@nmdp.org

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Transcribing HLA Typing Data Transcribing HLA Typing Data from Laboratory Reportsfrom Laboratory Reports

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Is it Serology or DNA?Is it Serology or DNA?

Serology

LymphotoxicityMicrolymphotoxicitySerology

DNALuminexMolecularPCRPCR-SSPSBTSSOSSOPDNA

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General Instructions General Instructions –– DNA TypingDNA Typing

One possible allele in each set of boxes

Use as many boxes as you have alleles to report

If more than six possible alleles, fill the boxes provided then write the remainder of the alleles in the available space on the page, clearly indicating the appropriate locus

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How to Read a Lab ReportHow to Read a Lab Report

“/” is used to separate alleles (example DRB1*0701/03 equals 0701 or 0703)

“-” is used to indicate a range of alleles (example B*0801-0803 equals 0801/0802/0803)

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DNADNA--Based Typing in Based Typing in FormsNetFormsNet

0201/09/43N/66/75/83N/89●

●

●

●

0301/01N/20/21N

●

0702/44

5701

0702

0602

04070701

The HLA field in FormsNetsupports up to 250 characters

HLA Laboratory Report

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DNADNA--Based TypingBased Typing

HLA Laboratory Report

Notice that the 02 is in front of the A*0289.

0 2 0 16 6

4 3 N0 9A*0 2 8 9

7 5 8 3 N

0 7 0 2

5 7 0 1

0 6 0 2

4 4

0 7 0 2

0 4 0 7

0 7 0 1

0 1

2 1 N2 00 3 0 1 0 1 N

Which Typing do I Use?Which Typing do I Use?

Remember to check the comments or footnotes on your laboratory report.

There is often further clarification of the HLA typing.

0 2 7 70 2 7 10 2 0 1

0 2 7 4 0 1 0 2 7 5

4 4 0 2 4 4 4 1

0 2 6 8

A*0281-85/0287-90/0293-96

1 8 0 1

0 5 0 1 0 5 1 3

0 7 0 1

0 2 7 10 2 7 7

Patient

Donor

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*0702*07@*1101*0301*0801*0702*3201*0201

Cw#2Cw#1DR#2DR#1B#2B#1A#2A#1

NT*0301*0201

DRB345#2DRB345#1DQ#2DQ#1

Laboratory Report

Enter the alpha/numeric data from the footnote on the forms and/or into FormsNet 2.0. Please note that without the key we cannot decipher your symbols.

@ = C*0701/0706/0718NT = Not Tested

What About Symbols @#$&%!What About Symbols @#$&%!

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Reporting NMDP Allele CodesReporting NMDP Allele Codes

02ANGA●

●

●

03XKS

1402

44AMUT

05AC0802

FormsNet

0 2 A N G A

0 3 X K S

1 4 0 2

4 4 A M U T

0 5 A C

0 8 0 2

Paper form

Directly transcribe any allele codes onto the form or into FormsNet.

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What to do with Positives??What to do with Positives??

Example

Either write or type “positive”or “pos”

Please do not use “+” or “-”

P o s

0 5 R V

0 6 B V AH

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Serologic Typing and DNA?Serologic Typing and DNA?ENTER BOTH!ENTER BOTH!

CUMULATIVE HLA TYPE RESULTS

MOLECULAR TYPING BY PCR-SSP, SBT AND/OR LUMINEX SSO

HLA Class I Antigens HLA Class II Antigens A B Cw DR DR51/52/53 DQ

A1 A24 B8 B35 Cw4 Cw7 DR1 DR17 DQ2 DQ5

HLA -A HLA -B HLA -Cw HLA-DPB1 A*0101 B*0801 Cw*0401 A*2402 B*3501 Cw*0701

HLA – DRB1 HLA – DRB3/4/5 HLA –DQB1

DRB1*0101 DQB1*0201 DRB1*0301 DQB1*0501

0 1 0 1

2 4 0 2

0 8 0 1

3 5 0 1

Serology

DNA

Serology in Serology in FormsNetFormsNet 2.02.0

●Drop down selection menu

Yes/No buttons

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Example Laboratory Reports for Example Laboratory Reports for Completing Form: Completing Form:

Confirmation of HLA Typing Form Confirmation of HLA Typing Form (Form 2005)(Form 2005)

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Example AExample A

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Example A Example A continuedcontinued

2 9 X X

2 4 X X

0 7 X X

4 4 0 3

0 7 X X

1 6 0 1

1 5 0 1

1 3 0 1

Positive

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Example BExample B

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Example B continuedExample B continued

1 5

0 1

0 2

0 7

4 4

0 5

0 7 0 2 1 01 7

0 7 0 1 1

1 5 0 1 1

0 1 0 1

1 5

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Example CExample C

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Example C continuedExample C continued

3

1 1

2 7

1

1

4

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Example DExample D

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Example D continuedExample D continued

0 8 0 1― 0 3

1 3 0 1― 0 3

0 6 0 2

0 7 0 1 0 7 0 6

0 3 0 1

0 7 0 1 0 3

0 3

0 1 0 1 0 3

0 1 0 1 ―0 5

0 2 0 1― 2 2 / 2 5 ― 2 7 / 2 9

3 0 0 1― 0 4 / 0 6

Example EExample E

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Example E continuedExample E continued

0 1 0 1

0 7 0 1

0 1

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SummarySummary

Form 2005 required on non-NMDP donors and recipients

Multiple donors = multiple forms

Transcribe data directly as reported on laboratory report*

Include all possible alleles for DNA-based typing

Report both DNA and Serology data, if available

*Remember to watch for footnotes

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Please remember:

If you have any questions about how to interpret a lab report, please contact either:Maria Brown 612/627-8186 mbrown2@nmdp.orgSteve Spellman 612/617-8334 sspellma@nmdp.org