flow crossmatching protocol

f o i/OxyA t© -erq-

Hospital of the University of Pennsylvania Department of Pathology and Laboratory MedicineDivision of Laboratory Medicine Histocompatibility Laboratory Section: 01-Flow Crossmatching________

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Version: 2.1 Current

Effective: 9/5/2014Subject: 01-Flow Crossmatching DC # :HLA.B.03.01

Title Flow Crossmatch

Prepared by Thanh-Mai Bui Date: 0 5 /1 6 /2 0 1 3

TABLE OF CONTENTS1.0 TEST INFORMATION..................................................2.0 ANALYTICAL PRINCIPLE..........................................3.0 SPECIMEN REQUIREMENTS....................................4.0 REAGENTS.....................................................................5.0 CALIBRATORS/STANDARDS...................................6.0 QUALITY CONTROL...................................................7.0 EQUIPMENT and SUPPLIES.....................................8.0 PROCEDURE.................................................................9.0 CALCULATIONS...........................................................10.0 REPORTING RESULTS AND REPEAT CRITERIA11.0 EXPECTED VALUES...................................................12.0 CLINICAL SIGNIFICANCE.........................................13.0 PROCEDURE NOTES..................................................14.0 LIMITATIONS OF METHOD....................................15.0 SAFETY..........................................................................16.0 RELATED DOCUMENTS...........................................17.0 REFERENCES..............................................................18.0 ADDENDA...................................................................

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Prin t Status; U ncontrolled W hen P rin ted

Jessica

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Jessica

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01-Flow Crossmatch page 2 of 30

1.0 TEST INFORMATION

A ssay M e th o d /In s tru m e n t O rd e r Code

Flow C rossm atch Flow C ytom etry /FC 500

FlowXMFlowXMABDXMDD FlowXM AutoFlowXM

Syn o n y m s /A b b re v ia tio n s __________________________AHG-CDC - A ntiglobulin augm en ted cytotoxicity assayDNA- D eoxyribonucleic AcidDSA - D onor Specific A ntibodyFITC - F lourescein IsothiocyanateGOL - Gift of Life Organ P rocurem en t O rganizationMFI-Mean Fluorescence In tensityPCR - Polym erase Chain ReactionSSP - Sequence Specific P rim ersUNOS - U nited N etw ork for Organ SharingRPM - Revolutions p e r m inuteRCF - Relative centrifugal force_____________________

Department _______________ __________ _H istocom patibility L aborato ry __________________ __

2 0 ANALYTICAL PRINCIPLE . . _ uThe Flow Cytom etry Crossm atch has been proved to be a m ore ^ n s i t iv e ec nique detecting dono r reactive an tibodies and to d ifferen tiate benign from harm fu l B cell an tibodies In th e th ree color sta in ing p rocedure , donor lym phocytes, p a tien t a FITC-conjugated an ti-hum an an tibody reag en t are incubated to allow th e anybody to b ind t o r i l l s and for th e conjugate to b ind to th e pa tien t's an tibody on th e ta rg e t cells. Additionally, PE an d PCS conjugated m onoclonal antibodies can be added to unequivocaUy identify th e na tu re of th e ta rg e t cell reacting w ith A e an tibod ies Cell fluorescence is th e n analyzed on the Flow Cytom eter. A positive flow cytom etric crossm atch is ind ica ted by a shift of the fluorescent in tensity of th e ta rg e t celhnd icatm g increased fluorescence an d g rea te r an tibody binding. The sam e p rocedu re can be utiliz to de term ine the p resence and specificity of HLA an tibod ies m a p a tie n t s serum . cell panels can b e ru n to determ ine the p resence o r absence of HLA an tibody o r individu cell panels can be analyzed to define HLA an tibody specificity.

3.0 SPECIMEN REQUIREMENTS3.1 P a t ie n t P r e p a r a t io n - N /A

Prin t Status: U ncontrolled W hen P rin ted

01-Flow Crossmatch page 3 o f30

S p ec im en T3q3e & H and ling '

C r ite r ia : : tT ype

-P re fe r r e dRecipient:

Recipient (optional] /D onor: :

Serum

Cells - P eripheral blood

-O th e r A ccep tab le D eceasedDonor:

Cells - Spleen, lym ph nodes

C ollec tion C o n ta in e r Serum Plain red top V acutainer tube

Cells(blood] Green top (Sodium H eparin] vacu ta iner

Cells (tissue] Yellow top (ACD] vacu ta iner Or S terile con ta iner

V olum e - O p tim u m Serum 10 ml (b lood collection volum e]

Cells (blood] 40 ml

Cells (tissue] m ultiple p ieces of sp leen o r m ultip le nodes

V olum e- M in im u m Serum 2 ml (b lood collection volum e]

Cells (blood] 10 ml

Cells (tissue] single p iece of sp leen o r a single sizeable node.

T r a n s p o r t C o n ta in e r & T e m p e ra tu re

Serum

Cells (blood]

20-25°C

20-25°C

Cells (tissue] sh o rt term : 20-25°C (up to 72 hrs] long te rm : 2-6°C

S ta b ility & S to ra g e R e q u ire m e n ts

RoomT em perature:

Serum : 10 days Cells (blood]: 48 hours

Refrigerated: Serum : 1 - 3 m on ths Cells (tissue]: 1 w eek H arvested lym phocytes: 1 w eek

Prin t Status: U ncontrolled W hen Prin ted

01-FIow Crossmatch page 4 of30

C ritè r ia / ' l ' - ï -Frozen: Serum : -70°C: 5 years

B lood/Lym phocytes: -70°C fo ra m inim um of 5 years.DNA: -70°C for a m inim um of 5 y ea rs if no o th e r donor tis su e /b lo o d is available.

T im in g C o n s id e ra tio n s N/A c'U n a c c e p ta b le S p e c im e n s & A ctions to T a k e

1. Sam ples th a t do n o t m eet lab o ra to ry labeling c riteria o r a re technically unsu itab le for testing:• Grossly hem olyzed (red to p vacutainer]• W rong vacu ta in e r type• Insufficient quan tity• O lder th an 10 days from d raw date p rio r t o .

p rocessing (serum )• Older th a n 48 hours from d raw date (green

o r yellow top vacu ta iner). However, deceased d o no r b loods o lder th an 48 hours m ay be p rocessed a t th e req u es t of th e GOL.

2. These sam ples a re accessioned into H isto trac as UNACC sam ples w ith a com m ent indicating the reason for rejection.

3. Serum th a t is g rea te r th an 30 days requ ires p rio r approval before use in a crossm atch

C o m p ro m is in g P hysica l C h a ra c te r is tic s

N/A

O th e r C o n s id e ra tio n s N/A

4 .0 REAGENTSRefer to the M aterial Safety Data Sheet (MSDS) supp lied w ith the reag en ts for com plete safety hazards. Refer to th e section in th is p rocedu re covering "SAFETY" for additional inform ation.

4 .1 R e a g e n t S u m m a ry

R e a g e n ts Supjplier & C atalog N u m b e r Q u a n tityRosette Sep Stem cell technologies -1 5 2 6 3 10 m L/boxP hosphate Buffered Saline (PBS)

G ib c o -14190144 500m L /bottle

Ficoll Paque Plus F isher H ealthcare - 45-001-749 100m L /bo ttle

Print Status: Uncontrolled When Printed

01-FIow Crossmatch page 5 of 30

H eat inactivated Fetal Calf Serum(FCS)

Gibco -1 6 1 4 0 -0 7 1 SOOm L /bottle

P ronase (P ro tease Type XIV Bacterial from Streptom yces Griseus")

Sigma Aldrich - PS 147 100 m g/vial

D eoxyribonuclease I(DNase) MP Biomedicals - 100S7S S m g /bo ttleH um an AB Serum (Off the Clof)

Gemini - 100318 100 mL bottle

Positive contro l se ru m In house control •

CD3 PCS M onoclonal A ntibody

Beckm an Coulter - IM263SU 1 m L/vial

CD19 PE M onoclonal A ntibody

Beckm an Coulter - IM268SU 2 m L/vial

FITCconjugated AffiniPure F(ab']a Fragm entG oat Anti- H um an IgG Fey Fragm ent specific

Jackson Im m unoR esearch Lab -109 -0 9 6 -1 7 0

1.0 m g/vial

Paraform aldehyde 16% Electron M icroscopy Sciences - 1S710

10 m L/vial

Flow Check Beckm an Coulter - 660S3S9 10 m LvialMESF beads Bangs Labs - SSS 7 mL vialRPMI Invitrogen - 22400097 100

m L /bottleSodium Azide ICN/MP Biom edicals - 1 0 2 8 9 1 2S gm /bo ttleA m m onium Chloride F isher Scientific 100

g m /bo ttleEDTA ICN/MP Biomedicals 100

g m /b o ttlePotassium Phosphate F isher Scientific 100

g m /b o ttlePenicillin-Streptom ycin Invitrogen - 1S1440122 100

m L /bo ttle

4.2 Reagent Preparation and StorageNOTES: Date and initial all reagen ts upon opening. Each con ta iner m u st be labeled w ith (1) substance nam e, (2) lo t num ber, (3] da te of p repara tion , (4) exp iration date, (5) initials of tech, (6) any special s to rage instructions; check for visible signs of degradation .

Refer to th e M aterial Safety Data Sheet (MSDS) for a com plete descrip tion of hazards. If a specific h azard is p resen t, it will be n o ted in th is p rocedure w hen the h azard is first encoun te red in a p rocedura l step.


01-Flow Crossmatch page 6 of30

Reagent Rosette Sep®R eagent is used to enrich lym phocytes from w hole blood. RosetteSep an tibody cocktail crosslinks u nw an ted cells in hum an w hole b lood to RBCs form ing im m unorosettes.This increases the density of th e unw an ted (ro se tted ] cells, so th a t they pelle t along w ith the free RBCs w hen centrifuged over a buoyan t density m edium .

Container 4 ml tu b e fbox of 5)Storage Store a t 4°CStability Stable until th e labeled expiration if s to re d according to

m anufactu rer's recom m endation.Preparation N/AQuality Control New Lots and Shipments

In-use reagent. Does no t req u ire a sep ara te QC.

Reagent Penniciilin-Streptomycin (Penn-Strep)Container BottleStorage Bulk bottle: -20°C

Aliquots: -20°CStability Stable until m an u fac tu rer’s exp iration datePreparation T haw bu lk bottle.

P repare 1.1 mL aliquo ts and sto re a t -20°C

Quality Control New Lots and Shipments

In-use re a g e n t Does no t requ ire a se p a ra te QC.

Reagent Fetal Calf Serum (Purchased Heat Inactivated)Container 500 mL bo ttleStorage Store a t-20°CStability Stable until th e labeled expiration if s to re d according to

m anufactu rer’s recom m endation.Preparation Before use:

Thaw a 500 mL bottle, and using sterile techniques, aliquot 5.25m l of th e FCS into 15mL ste rile tubes. S tore a t -20°C.


■ P repare one 2% FCS-PBS on one Flow W ash w ith the new lo t/sh ip m en t of FCS and use for T an d B cell isolation for flow crossm atch. T est in para lle l w ith cells iso lated for flow crossm atch using cu rre n t lo t FCS reagents.■ P repare 5% RPMI-FCS solution and ru n on a frozen cell tra y using AHG-CDC assay. No cell d e a th shou ld be observed.



Reagent Phosphate Buffered SalineContainer 500 mL bo ttleStorage Store a t room tem p era tu reStability • Stable until th e labeled exp ira tion if s to re d according

to m anufactu rer’s recom m endation .Preparation N/AQuality Contol New Lots and Shipments

T est new lots of PBS in paralle l w ith th e c u rre n t lo t during cell isolation for flow crossm atch.

Reagent 2%FCS-PBS solutionContainer 500 mL bo ttleStorage Store a t 4°CStability Expiration 2 m onths from p rep a ra tio n date.Preparation To a 500 mL bo ttle of PBS;

• Add 2-5.25mL Fetal Calf Serum (FCS) aliquots* Add 2 a liquots of l . lm L Pen-Strep

Quality Control In-use reagent. Does n o t req u ire a se p ara te QC.

Reagent Flow WashContainer 500 mL bo ttleStorage Store a t 4°CStability Expiration 2 m onths from p rep a ra tio n date.Preparation. To a 500 mL bo ttle of PBS:

• Add 5 mL sodium azide s to ck so lu tion (10% ].• Add 5.25 mL h e a t inactivated FCS f 1 aliquot].


Reagent Ficoll Paque PlusContainer Brown Glass BottleStorage Room T em peratu reStability • Stable until th e labeled exp iration if s to re d according to

m anufactu rer’s recom m endation .• Do n o t use if th e reag en t is cloudy, has a d istinct yellow

color, o r show s signs of contam ination .Preparation N/A____________________ ________________________________Quality Control New Lots and Shipments

In-use reagent. Does no t req u ire a se p ara te QC.

R e a g e n t PronasePrint Status: Uncontrolled When Printed


Container Brown bottleStorage Stock an d aliquots bo th s to red a t-20°C

Stability Stock: Stable until the labeled exp ira tion if s to red according to m anufactu rer's recom m endation. Aliquots: Stable for 1 y ea r from p re p a ra tio n date.

Preparation Dissolve Stock 100 m g and QS to 100 ml w ith PBS. Aliquot in to 0.5 m b in to 2mL sto rage tu b e label.


Perform flow crossm atch on one cell t re a te d th ree ways: o W ith no P ronase trea tm en t,• P ronase trea tm en t w ith c u rre n t p rep a ra tio n of

Pronase,• P ronase trea tm en t w ith new lo t/p re p a ra tio n of

Pronase.

Reagent Shock SolutionContainer 50 mL tubesStorage Store a t 4°CStability Expiration one year from p rep a ra tio n date.Preparation Add:

• B.Og Am m onium Chloride (NH4CL). 1.0gEDTACC10H14N208Na22H20)• O.lg Potassium Phosphate (KH2P04)

QS to 1 lite r w ith Type 1 w a te r, an d pH to 7.0 + /- 0.2 (very im portan ti


T est new p rep a ra tio n of Shock Solution in paralle l w ith cu rren t lo t during cell iso lation for flow crossm atch. W hen possible, please use living d o n o r cells.

Reagent , DNase - 500 KunitzContainer 2 mL b o ttle ___________________-Storage • Stock s to red a t 4°C in a dessica to r

• Aliquots from p rep a ra tio n are s to re d a t -20°C.

Stability DNase stock stable for 2 years from receive date, aliquots 6 m onths from p rep a ra tio n date.

Preparation R econstitu te 1 bo ttle of DNase w ith 20m l of PBS.To 1.5ml m icrocentrifuge tu b es add l.OmL of the reconstitu ted D N A S E . ______________ ______ _____


Perform flow crossm atch in para lle l w ith in use uN ase

I Reagent I Paraformaldehyde


01-FIow Crossmatch page 9 of30

Container Glass vialStorage Store a t 4°CStability • Stock: Stable until the labeled exp ira tion if s to red

according to m anu factu rer’s recom m endation.• W orking Solution: Expires one m on th from p repara tion

date.Preparation 1% Paraform aldehyde Fixative W orking solution:

ToaSOOmL PBS bottle:• Add 3 vials of 16% paraform adehyde.• Add 30|4.L of NaOH.0 A djust pH to 11-1A reco rd pH on bottle.


Reagent Sodium Azide - Health Hazard - refer to MSDS sheetContainer Foil covered 250 mL corning sto rage bo ttle

Storage Room tem p era tu reStability Stock: 4 years from da te of rece ip t

10% solution: One y ea r from p rep a ra tio n d a te ,Preparation 10% Sodium Azide solution:

25g of Sodium Azide (one stock bo ttle) QS to 250m L w ith PBS.

Quality Control New Lots and

In-use reagent. Does no t req u ire a sep ara te QC.

Shipments _________________________ _________________________________

Reagent FITC conjugated AffiniPure F (ah’) a Fragment Goat Anti- Human IgG Fcy Fragment specific

Container Brown vialStorage Store freeze d ried po w d er a t 4°C , w orking so lu tion 4 C

Stability Freeze d ried stock expires one y ea r from receive date, w orking solution 6 w eeks from p rep a ra tio n date.

Preparation Before reconstituting new lots of this reagent, please refer to the product insert to ensure that the following instructions still apply: reco n stitu te w ith 750 mL of t 5^ e I w ater.




• Each new lo t of IgG reag en t is quality controlled using a checkerboard titra tio n assay using seria l dilutions of the new an tibody vs. dilutions of p a tie n t pooled positive.

• The new lo t is te s ted in paralle l w ith th e cu rren t lot (the cu rren t lo t is te s te d a t its c u rre n t dilution).

o The optim al w orking dilution for th e assay should provide bo th sensitiv ity as w ell as an acceptable background level. All an tibod ies are u n d e r constan t QC and control evaluation during use.

Reagent CDS PCS Monoclonal AntibodyContainer Brown vialStorage Store a t 4°CStability Vials should be p ro tec ted from pro longed exposure to light.

M ust be used before th e m anufactu rer's expiration date.Preparation N/AQuality Control New Lots arid Shipments

New lots and sh ipm ents of m onoclonal an tibod ies are quality controlled by runn ing the old and new lo ts /sh ip m en ts in parallel.

Reagent CD19 PE Monoclonal AntibodyContainer Brown vialStorage Store a t 4°CStability Vials should be p ro tec ted from pro longed exposure to light.

M ust be used before the m anufactu rer's exp iration date.

Preparation N/AQuality Control New Lots and Shipments

New lots and sh ipm ents of m onoclonal an tibod ies are quality controlled by runn ing the old an d new lo ts /sh ip m en ts in parallel.

Reagent Human AB Serum (Off the Clot)Container Plastic bo ttle /v ia lsStorage Bulk: Store a t-70°C

Aliauots: S tored a t -20°CStability Use p rio r to m anufactu rer's expiration date.

Preparation The bo ttles a re thaw ed quickly and opened u n d er lam inar a ir hood. The se ru m is th en h ea t inactiva ted a t 56°C for 30 m inu tes and th en filtered. The serum is th en aliquoted into app rop ria te vials and sto red a t -20°C.




Refer to section 6.2 Control P repara tion and S to ra g e .

Reagent Pooled Positive Control fPPCIContainer Plastic bo ttle /v ia lsStorage Stock -70°C, aliquots -20°CStability Lab assigned expiration date: 4 years from p repara tion

date.Preparation Control is m ade in-house from pool of high PRA p a tien t

sera. A fter se rum is fully evaluated and app rop ria te dilutions are determ ined, the se ru m can be aliquoted and u sed for testing.

Quality Control Refer to section 6.2 Control P rep ara tio n and Storage.

Reagent Flow CheckContainer Plastic vialStorage 4°CStability • Stock: Expiration date as p e r m anufactu rer

• Aliquot: d iscard afte r 48 hoursPreparation 15 drops in plastic am ber flow tu b e d iscard a fte r 48 hours

Quality Control See procedure: A.05.06 Flow Cytom eter Calibration and M aintenance

Reagent MESF beadsContainer Brow n plastic vialStorage 4°CStability • Stock: Expiration date as p e r m anufactu rer. Keep

con ta iner closed and p ro tec ted from light.• Aliquot: d iscard afte r 48 hours

Preparation 2 d rops in O.SmL PBS in p lastic am b er flow tube. Discard a fte r 48 hours

Quality Control See procedure: A.05.06 Flow C ytom eter Calibration and M aintenance

5.0 CALIBRATORS/STANDARDS

5.1 C a lib ra to rs /S ta n d a rd s U sedCalibrator SupplierFlow Check Beckm an CoulterMESF Bangs Labs



5.2 C a lib ra to r P re p a ra t io n a n d S to ra g eNOTE: D ate and initial all ca lib ra to rs upon opening. Each con ta iner m ust be labeled w ith (1) substance nam e, [2) lo t num ber, (3) date of p repara tion , (4] exp ira tion date, [5) initials o f tech (6) any special sto rage instructions; check for visible signs of degradation.

C a lib ra to r F low C heckP r e p a ra t io n Vortex the stock bottle well

Add 15 d rops to an am ber flow tube.D iscard after 48 hours

S to rag e Store in the da rk a t 2-8°C

C a lib ra to r MESF b e a d sP r e p a r a t io n Add 2 d rops to 0.5mL PBS in am b er tube.

D iscard afte r 48 hoursS to rag e Store in the d a rk a t 2-8°C

5.3 C a lib ra tio n P ro c e d u re - see p rocedure A.05.06 Flow C ytom eter Calibration and M aintenance

6.0 QUALITY CONTROL

6.1 C o n tro ls U sedC on tro l S u p p lie rNegative Control GeminiPooled Positive Control P roduced by the labo ra to ry

6.2 C o n tro l P r e p a r a t io n a n d S to rag eNOTE: D ate and initial all contro ls upon opening. Each con ta iner should be labeled w ith (1) substance nam e, (2) lo t num ber, (3) da te of p repara tion , (4) exp iration date, (5) initials of tech, and (6] any special sto rage instructions; check for visible signs of degradation

N egative C o n tro l H u m an AB S e ru m (Off th e Clot)

Quality ControlPurchased in bulk, therefore, only new lots are quality controlled. The negative control QC may need to be repeated when a major FC500 repair is performed to validate the established control ranges.

1. W hen possible, m ultiple lo ts of H um an AB sera should be o rd ered for evaluation.

2. AB se ru m m ust be h ea t inactivated a t 56°C for 30 m inu tes p rio r to use.

3. Each QC lo t is te s ted in paralle l w ith th e cu rre n t lot using the flow crossm atch procedure:

a. A panel of cells m u st be te s ted for evaluation.b. The cell panel should include cells p rep ared from



periphera l blood, lym ph node and spleen.c. The cell panel should include m any different HLA

phenotypes.d. T an d B cell fluorescence of th e QC lo t is

com pared to th e cu rren t lo t4. QC lots a re also te s te d on a frozen cell tra y using the

CDC-AHG assay. Results:a. The fluorescence obtained from th e cell panel

shou ld reflect resu lts ob tained from negative p a tien t se ra in the flow crossm atch assay.

b. A sep ara te range shou ld be estab lished for bo th T and B cells calculating the m ean and + 2 s tan d ard deviations. The range can be specified as linear o r MESF values.

c. No reactiv ity (cell death ] shou ld be observed on the frozen cell tray.

5. Ongoing assessm en t of negative control values areperfo rm ed utilizing resu lts of th e SAB assays. The crossm atch resu lts of negative an tibody pa tien ts should be sim ilar to th e negative contro l value.________________

P o s itiv e C on tro l P o o led H um an S e ru m d e m o n s tra t in g HLA A n tib o d ie sP repara tion 1. Pooled se ra from 10-20 pa tien ts th a t dem onstra te a high

t i te r of class 1 a n d /o r class 11 HLA antibodies:a. Sera w ith b ro ad HLA specificities m u st be used.b. Sera m ust be filtered p rio r to tes ting to rem ove

aggregates w hich m ay clog th e flow cytom eter.c. Sera m ust be tes ted against a panel of cells w ith

know n HLA types to evaluate s tren g th of reactivity.

d. D eterm ine th e d ilu tion req u ired to achieve a AMESF shift of approxim ately 10,000- 60,000.

6.3 Frequency

T57pe o f R u nM in im um N ú m b e r o f QC : ’ - s a m p le s • =

QC P e r c e n t o f B atch Size \

T ested w ith eachrec ip ie n t/d o n o rcell

1 negative (tested in duplicate] 1 positive control

V ariable

6.4 T o le ra n c e L im its



The range estab lished for the negative and positive con tro l is a guideline. Not every cell te s ted will fall w ith in th is average range. Control values will provide an indication of a ssay sensitiv ity to assist w ith th e crossm atch in terp re ta tion . O ut-of-range controls do n o t necessarily invalidate th e assay, how ever, a pa tien t's an tibody p ro file /d iag n o sis /tran sp lan t history/H LA typing m u st be rev iew ed carefully before reporting crossm atch resu lts.

6.5 R ev iew P a t ie n t D a taReview pa tien t resu lts for unusual pa tterns, tren d s o r d istribu tions in pa tien t resu lts , such as an unusually high percen tage of abnorm al resu lts, o r unusually high percen tage of non-reactive, o r indeterm inate , o r reactive results.C om puter a ided tools should be u sed w hen available.

6 .6 D o c u m e n ta tio nRefer to d e p a rtm en t policies and p rocedures for QC docum en tation and to records m anagem en t p rog ram for reco rd re ten tio n requirem ents.

6 .7 Q u a lity A ssu ra n c e P ro g ra m __________________________________________________Instrument In stru m en t perform ance m ust be verified daily to ensu reControl Crossm atch resu lts a re optim al. S tandards are ru n to check

lase r output, fluidics, optim al a lignm ent and signal detection.F low Check: Defined rangeMESF B ack C alcu la tion E rro r : <15% (percentage m ay varyslightly depending on lo t of MESF in use].

7.0 EQUIPMENT a n d SUPPLIES7.1 A ssay P la tfo rm

Beckm an Coulter FC 500 Flow C ytom eter

7.2 E q u ip m e n tM icrocentrifugeCentrifugeFC500 Flow C ytom eter P ipettes: lOul, 200ul, lOOOul VortexIncubator: 23°C and 37°C37°C Bead Bath2-4°C R efrigeratorM icroscopeH em acytom eterP ipette AidScissorsScalpel

P rin t Status: U ncontrolled W hen Prin ted


Forceps

7.3 S u p p lie s50 ml Conical Polypropylene Centrifuge Tubes 100 X 15 m m T issue Culture Dishes Syringe: 1 ml and 3 ml 27 Vi G syringe needle 12 X 75 m m polystyrene cu lture tubes F isher tubesSerological P ipettes: 10ml, 25m l P ipette tips: 200ul, lOOOul M icrocentrifuge tubes M icroscope cover slips T ransfer P ipettes: 1.0ml, fine tip Cryovials

8.0 PROCEDURENOTES:1. For all p ro ced u res involving specim ens, b u ttoned lab coats, gloves, and face

p ro tec tion a re requ ired m inim um persona l p ro tective equipm ent. R eport all accidents to y o u r supervisor.

2. The package in se r t for a new lo t of k its m u st be rev iew ed for any changes before the k it is used.

8.1 L ym phocy te Iso la tio n (L ym ph N o d e s /S p le é n ]1. P reparing the tissue:

1. Trim fat off of several nodes w ith scissors o r c u t a Icm ^ of sp leen from the h ea lth ies t and least necro tic po rtion available.

2. Place n o d e /sp leen into a 100 m m tissue cu lture dish.2. Injecting the node/sp leen :

1. Using a syringe, in ject the n o d e /sp leen w ith fresh RPMI-fPen-Strep several tim es.

2. The m edium coming o u t of th e node should be cloudy w ith cells. The m edium com ing o u t of th e sp leen will be bloody.

3. Continue injecting the n o d es /sp leen until th e m edium com es o u t clear.4. A m inim um of 6 ml of lym phocyte enriched m edium should be collected.

3. 1. T ransfer the cell ex trac t into 6 fisher tubes an d sp in for 1 m inu te a t 1000 x g in the F isher centrifuge. Take off s u p e rn a ta n t

2. You shou ld now have 6 cell bu ttons.a. Use 3 for lym phocyte purification for flow crossm atch.b. Use 3 for m olecular typ ing/freezing.

i. Combine the 3 cell bu ttons w ith Im L of flow w ash.ii. Perform a cell count using a hem acytom eter. Optimal cell


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01-FIow Crossm atch page 16 of 30

concen tra tion for DNA extraction shou ld be 5x IQS to 1.5 x lO ^/m L lym phocytes. I.e.20 cells on five secondary squares in Im L of flow w ash is equal to 1 x 10^ cells.

iii. D ilute o r concen tra te th e cell p rep as need ed to obtain the optim al cell concentration.

iv. Place the cell so lu tion in a labeled cryovial for DNA extraction. See p rocedu res C.Ol- DNA EXTRACTION AND QUANTIFICATION.

3. Place u sed n o d e /sp le en in a labeled cryovial con tain ing RPMI W ash.4. S tore a t 2-6°C.

8.2 L y m p h o cy te Iso la tio n (P e r ip h e ra l B lood)1. Ros ette Sep T reatm ent:

1. For deceased donors only: Aliquot l-2 m l of p e rip h e ra l b lood in to a cryovial an d s e t aside for m olecular typ ing/freezing .

2. Add 8-lO m l o f p e rip h e ra l blood to each 50 ml conical tu b e . For each mL of b lood in the tube, 40pl of RosetteSep shou ld be added. Make su re to mix well.

3. Incubate for 20 m inu tes ,a t room tem pera tu re .4. A fter incubation, dilu te sam ple w ith an equal volum e of PBS + 2% FCS and

mix gently.2. LSM:

1. For RosetteSep tre a te d periphera l blood:a. Gently und erlay mbcture w ith 15 ml of lym phocyte sep ara tio n

m edium (LSM).b. Centrifuge for 20 m inu tes a t 400 x g (1180 rp m on th e RC3B) a t room

tem p era tu re . Ensure th e centrifuge se ttings allow for a slow s ta r t and a slow stop .

c. A fter centrifugation, rem ove the l)nnphoc3rte en riched layer from the LSM p lasm a in terface and tra n sfe r in to 50 ml conical tubes. To en su re com plete recovery, it is recom m ended to rem ove som e of the LSM along w ith th e lym phocyte cell layer.

d. A dd an equal volum e o f PBS+2%FCS to th e l5nnphocyte layer th a t w as collected an d centrifuge for 10 m inu tes a t 400 x g (1180 rpm on the RC3B) w ith the brake on.

2. For non-R osetteSep tre a te d periphera l blood:a. A dd 8-lO m l of p e rip h era l b lood to a 50 ml conical tube.b. Add PBS to th e periphera l b lood to achieve an approx im ate volum e of

35-40 ml.c. U nderlay the PBS+blood m ixture w ith 10 ml o f LSM.d. Centrifuge for 35 m inu tes a t 400 x g (1180 rp m on th e RC3B) a t room

tem p era tu re . Ensure the centrifuge se ttings allow for a slow s ta r t and a slow stop.



e. A fter centrifugation, rem ove the lym phocyte en riched layer from the LSM p lasm a in terface and tra n s fe r in to 50 ml conical tubes. To en su re com plete recovery, it is recom m ended to rem ove som e of the LSM along w ith th e lym phocyte cell layer.

f. Add an equal volum e of PBS to th e l5nnphocyte lay er th a t w as collected and centrifuge for 10 m inu tes a t 400 x g (1180 rpm on the RC3B) w ith th e b rake on.

3. Rem ove the superna tan t:a. If th e cell b u tto n is com pact: carefully, b u t quickly, p o u r off the

su p ern a tan t. W hile inverted , b lo t tubes w ith a p a p e r tow el.b. If th e cell b u tto n is n o t com pact o r if th e re is excessive red blood cell

contam ination: Use a p ipette to rem ove th e su p e rn a ta n t4. Com bine all th e cell b u ttons in th e conical tubes u sing 3m l of PBS. T ransfer

th e cell suspension in to labeled fisher tubes.5. Spin for 1 m inu te a t 1000 x g in th e F isher centrifuge.6. Take off s u p e rn a ta n t______________________________________________________ -

8.3 Lymphocyte Purification1. M onocyte D epletion (perform ed if source is sp leen o r non-R osetteSep blood).

1. R esuspend th e lym phocyte cell bu tto n w iü i 3 ml of RPMI W ash.2. Add cell suspension to 100x15 m m p e tr i dish. D istribute cell suspension

evenly across th e en tire surface of th e p e tr i dish.3. In c u b a te a tro o m te m p e ra tu re fo r 15 m inutes.4. Collect lym phocytes by tilting p e tri d ish and rinsing th e p late 4 o r 5 tim es

w ith th e cell suspension.5 T ransfer th is m onocyte-depleted cell suspension to (3) 1.0 ml fisher tubes.6. W ash cells 2 tim es w ith PBS. (Spin a t 500 x g in th e F isher centrifuge for 1

m im itp. This "soft sp in” will help to elim inate p la te le t contam ination^-----------

2. Red Blood Cell (RBC) Lyses (perform ed if cells a re co n tam ina ted w ith RBCsJ.1. W arm th e Shock so lu tion in th e 37°C bead b a th before use.2. I f th e re is a small am oun t o f re d b lood cells re -su sp en d the ly m p h o c^ e

pelle ts w ith 1 ml of Shock solution. If th e re is a large am o u n t of re d blood cells sep ara te th e cell b u tto n into m ultiple labeled fisher tubes. Add 1ml of shock to each labeled tube.

3. Incubate for 10 m inu tes a t 37°C.4. Spin th e tu b es a t 1000 x g in the F isher centrifuge for 1 m inute.5. Rem ove the su p e rn a ta n t and w ash 2 tim es w ith PBS. , , .6. A large concen tra tion of re d b lood cells has the capability of overw hdm m g

th e shock solution. Incom plete lysis of re d b lood cells is a sign th a t shock has b een overw helm ed. An a tte m p t to re-shock the cells shou ld be p e rfo rm e d —

3. Mini LSM (perfo rm ed if source m ateria l is from node o r sp leen)1. Fill 2 F isher tu b es ha lf full w ith LSM m edia.2. P u t 1-2 d rops of th e RPMI w ash onto the LSM in each tu b e ----------------- -----------


Ol-FIow Crossmatch page 18 of 30

\Wl ^

3. Gently layer your cell solution on top of the RPMl wash layer in each tube4. Centrifuge for 3 minutes at 1000 x g in the Fisher centrifuge using a slow

start-up (Set the speed setting to 0 then slowly adjust the speed to 1000 x g.)5. Carefully remove and save the layer (interface) in another labeled fisher

tube.6. Wash the cells from the layer 2 times with PBS for 1 minute at lOOOg and

discard the supernatant.4. Platelet Spins (optional): To remove platelet contamination:

1. Spin cell suspension at 500 X g in the Fisher centrifuge fo ri minute2. Remove the supernatant and resuspend with Flow wash. This "soft spin

will helo to eliminate platelet contamination.

8.4 Pronase Treatment1. Pronase Treatment (Always performed):

1. The optimal pronase to cell ratio is SOOul of pronase per 10 x 10 cells or approximately 200 cells across 5 secondary squares in ImL. This pronase to cell ratio is very important for the performance of the pronase treatment. If the cell concentration is too high pronase treatment will be less effective and may cause an increase in background reactivity in your flow crossmatch.

2. When performing the cell count using the hemacytometer make sure tocount the total cell concentration and not just the lymphocyte concentration. Pull enoughipronase to accommodate the number of cells that you have in your cell prep; Pronase treatment does not need to be performed on all of the cells in a prep if there is a large quantity of cells. Set-aside a quantity of the cell prep in a labeled fisher tube with the notation NOT pronase treated on the label.

3. Add Pronase to your cell button and pipette up and down gently to mix. Depending on the cell count you may have multiple tubes to pronase. Each fisher tube should only contain enough cells to be treated with 1 aliquot of pronase.

4. Incubate the pronase treated cells at 37°C for 30 minutes.5. To each fisher tube that was pronase treated: add 1 aliquot of DNAse (500

Kunitz/lml/aliquot) to the cell mixture and mix well to dissociate the clumps. Spin at 1000 x g for 1 minute and discard the supernatant.

6. Add 1ml of PBS and mix well, Spin at 1000 x g in the Fisher centrifuge for 1 minute, and discard the supernatant. Repeat this step for a total of 2 washes.

7. Combine the cell buttons for each patient using flow wash.

8.5 Flow Crossmatch Setup __________________ ____1. Counting and Adjusting Cells:

1 RpciicppnH fhe cell button with 1ml of Flow wash.2. Using a hem^fcytometer determine if there is a large number of non-

Ivmphocytes. I f there is perform steps in section 8.3 Lymphocyte


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Purifícatinn. A crossmatch which is performed in the presence of a large number of non-lymphocyte may exhibitfelse^negativà nr wpak rpartivity. The laree hunger of non-lvmphocvtes wilT&ind antibodies and decrease the antibody availability to the HLA antigen. If you cannot eliminate rnntaminating cells, vou must include these cells in the cell count.

rmint- anH pnrifieH T cell and R cell Ivmohocvtes to 7.50 X lO^.using th e^jTowWash b uff^ solution as the diluent flSO cells across 5 RBC squares),

4. I.ahel the fisher tube with "T/B dii" if the tube contains adjusted and diluted cells or with just "T/B" if the cells in the tube have not been adjusted.

2. Sample Preparation:1. Pull patient sera and verify the patient identifiers and sample accession

numbers.2. Check thé\?:nntrol Worksheet that is posted in the mam lab for the proper

_dilution of positive controls to be used. Pull the positive ccintr^l-and npp^ivp control

3. ^ r te ^ a l l controls and sera then spin all sera in the microline 12 mirmcenl-rifngèTor 5 minutes at lO.OOO rum f7,176 Xg).

4 Proporo yniir rocf fiihpc fnr crn<;smatrhing; You will need 2 negative control tubes. 1 positive control tube, ancí¿tube^ for each patient sera tested.

3. Labeling: " ■1. Label all tubes.(12 x 75 mm polystyrene tubes) with the cell label.2. Label 2 negative control tubes as3. Label 1 positive control tube as PPG. ,4. Label patient serum tubes with patient serum labels (each patient is run and

duplicate and should have 2 tubes per serum sample).a. Patient historical serum: On the serum label indicate one tube as the

1:1 dilution and the second tube as the 1:2 dilution. The historical dilutions are^o^run in duplicate.

4. Flow Crossmatch set-up:1. Add 40 pi of-patient serum or controls to their corresponding labeled tubes.9 Afifl 4-n |ii. of thp purified Ivmphocvte cell suspension that was adjusted

directly into the serum of each tube,9 Miv ijp and down with the pipette to make sure that the serum and cells have

been thoroughly mixed.4. Place the flow tubes in a centrifuge tube carrier5. Vortex tubes and incubate the tubes for 30 minutes at 23°C in the room

temperature incubator.6. Remove the tubes from the incubator after 30 minutes.7. Add 1 mL of Flow Wash to each tube and vortex.8. Centrifuge for 5 minutes at 400 x g (1180 rpm on the RC3B)9. Decant and blot the tops of the tubes with a folded paper towel while the

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10. Repeat wash steps 6-8 for a total of 3 washes.11. During the last spin you should make up the conjugate master mix.

5. Conjugate Master Mix:• The conjugate master mix contains: FITC Anti-lgG Conjugate, CD3-PC5, and

CD19-PE in a Flow Wash buffer diluent.• Each lot of FITC Anti-lgG Conjugate is tested to determine the optimum

working dilution and a FLOW CROSSMATCH CONJUGATE MASTER MIX TABLE is then created.

1. Identify the current lot of FITC Anti-lgG conjugate.2. An approved FLOW CROSSMATCH CONJUGATE MASTER MIX TABLE is

posted in the laboratory for use. If the lot is incorrect locate the correct FLOW CROSSMATCH CONJUGATE MASTER MIX TABLE in the QC file for the current lot.

3. Make your Conjugate Master Mix according to the tables:a. Prepare FITC/Flow Wash Buffer Mix.b. Prepare Conjugate Master Mix using the FlTC/Flow Wash Buffer Mix.

4. Add 50ul of the Conjugate Master Mix to each tube. Then vortex and in n ih ate the tubes for 20 minutes at 4°C in the dark. (Place centrifuge holders intotherefrigeratorj. -----

6. 2"'* Incubation and Wash:1. Remove the fubes from the fridge and add 1 mL of Flow Wash to each tube

and vortex. Centrifuge for 5 minutes at 400 x g (1180 rpm on the RC3B). Decant and blot the tops of the tubes with a folded paper towel while the tubes are inverted.

2. Repeat wash step for a total of 3 washes.3. Add 0.5 ml 1% Paraformaldehyde Fix to all tubes and vortex.4. The crossmatch is ready to be placed on the flow cytometer.

8.6 FC500 : Flow Crossmatch1. Instrument Start-up:

1. Log into the FC500 program under the user ID Crossmatch2. Startup - Follow the steps outlined in procedure A.05.06 - Flow Cytometer

Calibration and Maintenance.3. Flow Check - Follow the steps outlined in procedure A.05.06 - Flow

Cytometer Calibration and Maintenance.4. MESF - Follow the steps outlined in procedure A.05.06 - Flow Cytometer

Calibration and Maintenance.2. Creating a Protocol:

1. Clear the workspace. Then in the menu bar select FILE > OPEN PROTOCOL > QC_CrossmatchTemplate.PRO.

2. Once the default QC„CrossmatchTemplate.PRO has been loaded, 10 histograms will open up on the workspace on the left monitor. If you do not



have 10 histograms inform a supervisor.3. Save this default crossmatch protocol under a new file name. The "QC_” must

be in front of all file names. This identifier will prompt the FC500 to output the acquired data in the proper format.

4. The correct file name for donor UNOS AB1234 should look like this:QC_UNOS AB1234.PRO. If the protocol already exists you may add additional identifying characters such as your initials to make the file name unique.

5. Once saved the protocol name will appear in the Protocol column heading in the acquisition manager. If it does not show up under this column, the data will not be saved or imported properly. You will need re-create/re-open the protocol.

a. To re-open a protocol go to the menu bar and select FILE > OPEN PROTOCOL and in the window the opens locate your newly created protocol and select OPEN.

3. Creating a Worklist:1. Using the ADD TUBE button add the appropriate number of tubes to perform

the crossmatch.a. Add an additional tube for^ESI^A MESF tube MUST be run with

every crossmatch. If there is no MESF beads available foracquisition, water may be run in its place. Remember to make a comment on the resulting crossmatch report that MESF was not run.

2. The specific information related to your run needs to be entered into the following required.column headings of the worklist.:

3. Carousel No. = Enter the carousel number.4. Location = Enter the carousel position of the first tube. The remainder of

the rows will auto-fill with incrementing tube numbers based on the number entered in the first row. If specific tube locations are required these locations must be manually entered.

a. The first tube run in the crossmatch should not be a patient or control tube. The first tube should contain MESF or water. This is important when creating the crossmatch report. Failure to do this will result in incorrect data analysis on the crossmatch report. To correct this error refer to the procedure A.05.07 FC500 Troubleshooting Guide.

5. Cell Name = Enter the Donor UNOS or Cell Name and MRN6. Cell ID = Enter the Cell accession number7. Serum Name.= Enter the serum last name, first initial, and MRN # or if it is a

control MESF, NHS, or PPC. .a. For patients with historical samples: The first set of tubes should be

the patient's current sample. The second set of tubes should be the patient's historical sample. For the historical sample make sure that the dilution factor is included in the serum name. "The 1:2 historical sample should be run prior to running the 1:1 historical sample.

Example:H1.2 Doe 112345678



H l.l Doe J 123456788. Serum ID = E nter the Serum accession num ber o r if it is a control MESF,

NHS,orPPC.9. Check:

• The MESE tube is nam ed: MESE• The negative sam ples are nam ed: NHS• The positive sam ple is nam ed: PPG• All se ra th a t a re run in duplicate have the sam e accession num ber• For historical sam ples m ake sure th a t the dilution factor is included in

th e Serum Name and th a t the 1:2 historical sam ple is se t to run p rior to runn ing the 1:1 historical sample.

10. Save the w orklist. The file nam e for th e w orklist can be saved as the date and initials of the technician i.e. 082710MB.WLS

4. Running th e Crossm atch:1. Load th e corresponding sam ples into the ap p ro p ria te carousel num ber and

position.2. P ress the play bu tton to s ta r t running your crossm atch.3. The first tube should be your MESE tube. Stop the MESE tube w hen the y-

axis of the MESE p lo t reaches approxim ately 125.4. During the crossm atch acquisition the FC500 will c rea te an excel sheet w ith

th e acquired data (QC_output.xls) the ou tpu t nam e will be b ased on your pro tocol nam e.

5. During the acquisition of the negative control ad just your lym phocyte gate to cen te r a ro u n d your lym phocyte population. A djust your T and B cell quad ran ts to include your T and B cell populations. Large adjustm ents are no t norm ally required .

6. A fter the acquisition is com plete run a cleanse panel.7. Perform th e shu t dow n procedure for the FC500 as needed follow the steps

in p ro ced u re A.05.06 - Flow Cytom eter Calibration and M aintenance.4. FXM R eport Tem plate and general analysis:-

1. Open the QC_ouput.xls file th a t was created for your crossm atch.2. Select all of the data and copy.3. Open the @FXM R eport Template.xls file and select the PASTE DATA button

th a t is on the Report Tab.4. Once th e data is im ported check the following:

a. Are all of th e sam ples p resen t on the crossm atch sheet? If they are not re fe r to p rocedure A.05.07 FC500 Troubleshooting Guide for help.

b. NHS - If th e 2 negative control values are n o t consisten t w ith each o th e r o r if the negative control values a re significantly ou t of range inform a supervisor. The crossm atch m ay need to be repeated.

c. PPG - If the PPG is significantly ou t of range inform a supervisor. The crossm atch m ay need to be repeated.

d. Patien t resu lts - If the 2 resu lts for the pa tien t are no t consistent with



each o th er inform a supervisor. The crossm atch m ay need to be repeated .

e. Cell counts - If the cell counts for any of th e crossm atch sam ples are less than 500 inform a supervisor. The crossm atch m ay need to be repeated .

5. Fill in additional inform ation such as receive dates, expiration dates, lot num bers, type of cell source, type of crossm atch, and th e nam e and date of th e person setting up, running, and im porting the crossm atch.

6. Save the @FXM R eport Tem plate w ith the nam e o f yo u r crossm atch protocol w ith the addition o f ‘rep o rt’ a t the end of the nam e: I.e. QC_output - report.xls

7. If any of th e crossm atches contain unusual resu lts you m ust p rin t the PDFrep o rts for each tube of your crossm atch. The PDF’s will be tu rn ed into the sup erv iso r along w ith your final paperw ork._______________________________

8.7 H is to tra c Im p o r t1. D eceased Donors - Refer to procedure B.03.03-DECEASED DONOR

PROSPECTIVE CROSSMATCHING for im port steps.2. Living D onors and AutoCrossmatches:

Perform th e flow crossm atch analysis following section 10.1 INTERPRETATION OF DATA. Then save a copy of the flow crossm atch rep o rt in th e Histotrac Im port Folder.

3. Im port Living Donors and AutoCrossm atch into HistoTrac:1. Open the Flow XM Interface utility in HistoTrac.2. Select the crossm atch rep o rt from the window.3. Verify th a t each pa tien t sam ple has an associated DXM, FLOWXM, or

AUTOFLOWXM te s t in the TEST column. If the te s t colum n contains NO TEST th en a crossm atch te s t w as not o rdered o r could n o t be m atched w ith th e se ru m /ce ll accession num ber. O rder the appropria te te s t o r correct your accession num bers using p rocedure A.04.07 - FC500 TROUBLESHOOTING GUIDE before proceeding.

4. If the p a tien t has a historical sam ple the data should appear in the IMPORT FLOW RESULTS w indow im m ediately following the p a tien t’s cu rren t sam ple resu lts. The RESULTS column will have a b lank field associated w ith the Historical.

5. Select APPLY.6. In th e RESULTS SCREEN select the Flow Crossm atch w orkspace to access the

list o f p a tie n t tests for reviewing.7. Open each test, verify the results, and fill in th e following fields:

a. In th e upper left:



• RESULT: overall crossm atch result. If runn ing a pa tien t au to crossm atch this field shou ld be se t-to AUTOCROSSMATCH.

• CELL: Cell accession num ber• CROSSMATCH DATE: Crossm atch date

b. In th e low er resu lt area:• SERUM DATE: The date of the cu rren t o r of the historical.

• SAMPLE NUMBER: Serum accession num ber• RESULT: T or B cell crossm atch in te rp re ta tion

• DILUTION: dilution of the se ru m8. For historical sam ples the dilution should be se t to 1 :2 /1 :1 . Rem em ber

w hen se tting up your w orklist the 1:2 historical w as fu n p rio r to running the 1:1 historical. The data is im ported in th is o rd er also.

9. E n ter p a tien t specific com m ents in the com m ent box. If an overall c rossm atch com m ent needs to be en tered m ake a note on the crossm atch w orksheet. This crossm atch com m ent will be evaluated by the supervisor w hen the crossm atch is reported .

10. Once th e p a tien t results have been review ed check the Save Data and Results Com plete boxes. Rem em ber, once com pleted you can not m ake any changes. Select Save and Close. Results will go to th e sign-off section to be reviewed by a supervisor.

11. Perform steps 6-8 for each pa tien t in your crossm atch.

4. Hand in th e crossm atch w orksheets along w ith the p a tien t folder to the

superv isors.

9.0

10.0

CALCULATIONSMFI shift is calculated as a ratio [patien t M Fl/Negative MFI).

REPORTING RESULTS AND REPEAT CRITERIA10.1 In te rp re ta tio n of Data

Interpretation of Negative Control Results

The negative control should be run in duplicate to assess overall reproducibility of the assay. If the resu lts be tw een the tw o tubes are d iscrepan t (>40% difference! consult a superv isor to determ ine if one or bo th values should be used to calculate th e ratio.The range established for th e negative control is a guideline. Not every cell tes ted will fall w ithin this average range. Negative control values will provide an indication of assay sensitivity t o assist w ith the crossm atch in terpretation . A high negative control will



reduce sensitivity, w hereas, a low control value m ight resu lt in a false oositive crossm atch. O ut-of-ranse negative controls do no t necessarily invalidate the assay b u t should indicate the need for c loser evaluation of the resu lts a n d /o r review by th e d irector.

I n te rp re ta t io n o f P o s itiv e C on tro l R esu lts

The AMESF established for the oositive control is a guideline. Not every cell tested will fall w ith in th is range due to factors including antigen expression on a p articu la r cell surface o r having a low freq u en cy pbrniQi-ypp. V ariation in the positive control reactivity will also be observed w ith cells isolated from different sources. For exam ple, m uch low er reactivity is observed w ith lymph node p repara tions as com pared to peripheral blood lymphocytes. The posidve control will provide an indication o f a s sa v .5 ^ n s itM tv ^ assist w ith the crossm atch in terpreta tion . A low positive control m ay be indicative of a high cell concen tration resu lting in reduced sensitivity. If the values are significantly b e lo w ih e range, repeating the tes t may be indicated if the crossm atch is negative. An out-of-range positive control does not necessarily invalidate the assay b u t should indicate the need for closer evaluation of the resu lts a n d /o r review by the director.

Positive Crossmatch Cutoff s

• A definitive positive cutoff has no t been defined for reporting an incom patible crossm atch due to lim itations of the assay t^ l s e positiv^ reactions due to autoantibodies, n rnnase trea tm en t etc.l and consideration of the pa tien t's clinical s ta tus a t the tim e of the crossm atch. For exam ple, a ratio of 1.5 m ay be considered com natible for one na tien t fsensitization from blood products) b u t incom patible for ano ther pa tien t fsensitization due to p rio r transp lan t).

• Positive flow crossm atch ranges a re based on com parison of SAB MFI values and FXM reactivity. W eak crossm atch reactivity fl.3 -1 .5 ) for th e A, B, DR locus correla tes w ith an MFÏ range flOOO-3000). Stronger crossm atch reactivity m ay be observed if m ultiple w eak donor specific antibodies a re present. However, correlation of MFI and crossm atch reactivity is also dependen t on the specificity (HLA Locus) of the antibody. Anti-HLA antibodies d irected against C, DQA, DQB, DP locus tend to react weakly in th e crossm atch w ith MFI values in the 3000-5000 range.

• A pa tien t’s sensitization h isto ry m ust be evaluated to



determ ine immunological risk of going to tran sp lan t w ith w eakly reacting donòr specific antibodies. Factors which a re considered include clinical s ta tu s of the patient, sen sitiza tion /tran sp lan t history, w aiting tim e, curren t listing of unacceptable antigens, an d specificity of the w eakly reacting antibody. The D irector o r supervisory staff m em ber m ust be consulted on all equivocal/w eakly positive crossm atches if th e re is DSA present.

R atio R esu lt In te rp re ta t io n G u idelines

♦♦Based on consistency of screening data with FXM

results.1.0-1.2 Negative ' Com patible1.3-1.5 Equivocal: DSA

presen tConsultation required **Compatible w ith

DSA com m ent o r Incom patible

Negative: Non-HLA Compatible: Non-HLA com m ent

1.6-1.9 Wk Positive: DSA presen t

Consultation requ ired , **Compatible w ith

DSA com m ent o r Incom patible

■W kPositive: Non- HLA

■ fHlV/Lupus'Dx)

Compatible: Non-HLA com m ent

2.0-3.0 DSA p resen t o r Non- HLA

Incom patible - DSA is p resen to r Com patible with

the Non-HLA com m ent


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10.210.310.410.5

In te r p re ta t io nofAutocrossmatchResults

The purpose of the auto flow crossm atch (runn ing of recip ien t serum vs. recip ien t cells) is to identify pa tien ts w ith autoantibodies, im m une com plexes o r in terfering substances which m ay resu lt in non-HLA reactiv ity in the crossm atch. Non-HLA reactiv ity can in terfere w ith liv ing and deceased donor crossm atch in te rp re ta tion . An exam ple of w hen this inform ation w ould be utilized for crossm atch in terp reta tion : A highly sensitized p a tien t w ith a crossm atch ratio of j . 5 b u t no PSA an d a positive au toeressm ateh would m ost likely be rep o rted as com patible.______________________

Rounding - N/AU nits o f M e asu re - Linear Values (RLV) or MESF values Clinically R eportable Range (CRR) - N/A R e p e a t C rite r ia a n d R esu lting :A superv iso r should be inform ed and the crossm atch m ay need to be repeated if:

• NHS - values are no t consistent w ith the duplicate a n d /o r ou t of range.• P P C -v a lu es are o u t of range.• Patien t resu lts - values are not consistent w ith th e duplicates.* Cell counts - values are less than 500. ■• P atien t crossm atch results are not consisten t w ith HLA antibody profile.

11.0 EXPECTED VALUESN/A

12.0 CLINICAL SIGNIFICANCEA strongly positive crossm atch is a contradindication for tran sp lan t. Patients who have circulating an tibodies th a t recognize HLA antigens on a donor o rgan o r tissue may experience rap id and irreversib le destruction of the graft upon transp lan tation (hyperacute rejection).

13.0 PROCEDURE NOTES13.1 FDA Status: ASR



13.2Cell P repara tion and Count

, ’ ' : f . 1 Í • » ;

1. Quality cell p repara tions are critical to achieving optim al crossm atch results and therefore, m ust be evaluated w hen analyzing results.

2. Accurate cell counts include all cells in th e p repara tion including contam inating m onocytes and dead cells. These cells a re targets for HLA antibodies and therefore, m ust be counted to ensure the correct antibody.antigen ratio is achieved.

3. Cell p repara tions w ith viability >50% can still yield accurate crossm atch results. Dead lym phocytes can be excluded from th e analysis by changing the gate.

4. Cell p repara tions m ust be p late le t depleted . Platelets express class 1 antigen and can b ind HLA an tibody which will resu lt inreduction in sensitivity.

5. Abnorm al T and B cell ratios m ay indicate a m edical issue w ith the donor and should be reported . Normal ratios are:

a. Peripheral Blood: 80% T /2 0 % Bb. Lymph node and Spleen: 50% T /5 0 % B

6. Flow analysis m ust be perfo rm ed on > 500 T and B cells. Asuperv isor m ust be contacted w hen there is a low cell count to determ ine if rep e a t testing is required .

13.3Crossm atch Serum Selection

1. C urrent Sample: The labora to ry 's recom m endation is to use a nirrpnt sam nle th a t is < 3Ó days old for crossm atch. The tran sp lan t coord inator is notified w hen the sam ple date does n o t m eet the 30 day criteria. The tran sp lan t team m ust authorize use of the sam ple for crossm atch o r make a rrangem ents to have a new sam ple collected. The curren t sam ple is always tes ted n ea t and in duplicate.

2. Historical Sample: Use of a H istorical crossm atch sam ple may be indicated for:

a. High risk patients.b. Post tran sp lan t (highest CPRA se ru m selected)c. Post sensitization event if th e re appears to be an

im m ediate decline in reactiv ity (h ighest CPRA serum selected)

3. H istorical sam ples are tes ted neat and 1:2 dilutions4. Historical sam ples m ust bé < 5 years

14.0 LIMITATIONS OF METHOD■ The flow crossm atch cannot detect extrem ely low Jevels of HLA antibodies.■ Presence of IgM antibodies and im m une complexes m ay inhib it binding and,

therefore , reduce crossm atch sensitivity.

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■ False positive reactions m ay occur due to autoantibodies, therapeu tic agents e tc

■ Only IgG anti- HLA antibodies are detected in the flow crossm atch.■ C om plem ent and non-com plem ent binding an tibodies cannot be

d ifferentiated.■ Cell surface antigen expression can a lte r crossm atch reactivity.

14.1 Analytical M easurem ent Range (AMR) - N/A14.2 Precision - N/A14.3 Interfering Substances

,,, . _ 14.3.1 Rituxan is a CD20 m onoclonal an tibody adm in istered to tran sp lan tpa tien ts to p reven t the developm ent of HLA an tibody or reduce an tibodies in conjunction w ith IVIG and p lasm apheresis (a t HUP). Rituxan causes B cells to be positive in the crossm atch. T reatm en t with p ronase reduces the positivity b u t doeis no t elim inate the reactivity.

14.3.2 Thymoglobulin is an anti-lym phocyte reagen t used as an. im m unosuppressan t agent. Thym oglobulin causes a positive flow

crossm atch. Commercial beads can be purchased to rem ove . thym oglobulin.14.3.3 The labora to ry should he notified if a p a tien t is receiving rituxan or

thym oglobulin so th a t the appropria te ac tions/step s can be taken to en su re accurate crossm atch in terp reta tion .

14.4 Clinical Sensitiv ity /Spedficity /P redictive Values - N/A

15.0 SAFETYThe em ployee has d irect responsibility to avoid injury and illness a t work. Nearly all harm ful exposures to infectious substances and chemicals, and o th er injuries, can be avoided w ith effective tra in ing and consistent safe w ork practices.

Become fam iliar w ith th e Environm ental Health and Safety S tandard Operating P rocedures to lea rn requ irem ents on w orking safely and p ro tecting the environm ent from harm . A lthough lab w ork typically focuses on the hazards of w orking w ith specim ens and chemicals, we m ust also control o ther im portan t hazards.

• Slips, trips, and falls cause m any serious injuries. Please ensu re tha t spills are cleaned quickly (to avoid slippery floors) and ,tha t you can see and avoid obstacles in your path.

• Ergonom ic injuries resu lt from perform ing tasks w ith too m uch repetition, force, o r aw kw ard position. Ergonomic injuries include s tra ins and back injuries. Learn abou t ergonom ic hazards and how to p reven t th is type of injury.

• Scratches, lacerations, and needlesticks can resu lt in serious health consequences. A ttem pt to find ways to elim inate your risk w hen w orking w ith sh a rp m aterials.

R eport all accidents and injuries im m ediately to your supervisor.



16.0 RELATED DOCUMENTS 'N/A i

17.0 REFERENCES17.1 The ASHI L aboratory Manual, 3 rd Ed., (1996), A Nikaein, ed.. The American

Society for H istocom patibility and Im m unogenetics, NY17.2 Cook DJ, Terasaki PI, Iwaki Y, T erashita GY, and Lau M. An approach to

reducing early kidney transp lan t failure by flow cy tom etry cross matching, Clin. T ransplan tation , 1 :253 ,1987.

. 17.3 Vaidya, S, Cooper TY, Avandsalehi J, Barnes T, Brooks K, Hymel P, Noor M, Sellers R, Thoms A, Stewart D, Daller J, Fish J, Gugliuzza K, Bray R. Improved Flow Cytometric Detection of HLA Alloantibodies using Pronase: Potential Clinical Implications. Publication pending.

18.0 ADDENDA

Addendum Titie

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flow crossmatching protocol

Documents

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th e conjugate

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th e na tu

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th e pa tien ts