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User Group MeetingLong Beach, CAMarch 29, 2017

All Content © Immucor, Inc.

Immucor’s Initiative

• What our customers requested– Convenient Educational programs

– User Group Meetings

• How we responded–

–

– Competency Kit Electronic Entry

– User Group Meetings

– ImmuTech


Today’s Agenda: User Group Meeting

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Afternoon Agenda: ImmuTech Meeting


CE

• PACE: – Morning session: 3.0 hours

– Afternoon session: 2.5 hours

• California CE

• Complete meeting surveys & register for CE Credits via web addresses below:

Morning Session: https://www.surveymonkey.com/r/LongbeachUGMAfternoon Session: https://www.surveymonkey.com/r/LongBeachImmuTech

Mort Alzona

VP, Worldwide Manufacturing Technical Support and ValidationImmucor

Presentation Unavailable for Distribution

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Immucor Update on Service & Support

Harry SundstromManager, Instrument ServicesImmucor


U.S FIELD SERVICE MAP


SOUTHERN CALIFORNIA INSTRUMENT PLACEMENTS

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Technical Support Update• Department Overview

– Handle Instruments, Reagents, Specialty, LIS, ImmuLINK, Transplant

– 19 dedicated Technical Specialists

– Felicia Buck – Technical Support Manager

– Operate 24 / 7 – Never closes

– All are Blood Bankers and former users of Immucor Products

• Department Performance– Average > 3,000 inbound calls per month

– Average >4,000 outbound calls per month (follow up calls)

– Utilize Blud_Direct on 70% of the calls

• Correct 70% of those calls using Blud_Direct (49% rate)

– Overall fix rates exceed 80% for all platforms


What Happens When You Call

TS answers call live ~ 90% of time within 30

seconds

TS troubleshoots and resolves remotely via phone or blud_direct

If onsite service is needed, FSE dispatched

FSE contacts facility confirms issue and

provides on-site arrival time – often we can provide same day

service

Most issues are resolved within 1 business day

Appropriate instrument check to ensure operation before

departing

Service call report is provided at the completion of the

service call

Field Service representative will

contact your facility the next day to ensure

instrument is operating as expected

Immucor sends out randomly sampled

Customer Satisfaction survey link through e-

mail


Remote Support

Secure Remote Support via

“blud_direct”

Technical Support and FSE can connect

remotely to instruments to diagnose and

resolve issues 24/7 –secure - user initiated

Reaction image review, Error logs, remote

adjustment of parameters to enable

remote resolution

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Key Initiatives

• North Central and West FSE Team Leaders

• FSE participate on a Monthly Technical Call with our Field Service Specialists and Internal Technical Personnel

• FSE weekly look ahead calls

• Complete Instrument Preventative Maintenance within the calendar month of their due date

• Utilize Immucor University web-based training

• FSEs willing to assist lab staff with auditing ECHO start up Kits

A well stocked customer spares kit insures increased instrument uptime!

• Customer Satisfaction survey

We appreciate your service feedback with comments!

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All Content © Immucor, Inc. 14

Common Yet Avoidable:Tips for Keeping your Instrument in Top Shape

Joe Osuch MS, MT(ASCP) SBBCM

Area Technical ConsultantImmucor

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Objectives

• Explain the purpose of Technical Communications (CC)

• Review the Technical Communications that customers frequently have questions about

• Discuss and show what to look for to properly maintain your Echo

• Uncover some Technical Communications that may have gotten overlooked


Purpose of Technical Communications

• Technical Communications become part of the Operators Manual– Procedural changes or clarification – Example: Archive

Instructions

– Software upgrade – Example: Upgrade 1.4

– Hardware upgrade – Example: New Bracket for Cap and Seal

• Review other relevant documents for Instrument Operation:─ The Package Insert contains all relevant product information for

reagents

─ The Operators Manual is the official guide of an instrument


Technical Communications

• Where to find Technical Communications– Distributed by Immucor through eNotifications (email) or

mail

– On the Immucor Website ( www.immucor.com )

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Immucor Website– Technical Communications

– SDS Sheets

– Master Lists

– Shipping calendars

– Package Inserts

– Product Catalog

– LOINC codes

– SOP Models

– LEARN®

– Competency kit electronic entry• Tech-Chek, Self Check II, RiSE



• How to store Technical Communications – Place in Operator’s Manual

• Part #0087540

– Create a separate binder


Example of Technical Communications Binder

Divider Tabs:

Sample Information

QC Information

Maintenance

Parts Replacement

Probe

Image Interpretation

Software Updates

Miscellaneous

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• How to identify Technical Communications– Identification number

– Date implemented

• What to do with Technical Communication information– Have an implementation plan

– Ensure current and applicable

– Accessible to end users



Let’s review some of the Communications that customers frequently have questions about

We will also provide some examples of how to troubleshoot your instrument based on some common observations


Decontamination• Decontamination is an essential part of maintaining

an instrument’s performance – Operators perform decontamination to prevent and remove

bacteria and other contamination from the instrument– Excessive contamination can lead to poor instrument

performance, reactivity issues, and erroneous results

• Some common decontamination issues include:– RelyOn preparation and use of expired RelyOn– Proper positioning of PBS supply tubing– Decontamination/replacement of secondary PBS transport

container– Flush/Purge/Prime tasks performed as part of

Decontamination process– Recycling PBS in use prior to decontamination

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Use of RelyOnTM Multi-Purpose Disinfectant Cleaner for Decontamination of the Echo®

• CC-08-036-01

• Issue Date: 7/23/2008

• Prepare 1L of RelyOn by dissolving 2 tablets in 1 liter of tap water

• RelyOn expiration date• Certificate of Analysis

• Inferred from side of container

• Also used to wipe down the probe block

• Corrosive (do you have the SDS?)


RelyOn Expiration

The first 4 numbers printed on the container indicate the year and month of

manufacturing (YYMM). The expiration date is 2 years from the date of manufacturing. In this example, the date of manufacturing was

Feb 2015. The expiration date would be Feb. 2017


Position of PBS Supply Tubing

Position of tube should be in bottom of container, not on ledge

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Position of PBS Supply Tubing

Position of tube should be in bottom of container, not on ledge


More Common Observations

– Dirty or Malfunctioning Components• Inline filters• Peri-Pump• Syringes• Probe tubing and other tubing• Probe Splash Guard

– Tip: Maximize instrument performance by inspecting these items during monthly maintenance

• Fluid reservoirs are removed allowing for inspection of syringes and peri-pump

• Instrument cover is removed to wipe down the probe block allowing for inspection of probe, tubing, and other components


Discoloration in PBS Filter

What to do:• Replace inline filter• Initialize the instrument• Prime the instrument• Perform Decontamination

(as needed)

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Dirty or rusted Peri-Pump

• Contact Technical Support to request a new Peri-Pump

• Follow directions in the Operator’s Manual for replacing the Peri-Pump (pg. 11-56)

• Perform As-Needed Maintenance– Initialization– Fluidics Test

What to do:


Discoloration or Crystals on Syringes

• Replace Syringe – See directions in

Operator’s Manual, pg. 11-54

• Perform required maintenance procedures– Initialize– Prime Instrument– Probe Accuracy Test

• CC-08-011-01

What to do:


Discoloration in Tubing

• Discoloration in the intake tubing or excessive discoloration in waste tubing may be an indicator of improper decontamination

– Review decontamination processes

– Call service to replace tubing as needed

What to do:

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Discoloration in Probe

• Check for other possible contaminated areas in instrument

• Replace probe– Pg.11-37 in Operator’s Manual– CC-10-010-01

• Perform as-needed maintenance– Initialize– Calibrate probe– Prime Probe– Check probe alignment– Check probe vertical position– Probe accuracy test

• Run decontamination if changing probe due to growth or discoloration

What to do:

I1


Instructions for Removing and Cleaning the Galileo Echo® Probe Splash Guard

• CC-12-019-01

• Issue Date: 01/29/2013

• Provides guidance on how to clean between the Probe Block and Echo Probe Splash Guard

• Over time PBS can become crystalized due to splashing

• Common errors– 5150 Probe Crash (Status=5700)

– 5150 Motor Failed Positional Verify (Status=3103)

– 6630 Fluidics Test: Step 2 of 3 Failed (volume too low)

– 6650 Fluidics Test: Step 3 of 3 Failed (volume too low)

• Not part of routine maintenance


Salt Crystals on splash guard

• Follow directions outlined in CC-12-019-01

• Assure probe splash guard is being cleaned during monthly maintenance – Pg. 11-30 in the Operator’s

Manual• “apply and then remove the

working solution…on and off of the probe block…allow the working solution …to be in contact with the probe block for as long as required…”

What to do:

I1 I would not say "failure" to properly decontaminate. Maybe you could say something about how you should inspect your tubings for contamination - particularly if you're seeing excessive non-specific reactions. And, I would include some bullet points on what to do if you do see contamination in the lines or anywhere in the instrument. Maybe you should start with these slides and follow up with the tube placement and rely-on outdate as ways to troubleshoot contamination rather than just have it as part ofthe list of "problems"

See changes to notes on this slideImmucor, 4/22/2016

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Echo Archive Instructions Effective with Software Release 1.2

• CC-09-014-01

• Issue Date: 4/7/2009

• Must be performed at least every 7 days

• Use CC in conjunction with Operators Manual

• Acceptable Media include CD-R, DVD-R, and DVD+R

• Verify disc before deleting results

• Keep Event log


Archiving Issues

• Very Slow PC’s

• Increase in Echo software crashes and freezes– Ultimately can

lead to the need for a PC replacement

Symptoms: What to do:

• Review Archiving procedures

• Perform Archiving weekly

• Delete archived results


Interpreting QC Failure on the Galileo Echo®

• CC-13-023-02

• Issued 2/18/2014

• Guidance for interpreting QC failures on the Galileo Echo

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QC Failures – WB corQC

– WB corQC vials not re-mixed and re-spun each day of use

– Not allowing the QC material to come to room temperature before use

– Contaminated QC samples


QC Failures - Reagents – Reagents left on board continuously

– Pouring from one vial to another

– Re-use of disposable reagent caps (=contamination)

– Contaminated stirballs/no stirball added


QC Failures- Strips

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Strips – Things to check

• Open pouches or not sealed properly

• Strips left out of pouches

• Pre-loading strips on the instruments– Putting strips back in

pouch after being read

• Not checking humidity indicators in pouches each and every time strips are removed

• Humidity indicators are reversible



• Now lets review some Technical Communications that may have gotten overlooked


Important Notification Regarding Echo Antibody Screen and ID Interpretations

• CC-09-042-02

• Issue Date: 11/25/2009

• Instances where Echo may generate a negative result– Applies to antibody screen and ID

– Weak reactions near threshold of detection

• Perform visual verification before release of result

• Does not impact hemagglutination or Capture Select– Typing assays or Weak D, DAT, XM

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Guidance for Interpretation CC-12-020-01 CC-13-011-01

Date Issued: 1/18/2013 Date Issued: 4/22/2013

Defines a negative reaction up to a 4+ reaction

Degree of indicator cell adherence is key in assigning grades for positive reactions

Provides examples of equivocal images compared to negative

Provides examples of atypical negative reactions


Important Information regarding use of DAT Positive Control Cells on the Echo • CC-09-040-01

• DAT Positive Control Cells are limited to a maximum of 7 days of intermittent use

• 7 day expiration is monitored by instrument (Echo 1.4 SP1)

• Maximum on-board time of 16 hours followed by intervening period of refrigerated storage


Directions on Acceptable Strip Usage for Daily Residual Volume Maintenance Function on the Echo

• CC-12-004-01

• Capture Strips are to be used for Washer Residual Volume test

• No CMT

• Replace Capture Strips for Daily Washer Residual Volume once per week

• Monthly requires fresh strips CMT RS3

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Review of Objectives

• We are now able to:

– Understand the purpose of Technical Communications

– Identify the importance of following Technical Communications

– Describe what to look for to properly maintain your Echo

– Discuss some Technical Communications that may have been overlooked


THANK YOU!

Sheri Goertzen MT(ASCP)BB,CLS(CA), COA(ASQ)

Supervisor, Transfusion ServiceValley Children’s Healthcare

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Optimizing Automation and Manual Methods in Your Blood Bank

ImmuTech WorkshopMarch 29, 2017

Long Beach, CA

Sheri Goertzen,

MT(ASCP)BB, CLS(CA), CQA(ASQ)Valley Children’s Hospital

Madera, California

[email protected]

Objectives

1) Describe methods to effectively use both automation and manual methods to optimize testing and turnaround times.

2) Share ideas on how to keep a large number of generalist CLS competent in the blood bank.

3) Discuss the development of a decision tree for antibody identification when the solid phase antibody screen is positive.

4) Describe some simple ways to meet the CAP method comparison requirements.

A bit about us…

• Not-for-profit, independent children’s hospital serving central California – 356 beds

• Started 60 years ago by 5 young local mothers

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Located in the Heart of California, we are the 2nd

largest children’s hospital in California and treat more inpatient cases from these 9 counties than any other children’s hospital.

A bit about us…

• Average 6,000 transfusions per year

• AABB and CAP accredited

• CLS Training Program

• Magnet Recognition for Nursing Excellence

A bit about us…

• Pediatric Level II Trauma Center

• ECMO Program

• Resident program affiliation with Stanford Medical School

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Methodologies

• Echo 1 – acquired April 2009

• Echo 2 – acquired June 2012

• Back-up and Alternate Methods:– Manual Capture

– PeG Tube Testing

– NHance Tube Testing

• Max volume of specimen = 3 mL EDTA

Echo 1 and Echo 2

2 Echos are our Workhorses

• Donor Retypes

• Type & Screens

• AHG Crossmatches

• Antibody ID: Ready-ID, Extend I, Extend II

• Antigen Screening: C,c,E,e,K

• Interfaced – MediTech 5.67

• MediTech Bedside TAR

• Electronic Crossmatching

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Competency Assessment

• All 25 CLS working in blood bank are generalists

• How do you keep 25 Core Lab CLS competent to perform all the necessary testing in your blood bank?– Full antibody ID studies, including elutions,

adsorptions, phenotyping and titers

– Aliquot, irradiate, pool, volume reduce platelets, mix reconstituted whole blood for exchange transfusions, as well as wash RBC units if needed.

Competency Schedule –

Wet SamplesJanuary July

Type & ScreenCrossmatchDATAntigen Typing

Antibody IdentificationAntibody Titer

Annual cGMP Training & Post-Test• January

Annual Competency Observation:• May - June

Competency Schedule –Written Test

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Competency Program –Written Test

Ask for the reference for each answer.

Competency Program –Written Test

Ask for suggestions. Use these to improve your procedures/processes.

Provide feedback on each Comment/Suggestion.

Average TAT Data

Ongoing Monitors Target Actual

Newborn Workup > 90% completed < 90 min 94%

Average TAT < 80 min 63 min

Average STAT TAT < 60 min 46 min

Blood Type > 90% completed < 90 min 97%



Antibody Screen > 90% completed < 90 min 96%



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Antibody Identification Process

• Get referral specimens from several small rural hospitals for Antibody ID– Gel

– LISS

• High Risk Maternal/Fetal Center mothers

• Transported Maternal specimens

• Fair amount of Pediatric Antibody patients– WAIHA (panagglutinins, rare specificity ID’d)

– Chronically transfused

Antibody ID Decision Tree

PeG Screen

Positive Solid Phase Ab Screen

Solid Phase Panel(s)

Positive Negative

Identify Ab

Report Ab Specificity, provide Ag Neg units, AHG XM

Ab Screen Capture = Positive Report as “Nonspecific w/Solid Phase” (continue investigation in tube)

Yes

No

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PeG Screen

Positive Negative

Ab Screen Tube = Negative, AHG XM

PeG Panel(s)

Identify Ab

Report Ab Specificity, provide Ag Neg units, AHG XM

Ab Screen Tube = Positive Report as “Nonspecific w/Tube Testing”, AHG XM

… continued

Yes

No

Summary

• If we get Positive reactions with Capture,

• then Negative reactions in the Tube,

• we result the Capture screen as “Nonspecific with Solid Phase”

• and result the Tube Screen as “Negative”– We choose to require AHG crossmatching

– Some facilities do not, but with 25 rotators, it helps me sleep better at night…

Comparability of Instruments/ Methods

• Required by CAP

• CAP Checklist: COM.04250

If the laboratory uses more than one nonwaived instrument/method to test for a given analyte, the instruments/methods are checked against each other at least twice a year for comparability of results.

• Now applies to blood bank as well as the other clinical lab departments

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• NOTE: This requirement applies to tests performed on the same or different instrument makes/models or by different methods. The purpose of the requirement is to evaluate the relationship between test results using different methodologies, instruments, or testing sites. This comparison is required only for nonwaivedinstruments/methods accredited under a single CAP number. The laboratory must establish a written procedure for this check that includes acceptance criteria.


• Quality control data may be used for this comparison for tests performed on the same instrument platform, with both control materials and reagents of the same manufacturer and lot number. Otherwise, the use of human samples rather than stabilized commercial controls, is preferred to avoid potential matrix effects.


• Evidence of Compliance:

Written procedure for performing instrument/ method comparison AND

Records of comparability studies reflecting performance at least twice per year with appropriate specimen types

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Comparability Criteria

• **NEW** 04/21/2014

• CAP Checklist: COM.04300

Acceptability criteria are defined for comparability of instruments/methods used to test the same analyte, with records of action when the criteria are not met.

• Evidence of Compliance:Records of comparability studies with evidence of

review and action taken, as appropriate

Comparability of Instruments/ Methods• Method Comparison is performed twice a year,

comparing Echo1 vs. Echo2 vs. Manual Capture vs. Tube methods– ABO/Rh – No less than 3 specimens are

compared, each with different blood types, at least one should be Rh negative

– Antibody Screen – No less than 3 specimens are compared, at least one should be positive

– Antigen Typing – comparing tube to Echo, no less than 3 specimens

– Antibody ID – No less than 1 positive specimen is compared

Comparability of Instruments/ Methods• Interpretation/ Acceptance Criteria:

• Manual and Automated Capture methods are expected to correlate closely.

• Echo1 vs. Echo2 results are expected to correlate (match) closely.

• Capture vs. Tube methods are expected to show some variability between reactions due to the differences in the nature of the testing systems and enhancements.

• Corrective action must be taken and documented when criteria are not met.

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Method Comparison: ABO/Rh

Lot # ofSupplies/Reagents

TubeManualCapture

Echo 1 Echo 2Accept? *Yes or No

Date Tech

ABO/Rh

Specimen #

Capture Strip

Anti-A

Anti-B

Anti-D1 (ser.4)

Anti-D2 (ser.5)

Weak D

Rh Control

A1 Cells

B Cells

Interp. N/A

* Interpretation results must match closely between manual and automated Capture methods. Some variability is expected and acceptable between Capture and Tube methods due to the different nature of the test methods.

Reviewed_______________________ Date_______________ Instrument/Method Correlation Acceptable? Y / NMust be performed at least twice per year (CAP TRM.31450) and corrective action documented when criteria are not met.

Method Comparison: Ab Screen, Ag Typing, Ab ID

* Interpretation results must match closely between manual and automated Capture methods. Some variability is expected and acceptable between Capture and Tube methods due to the different nature of the test methods.

Reviewed_______________________ Date_______________ Instrument/Method Correlation Acceptable? Y / NMust be performed at least twice per year (CAP TRM.31450) and corrective action documented when criteria are not met.

Lot # ofSupplies/Reagents

TubeManualCapture

Echo 1 Echo 2Accept? *Yes or No

Date Tech

Ab Screen

Capture Strip

SC1 AHG

SC2 AHG

SC3 AHG

Interp.

Antigen Typing

Specimen #

Capture Strip

Antigen/Sera ___

Interp.

Ab ID

Specimen #

Tube

Capture

Interp.(attach panels)

Thank You!

• Contact info:

• Sheri Goertzen, MT(ASCP)BB, CLS, CQA(ASQ)

[email protected]

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Kim Lyon

Sr. Marketing Manager – TransfusionImmucor


Lori Gianetta BS MT(ASCP)

ImmuLINK Sales ManagerImmucor


Leila Cardeno CLS, MT(ASCP)

Blood Bank SupervisorSaint John’s Health CenterProvidence Health & Services

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Side-by-Side Evaluation ECHO®

to LumenaTM

85

Evaluation Sites

• Saint John’s Health Center, Providence Health & ServicesSanta Monica, CaliforniaJuly 18 – August 5, 2016

• Providence Portland Medical CenterPortland, OregonDecember 2016

86

Old and New Saint John’s

87

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Saint John’s Health Center

• 266 Beds• ED, Surgical, ICU, OB, NICU, Oncology,

Orthopedic, OP Transfusion• One ECHO®

• 8348 T&S• 3898 Unit Confirmation• 321 ABID• 256 AHG Crossmatch

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Providence Portland Medical Center

• 483 Beds• Diverse groups of patients including OB and

Oncology• Two ECHO®

• 7834 Type and Screen• BB wanted to see the improved functionality

of the Lumena in resolving indeterminate reactions

89

Room for Improvement

• Specificity

• Sensitivity

• Less equivocal results/indeterminate results

• Less reagent wastage

• Delay of blood availability

90

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Objectives

• Enumerate the improvements on the Lumena

• Demonstrate the difference in the specificity and sensitivity between the ECHO® and LumenaTM

• End-users observation

• Changes on workflow

91

Background

• Implemented ECHO in 2012 as the primary methodology

• Back-up: Gel and Tube/LISS

• On-Board Testing: Group, Screen, Confirm, ABID, AHG Crossmatch

• To be implemented: Cord blood work-up and Antigen testing

92

Evaluation Study Guidelines: Saint John’s

• Three weeks

• Perform Type and Screen on 200 specimens in the ECHO®

and then in the LumenaTM within 8 hours using the same lot of reagents

• NTD results on the Group and Confirm will be repeated using the Tube method

• Discrepant antibody screen results between the 2 instruments will be repeated using the AHG- LISS/Tube Method

• ABID will be performed on all positive Antibody Screen

93

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Evaluation Study Results: Saint John’s

• Total number of samples: 190 170 patient samples

(Type and Screen)20 donor samples

(Confirm)25 ABID

(Ready-ID/Extend I/Extend II)

94

Evaluation Study Results: Saint John’s• Group and Confirm Example

ECHO

LUMENA

95


• Group and Confirm

96

Instrument NTD Rate Cause

ECHO3.2% (6/190) Negative or weak reaction on

B cells

Lumena 1.1% (2/190)A Subgroup with Anti-A1Debris in well

No. of concordant samples 185

No. of discrepant samples 5

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Evaluation Study Results: Portland

97

• Total number of samples: 114• Group

Instrument NTD Rate Cause

ECHO 3.5% (4/114) HemolysisRouleaxControl IssueAntibody Interference

Lumena 5.3% (6/114) Rouleax

No. of concordant samples 105

No of discrepant samples 9

Evaluation Study Results: Saint John’s• Negative Antibody Screen Example

ECHO

LUMENA

98

Evaluation Study Results: Saint John’s• Positive Antibody Screen Example (Anti-C and Anti-E)

ECHO

LUMENA

Anti-E was evident in both ECHO and Lumena (Screen 2)Highlighted cells (Screen 1) are C-positive cells, called negative but visual positive in ECHO and 2+ in Lumena

99

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Evaluation Study Results: Saint John’s• ABID Example (Anti-C and Anti-E)

ECHO

LUMENA

Anti-E was evident in both ECHO and LumenaAnti-C reactivity was clear-cut in Lumena not in ECHO

100


• Positive Antibody Screen Example (Anti-Jka)ECHO

LUMENA

Highlighted cells (Screen 1) are heterozygous Jka positive cells

Negative in ECHO, but visually positive, Positive (1+) in Lumena

Screen 2 cells are homozygous Jka positive cells101

Evaluation Study Results: Saint John’s• ABID Example (Anti-Jka)

ECHO

LUMENA

Dosage was demonstrated clearly in the Lumena reactions (2+ and 3+)

102

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• Positive Antibody Screen: 25• Antibodies Identified

103

Antibody TotalAnti-C 1Anti-c 1Anti-D (Passive) 3Anti-E 3Anti-Fya 1Anti-Jka 4Anti-K 1Anti-S 2


• Sensitivity and Specificity

104

ECHO LUMENATP 11 TP 16

TN 164 TN 158

FP 4 FP 4FN 2 FN 0

INVALID 0 INVALID 3

TOTAL 181 TOTAL 181

Sensitivity (%) 84.62 Sensitivity (%) 100.00

Specificity (%) 97.58 Specificity (%) 97.53


105

• Sensitivity and SpecificityECHO Lumena

TP 11 TP 13

TN 101 TN 93

FP 3 FP 10

FN 1 FN 0

TOTAL 116 TOTAL 118

Sensitivity (%) 91.6 Sensitivity (%) 100

Specificity (%) 97.1 Specificity (%) 90.3

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• Indeterminate ABID Result

106

ECHO Lumena LISS/Tube Cause TotalEquivocal Invalid NP Icteric plasma 3

Visual positive

Negative Negative 3

Equivocal Equivocal Rouleaux at 4ºC Cold Agglutinin 2

Equivocal Equivocal Negative Non-specific reactivity 1


• False Positive/Negative Results

107

Instrument False Positive False Negative

ECHO Equivocal 3 Anti-D (Passive) 1

Lumena Artifact – Not repeated

7 None 0

Icteric 1

Buffy coat sampling 1

Equivocal 1

Assay Improvement

• Camera ResolutionSharper ImageLess equivocal/indeterminate resultsLess false negative

• SoftwareFaster response

108

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End-user Response

• Same operation, PM, QC = Minimal training• Better resolution and definition of images = easier to

interpret equivocal results as either positive or negative• More confident in reading reaction• Dosage is more evident• Increased specificity, without sacrificing sensitivity• Reproducibility of results• Increase efficiency and decrease unnecessary time to work-

up non-specific reactivity, simpler ABID algorithm• Less delay in providing RBC• Less reagent wastage

109

Changes?

• As a result of the side-by-side study, we made three changes in our ECHO workflowStopped over-reading

Stopped repeating positive antibody screen with equivocal result in the ECHO. Instead we use our back-up method, Gel. We use the Tube method to check for rouleaux or non-specific cold agglutinin.

Keep an eye on the residual volume to read between 0.08 – 0.12

110

Q & A

111

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Oksana Volod, MD, FCAP

Director, Coagulation Consultative ServiceCedars-Sinai Medical Center

‘’A Pathologist Perspective on

HIT Diagnosis ‘’

Faculty Name: Oksana Volod ,M.D.Faculty Institution: Cedars Sinai Medical Center, Los AngelesSpeaker Disclosure : None

Objectives

• Describe the features of Heparin Induced Thrombocytopenia (HIT) including:Mechanism

Diagnosis

• Review HIT Case Studies

• Discuss Workup Protocols used at Cedars Sinai to diagnose and treat HIT

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Heparin-Induced Thrombocytopenia

HIT & HITT *• HIT/HITT is an antibody-mediated adverse reaction

to heparin that can result in venous or arterial thrombosis.

• Diagnosis of HIT is based on both clinical and serological features.

VS• * HAT caused by nonimmunologic mechanisms

(mild direct platelet activation by heparin)

Onset within 4 days Incidence 5%- 30% Recovery 1-3 days

Mechanism of HIT

Heparin

PF4

PF4/Heparin Complex

Y

Anti-PF4/Heparin Antibody

PF4/Heparin/Ab Immune Complex

Platelet

4. IgG receptors

Activated Platelet

PF4

Thrombin

Thrombogenic Microparticles

ActivatedMonocyte

Tissue Factor

Heparin-like molecules

Damaged endothelial cells

5. Anti-PF4/Heparin

IgG Ab

6.

7.

8.

1. 3.2.

10.

9.

Heparin-like molecules

At most 5% to 30% of patients who form HIT –IgG, will develop clinical HIT depending upon the patient population

Anti-PF4/Heparin antibody (Ab) may form in up to 8% of patients receiving heparin

Mechanism of HIT1. Platelet factor-4 (PF4) from platelet α-granules binds to heparin chain to

form PF4/Heparin complex 2. Anti-PF4/Heparin antibody (Ab) may form in up to 8% of patients

receiving heparin

3. Ab binds to form PF4/Heparin/Ab immune complex -- detected by PF4 ELISA screening test

4. IgG receptors are found on the platelet surface

5. Pathogenic IgG class of anti-PF4/Heparin Ab forms in subset (5-30%) of these patients – this is called the HIT Ab

6. PF4/Heparin/HIT Ab complex binds to IgG receptor leading to platelet activation – detected by Serotonin Release Assay (SRA)

7. Activated platelet releases thrombogenic microparticles and PF4

8. Released PF4 leads to further formation of PF4/Heparin complexes and platelet activation, as well as neutralization of the anticoagulant effects of heparin

9. HIT Ab also reacts with PF4 bound to heparin-like molecules on endothelial cells and monocytes – releases tissue factor (TF)

10.TF initiates the coagulation cascade

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Incidence of HIT According to Patient Population and Type of Heparin

Exposure

CHEST / 141 / 2 / FEBRUARY, 2012 SUPPLEMENT

HIT/HITT : DiagnosisClinical

• Moderate Thrombocytopenia

• Thrombosis Venous ( DVT, PE,

adrenal) Arterial ( Limb, CVA, MI)• Appropriate timing• Other cause for

Thrombocytopenia are excluded

Pathological

• Heparin – dependent , platelet activating IgG

Detected by Antigen assay

( PF4 ELISA)

Confirmed by platelet activation assay (e.g. serotonin release assay (SRA))

Warkentin et al. Thromb Haemost 1998;79:1-7

Warkentin 4T Score

CHEST 2012; 141(2)(Suppl):e495S–e530S

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4T Score and HIT ProbabilityLow 4T score = low HIT probability ( 0%-

3%)*

High 4T score- 24% -61% of patients prove not to have HIT *

Isolated HIT antibodies are both frequent and not diagnostic of HIT

• Lo GK , Juhl D , Warkentin TE , Sigouin CS , Eichler P ,Greinacher A . Evaluation of pretest clinical score (4 T’s) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings . J ThrombHaemost . 2006 ; 4 ( 4 ): 759 – 765

• Pouplard C , Gueret P , Fouassier M , et al . Prospective evaluation of the ‘4Ts’ score and particle gel immunoassay specific to heparin/PF4 for the diagnosis of heparin-induced thrombocytopenia [see comment] . J Thromb Haemost . 2007 ; 5 ( 7 ): 1373 - 1379

HITLaboratory Testing

Antigen Assays

Detect HIT antibodies

Functional Assays

Detect presence of platelet activation by HIT antibodies

ELISA SRAHIPA

Heparin Antibody Assays• Survey of Coag labs in North America ID 8 different assays and wide

discrepancies in practice between centers using the same assay *

*Price EA , Hayward CP , Moffat KA , Moore JC , Warkentin TE , Zehnder JL . Laboratory testing for HIT is inconsistent in North America: a survey of North American specialized coagulation laboratories . Thromb Haemost . 2007 ; 98 ( 6 ): 1357 - 1361 .

Test Advantages Disadvantages

ParticleImmuno Filtration Assay (PIFA )

• POC assay• Quick TAT ( 15 min)

• Sensitivity 94-95%• Specificity 88-92%• Results NOT “quantifiable”

Immunoassays(PF4 ELISA)

• Sensitivity 100%• Technically easy• TAT 3.5 hours

Specificity 82-85%

Serotonin Release( SRA)

• High Sensitivity and Specificity

• FP rare

• Technically demanding • Require radioisotopes• Performed in specialized

laboratories

Platelet Aggregation

High Specificity • Low Sensitivity• Technique dependant

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Rapid AssaysPIFA HemosIL HIT-Ab (PF4-H)

PF4 ELISA Recognize binding of antibodies to PF4/polyanion complexes Detects antibodies presence

PatientPlasma or

SerumSample

PF4 coated ELISA plate

Tagged goat anti-human Ig

Detect absorbance

Add substrate

Solid-phase Anti-PF4/heparin-ELISA “Immunoassay” PF4 ELISA

2015 CAP Proficiency Test

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Load Dense GranulesWith 14C-Serotonin

Platelet

+

SerumMeasure 14C-Serotonin

Released

Heparin - Low

No Release

14C - Serotonin Release AssayPlatelet Activation Assay

HIT WORK UP AT CSMC

Historical Perspective

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CS Medicine Initiative

• Our 2011 audit (6 months, 493 cases) showed that we had:

• Significant number of borderline/low positive/positive results that were not HIT (91/103 cases)

• Too many SRA were sent out for confirmation, even on very negative PF4 ELISA results (176 /493)

• During that period of time we had only 12 confirmed HIT PF4 ELISA results 2.4% , which were confirmed by SRA.

• The results of the 2011 audit lead to a 2012 Cedars-Sinai Medicine Initiative project

2012 CS HIT ProjectCost-effective HIT diagnosis: utilizing IgG-specific PF4 immunoassays reduces the number of confirmatory Serotonin Release Assays without missing true HIT.

Study Aim:

Compare polyspecific vs. IgG-specific PF4 ELISA assays To determine whether the IgG specific assay can reduce confirmatory SRA

testing (expensive send-out with long turn-around-time) Without missing true HIT and/or unnecessary treatment with DTIs

Methods: 453 HIT work-ups (4T’s score, polyspecific PF4) were reviewed (05/2011-02/2012), including 86 work-ups on 49 patients with polyspecific OD ≥ 0.4

Study Conclusion

22 of 29 “borderline” poly-ELISAs were negative by the IgG-ELISA

IgG-ELISA reduced the number of “borderline” cases by 75%

Positive IgG ELISA results need to be confirmed by platelet activation assay ( SRA)

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Predictivity of (PF4)-ELISA for a Strong-Positive SRA

Negative IgG PF4 ELISA and SRA will miss IgA and IgM Antibodies

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HIT Panel Work Up at CSMC

Cases

Case 1 – Borderline PF4 IgG• 43 yo African American male with acute on chronic CHF• Heparin exposure on 7/4/2015 - 163 K• Thrombocytopenia on 7/5/2015 - 88 K• There was prior heparin Exposure in May • Switched to Argatroban• No thrombosis

W4T score – 3 pointsPF4 IgG – 0.64SRA – 69%

Interpretation : Rapid HIT onset in a patient with prior heparin exposure

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Case 2 – Strongly Positive PF4 IgG , Initial Neg SRA

• 55 YO male admitted with massive MI , VSD• Heparin exposure 12/21/2015 – 227K• Thrombocytopenia 12/28/2-15 – 119K…74K• Thrombosis - Yes (12/31)• 1/1 taken to OR for VSD repair • Treated with Argatroban

HIT score - 7 points12/29 - PF4 IgG –2.57

SRA - 1%1/4 - PF4 IgG –2.33

SRA - 87%

Interpretation: HIT with delayed SRA reactivity

Role of Plasma Exchange in HIT Treatment

EIA IgG

SRA

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Case 3 – Stroke During Hemodialysis

• 53 yo Female with ESRD , history of multiple chronic clots

• s/p TPA• ? Prior history of HIT• Started on Argatroban

PF4 IgG 0.03 ( CSMC)PF4 IgM 0.05 ( BCW)PF4 IgA – Neg

Case 3 : 2009 - Positive Poly PF4- NegSRA

• Multiple medical problems • Received Heparin during 07/14 -07/24 admission (

Platelets 314K)• Readmitted on 7/31 with Platelets 39K• 8/8 - Acute DVT• Started on Argatroban

Polyclonal PF4 8/14 – 2.36BCW: SRA- 0% Strong IgG ( not inhibited

by heparin) Strong IgM – reacting

with heparin)

Polyclonal PF4 8/22 - 1.34BCW :• SRA - 0% • Strong IgG ( not inhibited

by heparin)• Absent IgM Interpretation : IgM antibodies mediated HIT

Natural History of HIT Antibodies

• Anti-heparin/PF4 antibodies are frequently not detectable at a median of 50-80 days after HIT initially documented

• Recurrent HIT does not necessarily recur upon re-exposure to heparin

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HIT: Take Home Points• HIT is a clinico- pathologic diagnosis

• Advantages and limitations of each assay performed are necessary to be aware of:

IgG-ELISA reduces the number of “borderline” cases Positive IgG ELISA needs confirmation with functional assay (

SRA) Both will detect IgG antibodies only

• If there is a strong clinical probability of HIT , repeat testing is recommended in 2-3 days

• If Thrombocytopenia persists > 4 days following cardiac surgery , HIT work up is recommended

• Dedicated multidisciplinary team ( pathology, pharmacy, blood bank, hemonc) is needed for appropriate diagnosis and management of HIT

CHEST 2012; 141(2)(Suppl):e495S–e530S

Thrombocytopenia Following Cardiac Surgery

• 50 % of patients will develop HIT antibodies

• 1-2 % will develop HIT• Consider HIT if :

1. Fall begins > 4 days postoperatively

2. Thrombocytopenia that persists for > 4 days after surgery

CHEST 2012; 141(2)(Suppl):e495S–e530S

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Jay Hudgins, DO

Assistant Medical DirectorKeck Medical Center of USC

Rh Group: A Review, and the Identification of Genetic Variants

discovered by atypical typing results on the NEO/Echo

PlatformsJay Hudgins D.O., M.S.

March 29th 2017

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Financial Disclosures

• None

4/13/2017

The Rhesus (Rh) Blood Group

The Rh(D) Antigen

• Rh is the most complex system, with over 45 antigens

• The complexity of the Rh blood group Antigens is due to the highly polymorphic genes that encode them.

• Discovered in 1940 after work on Rhesus monkeys

• The 2nd most important after ABO in the crossmatch test

• Only the most clinically significant Atigenswill be discussed

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Rh Genetics

• The genes that control the system are autosomal co‐dominant – located on the short arm of chromosome 1.

• RHD and RHCE

• The antigens of the Rh blood group are glycoproteins.

• The RHD gene encodes the D antigen• A large transmembrane protein with external and internal domains– Encoded by 10 exons

The Rh(D) AntigenProtein Structure Genetic Arrangement

RHD gene deletion occurred in the Rhesus Box Blood 2000 95(12):3662‐3668

The Rh blood group system: a review Blood 2000 95(2):375‐387

Rh Antigen Frequency

• D antigen – 85% Rh Positive

• d antigen – 15% Rh Negative

• C antigen – 70%

• c antigen – 80%

• E antigen – 30%

• e antigen – 98%

The presence or absence of D Ag determines if the person is Rh+ or Rh‐

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Nomenclature of the RH system

3 Different nomenclatures:

1‐ Fisher‐Race

2‐Weiner

3‐ Rosenfield Nomenclature

Fisher-Race Theory

• This theory is named after the two British workers who proposed it in the 1940ʹs.

• The theory is useful to explain routine inheritance of D, C, E, c, and e genes.

• The main tenets of the theory are as follows:

– Rh inheritance is controlled by 3 closely linked loci on each chromosome of a homologous pair.

– Each locus has its own set of alleles.

• The 3 loci are so closely linked that crossing‐over does NOT occur, and the 3 genes on one chromosome are always inherited together.

Br Med J. 1956 Apr 14; 1(4971): 862, 863.



1‐ Fisher‐Race

2‐Weiner

3‐ Rosenfield Nomenclature

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Wiener Theory

• Developed by Dr. Alexander Wiener

• There is one Rh locus at which occurs one Rh gene, but this gene has multiple alleles.

• The three factors are analogous to C, D, and e respectively– R2 (cDE in Fisher‐Race)

– Ro (cDe in Fisher‐Race)

– The d gene does not exist in Wienerʹs theory.

Heredity of the Rh Factor. Wiener AS, Sonn EB. Genetics. 1943 Mar;28(2):157‐61.

Wiener Theory

• The main difference between the Fisher‐Race and Wiener theories is that the:

– Fisher‐Race theory has three closely linked loci

– The Wiener theory has only one gene locus at which multiple alleles occur.


Wiener Theory

• Wiener further theorized that 8major genes led to different combinations of antigens (D, C, E, c, e):

– R0, R1, R2, Rz

– r, r′, r″, ry


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1‐ Fisher‐Race

2‐Weiner

3‐ Rosenfield

Rosenfield Nomenclature• Each antigen assigned a number

• Rh 1 = D

• Rh 2 = C

• Rh 3 = E

• Rh 4 = c

• Rh 5 = e

• In writing the phenotype, the prefix “Rh” is followed by colon, then number (if negative, number is preceded by ‐)

• e.g. D+, C+, E‐, c+, e+ is written as

Rh:1,2,‐3,4,5

Rh nomenclature. Transfusion. 1979 Jul‐Aug;19(4):487.

Why Do We Care?

• The D antigen is extremely immunogenic.

• It causes the production of anti‐D in up to 70% of Rh negative individuals exposed to D antigen. – Anti‐D is the most common (Non‐ABO) cause of severe HDN.

• The C/c and E/e antigens are not as immunogenic.– But the genes are highly interrelated

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Rh Complex :The relationship between genes and proteins

Rh ComplexProtein Structure


The Rh complex

• Rh proteins carry Rh antigens but are onlyexpressed on the erythrocyte surface if RHAG is present.

• Homology (~40%) of the Rh proteins and RHAG indicates an ancestral relationship.

• The complex of the Rh protein family consist of a tetramer:– 2 RHAG molecules and 2 RHCcEe or RHD protein molecules.

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Primitive Rh System

• Sequencing the intergenic region of the two RH genes suggests that the order of the genes are RHD‐RHCE.

• The primordial human Rh haplotype is believed to be Dce, and the other 7 common Rh haplotypes most likely each arose from this gene complex by a single genetic event.

Sequence analysis of the spacer region between the RHD and RHCE genes.Biochem Biophys Res Commun. 1999;263:378-383.

RHD and RHCE

• Analysis of the primary amino acid sequences (from cDNAs) shows that the first 41 N‐terminal amino acids of RHD and RHCE/e are identical.

• RHD differs from the common forms of RHCE by only 30 to 35 amino acids.

• Despite the high degree of homology:

– RHCcEe proteins do not express any D epitopes

– RHD protein does not express C or e antigens.cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh

(Rhesus)‐blood‐group‐antigen expression. Biochem J. 1990;271:821‐825.

Rh Genetic Diversity

• The RH genes are a source of massive diversity.

• Combinations of different genetic rearrangements are abundant among all racial groups


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RHD: Genetic Variation and Serologic Outcomes

RhD and RhCE Gene Arrangement

RHD gene deletion occurred in the Rhesus boxBlood 2000 95(12):3662‐3668

Rh Negative

RHD gene deletion occurred in the Rhesus boxBlood 2000 95(12):3662‐3668

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Rh Negative

Expert Reviews in Molecular Medicine: 2006, 8(13)

RHD Antigen

• High genetic variability

• Increased interactivity with an adjacent homologous gene

• Impacts the expression of the D antigen resulting in:

– Weak D or Variant D phenotypes

Weak D vs Variant D

• Some D‐positive RBCs do not agglutinate when exposed to reagent Anti‐D.

– These require further testing to determine the D status of the patient.

• Traditional Weak D testing cannot determine the difference between a Weak D or Variant D phenotype.

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Classes of Weak D Expression

• Biochemical Hindrance (Ceppellini effect)

• Variant D

• Weak D w/o alloimmunization

• Other

Weak D due to Biochemical Hindrance (Ceppellini Effect)

• Genetic and Biochemical interference:

• Genetic:– C allele in trans position to D allele

• Dce/dCe, DcE/dCEIn both of these cases the C allele is in the trans position in relation to the D allele.

• Biochemical:– D antigen is normal, C antigen appears to be crowding the D antigen. (Steric hindrance)

• Does NOT happen when C is in cis position– Example: DCe/dce

Ceppellini et al. An interaction between alleles at the Rh locus in man which weakens the reactivity of the Rh0 factor (D

u). Proc Natl Acad Sci U S A. 1955;41:283.

Variant D

• Majority of variant D phenotypes are due to hybridization of the RHD and RHCE genes.

– Loss of epitopes of wild‐type D antigen

• Results in anti‐D alloimmunization


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Weak D (Inherited)

• Various genetic variations in the RhD gene can result in decreased expression of “wild‐type” D antigen.– Unique single nucleotide polymorphisms(SNPs) result in weak D types 1‐84.

• Weak D types 1‐3 represent 90% of weak D types in people of European decent.

– Insertions, deletion ,and defects of transcription or pre‐mRNA processing.

• Weak Ds 1‐3 are the only weak Ds that do not carry a risk of alloimmunization.

Weak/Variant D and Serology

• RBCs with some weak/variant D antigens may not be agglutinated by all monoclonal anti‐D.

• Therefore it is the responsibility of the Transfusion Service to determine cutoffs for weak reactivity when testing on our automated platforms.

Rh in Gel and Tube

• Investigation of 10 Rh discrepancies.

• Samples were identified by discrepancies between newly implemented automated gel technology (ProVue) and historical records.

• The gel Rh typing test indicated that the patient was D+ and historical record was D–.

Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques

Denome et al. TRANSFUSION 2008;48:473‐478

Mount Sinai Hospital, Laboratory Medicine and Pathobiology,

University of Toronto, and Research & Development, Canadian

Blood Services, Toronto, Ontario, Canada; and the Department

of Pathology, University of Michigan Medical School, Ann Arbor, Michigan.

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Rh in Gel and Tube

• Denome et al.

• Additional discrepancies were noted when an IS tube test was performed to confirm the Rh type.

• Serologic analysis was performed using anti‐D reagents:– Gamma‐clone (Immucor, Inc.)

– BioClone (Ortho Clinical diagnostic)

– Panel of monoclonal antibodies indicated as A through L (Alba Bioscience)

Rh in Gel and Tube

• Denome et al.

• Molecular analysis

– Discrepant IS tube test result were analyzed for partial D variants.

• Samples with RHD‐specific exons were further analyzed for the presence of weak D Type 1‐3 by PCR restriction fragment length polymorphism (RFLP) assay

Rh in Gel and Tube

• Denome et al.

• Molecular analysis

– Samples that could not be assigned an RHD allele underwent:

• Direct automated sequencing of exon 5

• Direct sequence analysis to evaluate the presence of the DAR allele (exon 7)

• Comparative analysis with the reference RHD sequence

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Rh in Gel and TubeDenome et al.

Results:

Rh in Gel and Tube

• Denome et al.• Results:

– Monoclonal anti‐D reagents did not distinguish between partial and weak D Types 1 ‐ 3

– Weak D Types 1 and 2 do not show consistent reactivity with FDA‐approved reagents and technology.

– It is recommended that patient be considered Rh(‐) if:

• Immediate‐spin tube test cutoff score of ≤ 5 (i.e., 1+ agglutination)

• Or by gel technology with a score ≤ 8 (i.e., 2+ hemagglutination)

– Tube test anti‐D reagents react poorly with some Weak D Types.

– Molecular tests that distinguish common partial and Weak D types provide the solution to resolving D antigen discrepancies.

Rh Typing on Echo/NEO

Galileo Echo Training Guide ECO‐003‐200 Lesson 6 Module 3 pg 3‐13

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Echo Interpretation

Galileo Echo Training Guide ECO‐003‐200 Lesson 6 Module 3 pg 3‐13

Neo Interpretation

Note: Assays that include the use of two different anti‐D reagents and yield a positive result with one anti‐D reagent, but a negative result with the other anti‐D reagent for a given sample , will generate an NTD result related to the Rh(D) testing

NEO Operator Manual, NEO‐001‐102: ATT1‐35

Rh Typing on Echo/Neo

• Similar to tube testing

• No direct guidance on how to interpret weak or equivocal reactions

• No literature available to direct interpretation in the hospital blood bank

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RhD Serologic Discrepancies using Microtiter Well Agglutination: The

Search for Allelic Variants• Hudgins J., Tuma C., Matsushita C., Shulman I. 2017 (Manuscript)

• Thesis:– The identification of individuals with divergent epitopes of the D

polypeptide can only be discovered if interpretive guidance is provided for inconsistent and/or weak D typing results on the NEO and Echo platforms.

• Sample Selection: – NEO: Reactivity <3+/3+ for both series D4 and series D5 anti‐D.– ECHO: Reactivity < 2+/2+ for both series D4 and series D5. – In addition

• Any specimen typed by either platform as equivocal ʺ?ʺ or < 2+ reactions in either anti‐D series.

• Patients with discrepant historical types.

LAC+USC Study

• 41,170 samples from Jun. 2015 – Sept. 2016– 82 weak or discrepant samples were identified and tested.

• 1 sample was disregarded as DNA amplification was not possible.

• Samples were then sent for molecular testing:

– DNA extracted on Qiacube, and quantified on Nanodrop.

– Genotyping assays

• RHD BeadChip (RUO): identifies RHD variants, commercially available.

• RHDxp: Prototype; not available commercially.

• RHD Zygosity: Prototype; not available commercially.

• RHCE BeadChip (RUO): identifies RHCE major antigens and variants, commercially

available.

LAC +USC Study Results

ExtractedDNA

Report Result*(51/81, 63%)

RHD BeadChip Variant Detected?

Yes

NoRHDxp variant

Detected?

Yes

Report Result(1/81, 1.2 %)

NoCeppellini effect? RHCE BeadChip &

RhD Zygosity

Yes

Report Result(16/81, 19.8%)

*Phenotypic variant results were further classified into phenotypic classes.

No Report as D+(13/81, 16.0%)

Of the 52 samples that resulted from a SNP that resulted in weak D or a variant D antigen,35 of 50 (68%), were variant D proteins with the potential to cause alloimmunization.

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LAC + USC Study

• Conclusions:– Any patient that reacts ≤1+ by NEO/Echo should be treated as Rh negative.

– High risk populations (i.e. women of child bearing age or allogeneic stem cell transplant) should be identified and tested for molecular variation.

– Pregnant patients that reacts ≤1+ by NEO/Echo in either or both of the D4/D5 series is candidate for RhIg, until resolved by molecular typing.

References

• The Rh blood group system: a review Blood 2000 95(2):375‐387• RHD gene deletion occurred in the Rhesus box Blood 2000 95(12):3662‐3668• Br Med J. 1956 Apr 14; 1(4971): 862, 863• Heredity of the Rh Factor. Wiener AS, Sonn EB. Genetics. 1943

Mar;28(2):157‐61.• Sequence analysis of the spacer region between

the RHD and RHCE genes. Biochem Biophys Res Commun. 1999;263:378‐383.

• cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)‐blood‐group‐antigen expression. Biochem J. 1990;271:821‐825.

• Rh nomenclature. Transfusion. 1979 Jul‐Aug;19(4):487.• Expert Reviews in Molecular Medicine: 2006, 8(13)• Denome et al. Rh discrepancies caused by variable reactivity of partial and

weak D types with different serologic techniques. TRANSFUSION 2008;48:473‐478.

• Galileo Echo Training Guide ECO‐003‐200 Lesson 6 Module 3 pg 3‐13• NEO Operator Manual, NEO‐001‐102: ATT1‐35

Acknowledgements

• Cheryl Matsushita (Asst. Blood Bank Supervisor)

• Chris Tuma (Blood Bank Supervisor)

• Dr. Ira Shulman (LAC+USC Medical Director)

• Dr. Sukanta Banerjee (Immucor genomics lab)

• Immucor, Inc.

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Janet Bowker MT(ASCP)

Transfusion Service Lead TechnologistProvidence Saint Patrick Hospital

Immunohematology Case Studies

Case Study presented for Immucor User-Group by: Janet Bowker, MT (ASCP)

Transfusion Service Lead TechnologistProvidence Saint Patrick Hospital

Missoula, Montana

Providence Saint Patrick Hospital

Serving the Missoula valley and surrounding areas since 1873.

Licensed for 253 beds

Level II Trauma Center

Home of the International Heart Institute (IHI) & Montana Cancer Center

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SPH Transfusion Service 4619 blood products transfused in 2016

4078 antibody screens performed in 2016

ABS live in 2005

Galileo Echo live in 2008

Case Study #1(A lesson in weakness)

70 year old multiparous female admitted for open heart surgery on 12/5/12

12/15 ICU orders 2 units PC’s to be transfused– Patient is 9 days post open heart surgery– Multiple complications

Previous history includes a negative screen on 12/5/12 and no transfused products.

12/15/12Type & Screen performed on Echo

Screen cell II yields a 1+ reaction.Most likely candidates are E or K.

0

0

+

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Echo 14-cell antibody panel

0

0

00

0

00

00

000

0

+

Manual 16-cell panel (LISS & PEG)

37 AHG

cc ccPEG

2+ w+ 2+

0

0

00000

0 2+ 1+

2+

2+

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Case Study #1 - Conclusion

Patient has a weak Anti-E.

She was successfully transfused two units of E, c antigen negative RBC’s.

Different methodologies were important in identifying this antibody.

Case Study #2(A lesson in education)

5/14/10 patient arrives in ED with a GI bleed

– 51 year old female with cerebral palsy

– Hgb at admission is 6.9 g/dL

– History of anti-E, anti-K, anti-Jkb

– Last transfusion was 7 months prior to this admission

– Type & Screen ordered

Case Study #2 (cont.)

Echo type & screen– A+ patient with positive antibody screen.

Echo Ready-ID panel = anti-Jkb

– There was one cell showing unexpected reactivity that wasn’t consistent with the known anti-E, -K, or -Jkb.

Manual peg antibody ID was consistent with anti-E, -K, -Jkb. – All other abs to significant ag’s were ruled out.

By the time antigen negative units were found and crossmatched, the patient’s hgb had dropped to 3.3 g/dL. 3 RBC’s and 4 FFP were transfused.

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5/15– Patient hgb now at 9.1 g/dL and GI bleed has

been successfully treated.– Patient is discharged 2 days later.

5/20– Patient arrives back in the ED, again with a GI

bleed… Hgb is 6.7 g/dL upon arrival Two RBC’s ordered for transfusion STAT Physician made aware of complex workup and

delay in crossmatch-compatible blood availability.


Echo was down for PM servicing.

Manual screen & antibody ID were performed.– One unexpected reactive cell - consistent with Yt(b+).

A second Yt(b+) positive cell found was also reactive.

– All abs to significant ag’s were ruled out with the exception of S. Patient was typed and found to be S-antigen positive.

Two units of E, K, JKb antigen negative RBC’s were found to be crossmatch compatible and transfused late that evening.

And then…

EEEEKKKK! A Transfusion Reaction!!

When the 2nd RBC was almost completely transfused, the RN noticed the patient’s urine had turned red in the foley bag!

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Suspected transfusion reaction workup performed:– Clerical checks all match (right unit to correct patient).– Pre & post-tx samples are both A positive.– Post-tx sample shows moderate hemolysis!– Pre & post-tx DAT’s are both negative?? – Post-tx antibody screen matches initial testing.– Post-tx antibody identification (on ECHO) now shows two cells

with unexpected reactivity.– Crossmatches are repeat compatible with pre-tx sample.– IV fluids used during transfusion were correct (normal saline).

Sample is immediately sent out to the SLC ARC reference lab for a STAT elution and further workup.

In the meantime, the patient’s hgb has dropped from 6.7 to 6.4 g/dL.


ARC reference lab workup reveals:– DAT – IgG microscopically positive– Anti-Fyb (in eluate only)– Anti-Doa (in eluate only)– Probable Anti-Bg– Undetermined reactivity – 1 of 6 cells crossmatched still showed

unexplained reactivity.

Patient is now compatible with approximately 1% of the population. (Anti-E, -K, -Jkb, -Fyb, -Doa)

4 antigen-negative, crossmatch-compatible cells are shipped to Missoula for transfusion.

1 unit of RBC’s transfused on 5/21 & 5/23 w/o reaction


5/27 – patient still admitted, order to transfuse RBC’s received by blood bank.

Samples sent to SLC ARC reference lab for workup and crossmatching.– Anti-E, -Fyb, -Jkb, -Doa confirmed– Anti-K not demonstrated– Undetermined reactivity consistent with Anti-Bg present.

Patient transfused one unit RBC’s on 5/28 w/o reaction.

Patient discharged on 6/1.

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11/9 Patient returns with GI bleed!– 12.5 hgb at 1807 and drops to 7.1 by 11/10 0410. – 2 units RBC’s ordered to be available 11/10.– Sample drawn and sent to SLC ARC.

ARC results:– History of anti-E, -K, -Jkb, -Fyb, -Doa

– Probable anti-Bg not reconfirmed.– New anti-Kpa, -Lua, and possible anti-Sla (Knops

system)

2 units RBC’s found thru the rare donor program transfused on 11/13.


11/16 – Patient still admitted, GI bleed controlled, hgb dropping.– Provider calls blood bank to request 2 more

RBC’s be available. – Also mentions “It seems like she’s

hemolyzing, but we don’t know why”.

STOP TRANSFUSING THE PATIENT!!

Hemoglobin stabilizes and patient goes home.

Case Study #2 - conclusion

ARC ref lab recommended that patient donate blood for future use.– Possibly eligible for artificial blood products.

Education of patient is important!– Patient was reacting to every unit transfused.

Education of the techs is important!– Techs need to know when to notify the med. director.

Education of providers is important!– PA learned a lot with this case!

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Case study #3(A lesson in assumption)

Multiparous female admitted with a GI bleed.– No history of transfusion at our facility or surrounding hospitals– 2 units of RBC’s ordered to be transfused ASAP.– Type & Screen performed

Patient is O positive Positive antibody screen on Echo (SCIII).

ECHO

00+

Case Study #3

000+++++

+++++++0

Cells 1,2 & 3 are negative, all others are positive..

000000+0

0000

0000

Case Study #3

All cells are negative (except positive control)

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Nightshift conclusion All significant antibodies can be ruled out with the

Extend-I panel. Manual LISS screen was performed and it was negative. Cold screen was negative. Two units of RBC’s were found to be AHG-crossmatch

compatible. The unresolved reactivity was called ‘non-specific

reactivity’.

Supervisory review the next morning:– Hmmmm… this is suspicious, let’s run the

Extend-II panel.

+++++++0

++++++++

Case Study #3

All cells are positive (except negative control)

Let’s look at the whole picture

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Cells 1,2 & 3 are negative, all others are positive..

All cells are negative (except positive control)

All cells are positive (except negative control)

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Conclusion – Case Study #3 Anti-f

– f antigen occurs when RBC’s have c and e antigens in the same haplotype (in cis), for example, R1r(DCe/dce).

– Antigen is not expressed when c and e occur on separate haplotypes (in trans), e.g., R1R2 (DCe/DcE).

Patient’s phenotype: C+,E+,c+,e+– Possible genotype: R1R2 (DCe/DcE)– Lacks the ce combo on the same haplotype– f-negative and capable of form anti-f

Never assume reactivity is non-specific, always be on the look-out for patterned reactivity.

Case Study #4 (a lesson in communication)

84 year old female– Diagnosed with endometrial cancer– Scheduled for hysterectomy on 11/3/16– Previous type & screen (7/6/16) A pos with neg antibody screen

11/3/16 – patient in pre-op– Type and screen ordered


Type & Screen performed on Echo– A pos, Abs positive 1+/2+ on all three cells.

Patient states she has never been transfused.

SURGERY DELAYED!!

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– Ready-ID panel shows 5 negative cells all others are ‘?’, 1+ or 2+.

– Extend-I panel shows only 4 reactive cells Able to r/o all antibodies to significant antigens.

– Manual LISS abs = weak reactivity in one cell at AHG only.

– DAT = negative

– Two RBC’s crossmatched and found to be compatible at AHG.

SURGERY CAN PROCEED!

Case Study #4 (conclusion) What’s causing this reactivity?

Further investigation revealed another diagnosis…

MULTIPLE MYELOMA!

Patient was treated with Daratumumab/Darzalex (anti-CD38) from 7/2016 until 10/2016.

Established interdepartmental workflow failed.– T&S was ordered on day treatment started, but pharmacy

failed to tell BB of Daratumumab treatment.

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In Conclusion…

Automation is extremely helpful in managing workload, but it does result in another level of complexity on occasion.

You don’t have to feel like you’ve bitten off more than you can chew...you just have to engage the brain and use your knowledge! Automation does not give us permission to turn off our thinking skills.

Slow down, think about all the tools you have available and what information they might reveal if used.

Communication is important within the lab, with other areas and providers, and especially with patients!

Be okay with the fact that there is not one perfect methodology.

Thank You!

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