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Seeing Beyond Limits
2020 Webinar Schedule
Link to register: https://app.livestorm.co/immucor
Seeing Beyond Limits
2020 “Mini” WebinarsShort,10-minute educational videos with an industry leader.
Link to register: https://app.livestorm.co/immucor
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learn.immucor.com
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learn.immucor.com
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Continuing Education
• PACE, Florida and California DHS
• 1.0 Contact Hours
• Each attendee must register to receive CE at:
https://www.surveymonkey.com/r/VariantD
• Registration deadline is August 28, 2020
• Certificates will be sent via email only to those who have registered September 4, 2020
The Rh System
Regina Castor BS MT(ASCP)SBB cm
Area Technical Consultant – East Region
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Objectives
• Review characteristics of the Rh antigens
• Discuss the genetics of the Rh system as it relates to antigen expression
• Describe the difficulties experienced with testing for Rh antigens
• Explore some of the reasons for typing discrepancies
• Present the proposed typing algorithm introduced by AABB/CAP for RHD genotyping
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Rh System
• Composed of 2 genes• RHD and RHCE
• Responsible for 50+ antigens
• Both code for 417 amino acids
• Closely linked
• 97% sequence identity is shared
• Several nomenclatures• Fisher-Race (ex: DCe)
• Weiner (ex: R1)
• Large molecular weight proteins
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Rh Genetics Source: “Discrepancies in RhD Typing: Picking up the Rhesus Pieces” Sue Johnson
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Rh Antigens
• Found only on red blood cells
• Well developed at birth
• HDFN
• Enhanced with enzymes
• Ammonium channel transporter
• Important for RBC structural integrity
• Highly immunogenic• HTR
• Combination antigens possible
• Wide amount of genetic variation
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Rh Antigens
“Rh-positive” or “Rh-negative”
D+ or D –
85% 15%
Caucasians
African
Americans Asians
D: 85% 92% 99%
C: 68% 27% 93%
E: 29% 22% 39%
c: 80% 96% 47%
e: 98% 98% 96%
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Genetic Variation
• >500 RHD alleles reported involving amino acid changes
• Estimated 1% have an allele that codes for altered D expression (European ethnicity)
• Higher rates in African ethnicity
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Genetic Variation
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Genetic Variation
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Genetic Variation
• Altered D categories • Weak D
• Quantitative difference in the amount of D antigens present on the cell
• Partial D*• Qualitative difference in the expressed antigen• “mosaic”
• Del*• Extremely low levels of antigen present cannot be detected
by routine serologic testing methods
• Nonfunctional RHD*• Gene does not encode full protein and are non-functional• Aka D null
*Capable of making Anti-D
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Genetic Variation
• Altered D categories • Weak D
• Quantitative difference in the amount of D antigens present on the cell
• Partial D*• Qualitative difference in the expressed antigen• “mosaic”
• Del*• Extremely low levels of antigen present cannot be detected
by routine serologic testing methods
• Nonfunctional RHD*• Gene does not encode full protein and are non-functional• Aka D null
*Capable of making Anti-D
Sometimes the term “Weak D” is used as a generic term to describe serologic results as opposed to a specific group of RhD variants
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“Rh-positive”
D+
D+, Partial D, Partial weak D, Weak D, D –
“Rh-negative”
D –WEAK D+
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Typing and Testing Discrepancies
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Reasons for Typing Discrepancies
• Genetic variants (as previously discussed)• Decreased quantity of Rh antigens
• Missing epitopes
• Both qualitative and quantitative differences
• Reagent differences• Polyclonal vs. Monoclonal
• Different manufacturers
• Different clones detect different epitopes
• May detect at different phases of testing
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Typing Discrepancies
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Reasons for Typing Discrepancies
• Differences in testing methods• Serological vs. Molecular
• Serologic methods cannot distinguish between Weak D and Partial D types (those who can make Anti-D and those who cannot)
• Hemagglutination, Gel, Solid Phase
• Manual vs. Automation
• Why the testing is being performed vs. interpretation• Donor testing
• Transfusion recipient
• Neonatal typing/RhIG Administration
• Age of the patient
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Testing Algorithms Lack of standard practice
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Testing Algorithms
• 2012 CAP survey: • Number of transfusion services performing a
serological weak D test on patients from 58.2% to 19.8% as a strategy to manage those with a weak D as Rh negative
• 2014 CAP survey:• How do you report the patient Rh type when it is
weak or variable?• RhD positive (47 %)
• RhD negative (11 %)
• If female or OB, report as RhD negative (some)
• Weak D positive (30 %)
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Transfusion Volume 55, Issue 3, pages 680-689, 1 DEC 2014 DOI: 10.1111/trf.12941 http://onlinelibrary.wiley.com/doi/10.1111/trf.12941/full#trf12941-fig-0002
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AABB/CAP issue joint statement on phasing-in RHD Genotyping for pregnant women and other females of childbearing potential with a serologic weak D
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Guidance Updated in 2020
Type 4.0 and 4.1 added to those that
can be treated as Rh +
“Use of two different serologic methods or Anti-D reagents have been shown to be an
effective practice for identifying those patients who
will benefit from RHDgenotyping”
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• Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations.
• The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor.
Identification and Management of patients
with Weak D and Partial D phenotype
A risk-based approach in a daily practice
NYP Lower Manhattan Hospital, New York
3 years experience
Daniel Alvarado MLS(ASCP)CMSBBCM, CQA(ASQ)Lisa Huynh MLS(ASCP)
Ljiljana Vasovic MD
Serologic weak D phenotype• Strength of reaction weaker than expected • Inconclusive Result • Discrepant Result vs. Historical or vs. other institution
Send for RHD genotyping• Result as per tube testing (or INV)• Manage as Rh Negative
genotyping result: Weak D type 1, 2, or 3 • Not at risk for anti-D • Not candidate for RhIG• Result as D positive
genotyping result: Weak D type 1, 2, or 3 Not detected • May be at risk for anti-D • Give Rh(D) negative blood • Candidate for RhIG• Result as D negative
@ LMH
Rh D Discrepancy Analysis / Partial D Analysis
• 6mL EDTA whole blood (lavender or pink top)
Rh D Genotyping
Manual Tube Weak D Cells Testing
Solid Phase ECHO or Manual Tube D Testing
Immucor Anti-D (Series 4) (Monoclonal Blend) Blood Grouping Reagent is prepared by blending IgM monoclonal anti-D
secreted by a human/murine heterohybridoma (MS201) with IgG anti-D heterohybridoma (MS26)
Immucor Anti-D (Series 5) (Monoclonal Blend) Blood Grouping Reagent is prepared by blending IgM monoclonal anti-D
secreted by a human/murine heterohybridoma (TH28) with IgG anti-D of another heterohybridoma (MS26).
the director Connie Westhoff, SBB, PhD
63 2+ ? 3+/3+ 3+/3+ hemizygote RHD*DFL
Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING
Anti-D
(series4)
Anti-D
(series5)
Anti-D
(series4)
Anti-D
(series5)
RHD alleles PREDICTED RHD PHENOTYPE 2nd alleleN
25 y.o. white female
LIS Comments :Day 0: Please Give O Neg RBCS till results from NYBC for D typing molecular are received.
Sample sent to the Molecular lab.Day +10 : Patient must receive RhIg (Rhogam).
Molecular Results for D Antigen: RHD*weak D type 1 Val207Gly
Predicted RHD Phenotype: D+ (weak partial)The patient has a RHD allele first reported as a weak D but now known to also encode a partial phenotype, hence the terminology "weak partial D phenotype". The RBCs may have weaker than expected D antigen expression depending on the reagent and method used.
Allo anti-D has been observed in patients with this RHD genotype who are on chronic transfusion therapy.
The risk for Anti-D in pregnancy is unknown and for transfusion appears to be far less than for other partial D phenotypes. Females of child bearing potential with a partial D phenotype are better served as Rh negative for transfusion and candidates for Rh immune globulin (if they have not produced Anti-D).
27 y.o. female delivered a healthy male child,Rh testing: serologic weak D phenotype
Solid Phase ECHO TUBE TESTING IS/AHG
Anti-D (series4) Anti-D (series5) Anti-D (series4) Anti-D (series5)
1+ 0 1+/3+ 1+/3+
RHD alleles
RHD genotype: RHD*DAR encodes amino acid changes 201Arg, 223Val, and 342Thr. Inactive RHD Ψ
D phenotype: D+ (partial)
COMMENTS: The patient has a partial D phenotype associated with risk for anti-D, the RBCs may have weaker than expected D antigen expression depending on the reagent and method used. Females of child bearing potential with a partial D phenotype are better served as Rh negative for transfusion and candidates for Rh immune globulin (if they have not produced active anti-D). (Transfusion 2015:55:680-89)
Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING
Anti-D
(series4)
Anti-D
(series5)
Anti-D
(series4)
Anti-D
(series5)
RHD alleles PREDICTED RHD PHENOTYPE 2nd alleleN
61 1+ ? 2+/2+ 2+/2+ RHD alleles RHD*DAR Inactive RHD Ψ
22 y.o. black female
Specimen sent to NYBC Molecular Lab for D Variant Testing on 03/14/2018.
RHD with no changes associated with common or partial D or weak D.PREDICTED RHD Phenotype: D+
Gene sequencing did not identify any changes from conventional. The patient is predicted to have two RHD alleles, and to have normal expression of D antigen and to not be at risk for anti-D, The results are consistent with massive transfusion with D- donor units.
50 y.o. Asian female Hx of A Pos
MHX: Beta thalassemia. consistent with massive transfusion with D- donor units at other institution
Solid Phase ECHO TUBE TESTING IS/AHG
Anti-D (series4) Anti-D (series5) Anti-D (series4) Anti-D (series5)
0 0 0/w+ 0/w+
PREDICTED RHD PHENOTYPE Designation n %D+ RHD 4 6.0%
D+ (partial) RHD*DAR 12 17.9%
RHD*DAU4 3 4.5%
RHD*DCS1 2 3.0%
RHD*DFL 1 1.5%
RHD*DOL 1 1.5%
D+ (weak partial ) RHD*weak partial D type 4.0 25 37.3%
D+ (weak) RHD*weak D type 1 6 9.0%
RHD*weak D type 122 1 1.5%
RHD*weak D type 2 3 4.5%
RHD*weak D type 3 8 11.9%
RHD*weak D type 841c 1 1.5%
GENDER
PREDICTED RHD PHENOTYPE Female Male Grand Total
D+ 2 2 4
D+ (partial) 17 2 19
D+ (weak partial ) 16 9 25
D+ (weak) 12 7 19
Grand Total 47 20 67
PREDICTED RHD PHENOTYPE Female Male Grand Total
D+ 3% 3% 6%
D+ (partial) 25% 3% 28%
D+ (weak partial ) 24% 13% 37%
D+ (weak) 18% 10% 28%
Grand Total 70% 30% 100%
At LMH GENDER Female Male
PREGNANCY STATUS No Yes N/A
PREDICTED RHD PHENOTYPE
D+ 2 2
D+ (partial) 9 8 2
D+ (weak partial ) 9 7 9
D+ (weak) 3 9 7
Grand Total 23 24 20
GENDER Female Male
PREGNANCY STATUS No Yes N/A
PREDICTED RHD PHENOTYPE
D+ 3% 0% 3%
D+ (partial) 13% 12% 3%
D+ (weak partial ) 13% 10% 13%
D+ (weak) 4% 13% 10%
Grand Total 34% 36% 30%
Average of AGE Female Male Grand Total
PREGNANCY STATUS No Yes N/A
D+ 70.0 45.5 57.75
D+ (partial) 43.1 30.1 49.5 38.31578947
D+ (weak partial ) 46.7 28.9 59.7 46.36
D+ (weak) 36.3 34.9 51.1 41.10526316
Grand Total 46.0 31.5 54.3 43.26865672
PREDICTED RHD PHENOTYPE White Black Asian
Pacific
Islander Other Refused U/K
Grand
Total
D+ 2 1 1 4
D+ (partial) 3 11 3 1 1 19
D+ (weak partial ) 2 14 1 5 1 2 25
D+ (weak) 12 1 1 3 1 1 19
Grand Total 17 27 2 2 12 3 4 67
PREDICTED RHD PHENOTYPE White Black Asian
Pacific
Islander Other Refused U/K
Grand
Total
D+ 0% 3% 1% 0% 1% 0% 0% 6%
D+ (partial) 4% 16% 0% 0% 4% 1% 1% 28%
D+ (weak partial ) 3% 21% 0% 1% 7% 1% 3% 37%
D+ (weak) 18% 0% 1% 1% 4% 1% 1% 28%
Grand Total 25% 40% 3% 3% 18% 4% 6% 100%
Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING
Anti-D
(series4)
Anti-D
(series5)
Anti-D
(series4)
Anti-D
(series5)
RHD alleles PREDICTED RHD PHENOTYPE Amino Acid Change 2nd alleles
1+ 0 1+/3+ 1+/3+N Anti-D Anti-D Anti-D Anti-D RHD alleles PREDICTED RHD PHENOTYPE Amino Acid Change 2nd alleles 1 0 0 0/3+ 0/3+ hemizygote RHD*weak D type 1 Val207Gly
2 0 0 1+/2+ 0/1+ hemizygote RHD*weak D type 1 Val207Gly
3 ? 0 0/3 0/2+ hemizygote RHD*weak D type 1 Val207Gly
4 ? 0 0/2+ 0/W+ hemizygote RHD*weak D type 1 Val207Gly
5 ? 0 1+/3+ 0/3+ hemizygote RHD*weak D type 1 Val207Gly
6 1+ 0 2+/3+ 2+/3+ hemizygote RHD*weak D type 1 Val207Gly
7 0 0 0/2+ 0/1+ hemizygote RHD*weak D type 2 Gly385Ala
8 0 0 0/2+ 0/2+ hemizygote RHD*weak D type 2 Gly385Ala
9 0 0 2+/2+ 2+/2+ hemizygote RHD*weak D type 2 Gly385Ala
10 0 0 3+/4+ 3+/4+ hemizygote RHD*weak D type 3 Ser 3 Cys
11 ? 0 2+/3+ 2+/3+ hemizygote RHD*weak D type 3 Ser 3 Cys
12 ? ? 1+/4+ 1+/4+ hemizygote RHD*weak D type 3 Ser 3 Cys
13 1+ ? 2+/2+ 2+/2+ hemizygote RHD*weak D type 3 Ser 3 Cys
14 1+ ? 2+/3+ 2+/3+ hemizygote RHD*weak D type 3 Ser 3 Cys
15 1+ ? 2+/3+ 2+/3+ hemizygote RHD*weak D type 3 Ser 3 Cys
16 1+ ? 2+/3+ 2+/3+ hemizygote RHD*weak D type 3 Ser 3 Cys
17 1+ ? 2+/3+ 0/3+ hemizygote RHD*weak D type 3 Ser 3 Cys
18 1+ ? 2+/2+ 2+/2+ hemizygote RHD*weak D type 841c Val281Leu
19 0 ? 2+/2+ 2+/2+ hemizygote RHD*weak D type 122 Arg70Trp
N
Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING
Anti-D
(series4)
Anti-D
(series5)
Anti-D
(series4)
Anti-D
(series5)
RHD alleles PREDICTED RHD PHENOTYPE Amino Acid Change 2nd alleles
20 0 0 2+/3+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
21 0 0 3+/3+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
22 ? ? 3+/4+ 2+/4+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
23 1+ 0 1+/3+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
24 1+ 0 2+/3+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
25 1+ 0 2+/3+ 2+/3+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223ValHYBRID RHD*DIIIa-
CE(4-7)-D
26 1+ 0 2+/3+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
27 1+ 0 3+/4+ 2+/4+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
28 1+ 0 4+/4+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
29 1+ ? 0/2+ 0/1+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
30 1+ ? 0/4+ 0/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
31 1+ ? 1+/4+ 1+/4+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223Val RHD*330delGT
32 1+ ? 2+/2+ 0/2+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
33 1+ ? 2+/3+ 1+/2+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
34 1+ ? 2+/3+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
35 1+ ? 2+/4+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
36 1+ ? 2+/2+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
37 1+ ? 2+/3+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
38 1+ ? 2+/2+ 2+/4+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
39 1+ ? 2+/4+ 2+/4+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223Val Inactive RHD Ψ
40 1+ ? 3+/3+ 2+/2+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
41 1+ ? 3+/3+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
42 1+ ? 3+/4+ 3+/4+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223Val Inactive RHD Ψ
43 1+ ? 4+/4+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val
44 1+ ? 2+/2+ 2+/2+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223ValHybrid MID*D111a-
CE(4- 7) -D
N
Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING
Anti-D
(series4)
Anti-D
(series5)
Anti-D
(series4)
Anti-D
(series5)
RHD alleles PREDICTED RHD PHENOTYPE Amino Acid Change 2nd alleles
45 0 0 0/2+ 0/2+ RHD alleles RHD*DAR 201Arg,223Val and 342Thr Inactive RHD Ψ
46 0 1+ 1+/3+ 2+/4+ hemizygote RHD*DAU4 233Lys and 379Met
47 0 3+ 2+/3+ 3+/3+ hemizygote RHD*DAU4 233Lys and 379Met
48 0 4+ 3+/3+ 4+/4+ hemizygote RHD*DAU4 233Lys and 379Met
49 ? 0 0/4+ 0/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
50 ? 0 1+/2+ 1+/2+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
51 ? 0 2+/3+ 0/3+ hemizygote RHD*DCS1 223Val and 226Pro
52 ? 0 3+/4+ 2+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
53 1+ 0 w+/4+ w+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
54 1+ 0 2+/4+ 1+/3+ RHD alleles RHD*DOL 170Thr and 223Val partial, DAK+
55 1+ 0 2+/3+ 2+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
56 1+ 0 2+/3+ 2+/4+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
57 1+ 0 2+/4+ 2+/4+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
58 1+ 0 2+/3+ w+/2+ hemizygote RHD*DCS1 223Val and 226Pro
59 1+ ? w+/4+ w+/4+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
60 1+ ? 2+/3+ 1+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
61 1+ ? 2+/2+ 2+/2+ RHD alleles RHD*DAR 201Arg,223Val and 342Thr Inactive RHD Ψ
62 1+ ? 2+/2+ 2+/2+ hemizygote RHD*DAR 201Arg,223Val and 342Thr
63 2+ ? 3+/3+ 3+/3+ hemizygote RHD*DFL Tyr165Cys
N
Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING
Anti-D
(series4)
Anti-D
(series5)
Anti-D
(series4)
Anti-D
(series5)
RHD alleles PREDICTED RHD PHENOTYPE 2nd allele
1+ 0 1+/3+ 1+/3+N Anti-D Anti-D Anti-D Anti-D RHD alleles PREDICTED RHD PHENOTYPE 2nd alleles note Hx
64 0 0 0/w+ 0/w+ homozygote RHD
weak D performed due
to Hx Rh Pos
ex: Tx Rh neg Beta thalassemia. consistent with
massive transfusion with D- donor units
65 1+ 0 0/3+ 0/3+ homozygote RHD
weak D performed due
to Hx Rh Pos fall rib fracture
66 1+ 1+ N/A N/A homozygote RHD RHD
Anti-D African American male; sickle cell disease.
Transfused. Anti-D identified at another facility.
67 3+ 4+ N/A N/A homozygote RHD RHD
Anti-D African American male; sickle cell disease.
Possible auto or allo anti-D.
N note Hx
Thank you Questions?
www.aabb.org 20
Daniel Alvarado MLS(ASCP)CMSBBCM, Lisa Huynh MLS(ASCP)Meta Morrison MLS(ASCP)
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Continuing Education
• PACE, Florida and California DHS
• 1.0 Contact Hours
• Each attendee must register to receive CE at:
https://www.surveymonkey.com/r/VariantD
• Registration deadline is August 28, 2020
• Certificates will be sent via email only to those who have registered September 4, 2020
22All Content © Immucor
24All Content © Immucor
Thank you!
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