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Application of the CLSI EP15-A3 Guideline as an Alternative Troubleshooting

Tool for Verification of Assay Precision

Article in American Journal of Clinical Pathology · September 2019

DOI: 10.1093/ajcp/aqz117.007

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Mayo Foundation for Medical Education and Research
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AJCP / Meeting Abstracts
of patients with infectious mononucleosis and occurs
in approximately 1% to 3% of these patients. The path-
ogenesis of EBV-related autoimmune hemolytic anemia
is unknown, with a suspicion for inadequate B-cell func-
tion after infection. Appropriate laboratory testing and
analysis are critical for rapid diagnosis in these patients
to ensure adequate treatment with no long-term sequelae.
Application of the CLSI EP15-A3 Guideline as an
Alternative Troubleshooting Tool for Verification of Assay
Precision
Jose Jara Aguirre, MD1, Karl Ness, MLS1,
Alicia Algeciras-Schimnich1; 1Mayo Clinic
Introduction: The CLSI EP15-A3 guideline “User
Verification of Precision and Estimation of Bias” provides
a simple experimental approach to estimate a method’s
imprecision and bias. The objective is to determine if the
laboratory precision performance of repeatability (SR)
and within-laboratory imprecision (SWL) are in accord-
ance to the manufacturer specification claims (MSCs).
Objectives: Evaluate the utility of the EP15-A3 protocol
to verify method precision during a troubleshooting in-
vestigation and after major instrument maintenance,
using a carcinoembryonic antigen (CEA) immunoassay
as an example.
Methods: CEA was performed on the Beckman Coulter
DxI (Beckman Coulter, Brea, CA). Quality control (QC)
levels (L1: 2.89; L2: 21.10; L3: 39.10 ng/mL) (Bio-Rad
Laboratories, Irvine, CA) were used. Each QC level was
measured before and after instrument maintenance as fol-
lows: five replicates per run, one run per day, and during
5 days. Imprecision estimates (IEs) for SR (%CVR) and SWL
(%CVWL) were calculated by one-way analysis of variance
using Microsoft Excel Analyse-it software. Estimated im-
precision was compared to MSC and desirable impreci-
sion specifications based on biological variation (BV).
Results: A change in the analytical performance of CEA
was detected by a decreased sigma-metric indicator. After
a bias problem was ruled out, the observed %CVR for L1,
L2, and L3 were 7.2%, 3.7%, and 4.8%, respectively. The
%CVWL were 8.3%, 5.0%, and 5.5%, which exceeded the
MSC of %CVWL ~4.0% to 4.5%. After a laboratory inves-
tigation, major instrument maintenance was performed
by the manufacturer. The %CVR and %CVWL estimates for
L1, L2, and L3 after maintenance were 3.2%, 3.8%, 3.5%
and 3.9%, 4.2%, 4.0%, respectively. After maintenance,
the CEA performance was consistent with the MSC for
each of the levels analyzed and within the BV impression
goal of %CV ≤6.4.
Conclusion: CLSI EP15-A3 guideline is an alternative
troubleshooting tool that can be used to investigate and
verify method precision performance before and after sig-
nificant instrument maintenance.
S88 Am J Clin Pathol 2019;152:S85-S91
DOI: 10.1093/ajcp/aqz117
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Paroxysmal Nocturnal Hemoglobinuria: Grasping the
Flow of Diagnostic Dilemmas
Phuong-Lan Nguyen, MD1; 1University of Kentucky
Healthcare
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare
life-threatening condition due to an acquired somatic mu-
Downloaded from https://academic.oup.com/ajcp/article-abstract/152/Supplement_1/S88/5567864 by Pfizer Inc user on 13 November 2019
tation of the PIGA gene, leading to nonmalignant clonal
expansion of hematopoietic stem cells, which are deficient
in glycosyl phosphatidylinositol-anchored proteins (GPI-
APs). Fluorescein-labeled proaerolysin (FLAER) and
flow cytometry are key tools in the diagnosis of PNH.
While clonal detection of PNH in both tests has a sen-
sitive diagnostic threshold of 0.01% in erythrocytes and
0.05% to 1% in leukocytes, one must be cautious in ruling
out the possibilities of myelodysplastic syndrome (MDS)
or aplastic anemia. We propose guidelines in the differ-
ential diagnosis and evaluation of PNH from these and
other hematologic disorders that can arise from GPI-AP
deficient cells. These guidelines are based on a meta-
analysis of five research literature sources, including four
case studies. We also compare and contrast our limits of
quantification of the in-house PNH assay at University
of Kentucky Healthcare with those of an interlaboratory
validation of 11 institutions within the United Kingdom.
Our report advocates for thorough evaluation of mul-
tiple laboratory and clinical variables affecting sensitivity
and accuracy of flow cytometry and FLAER in PNH.
Furthermore, we recommend lowering of the in-house
limit of quantification from the current 1% to 0.01%. This
allows for the critical consideration of conditions such as
MDS and aplastic anemia and their disease courses, all of
which can present with PNH clones as low as 0.01% on
flow cytometry and FLAER.
Primary Chondroblastic Osteosarcoma of the Breast in a
Teenager: What Is Changing?
James Yahaya, DDS1; 1Makerere University
Background: Nonepithelial tumors of the breast are ex-
tremely rare and have an incidence of less than 1%. The
most common nonepithelial breast tumor is phyllodes
tumor (PT), which accounts for 61%. Primary osteosar-
comas of the breast contribute up to only 12.5% of all
the extraosseous osteosarcomas, and among young fe-
males, they are extremely rare, especially in those without
a previous history of primary bone osteosarcoma. A case
of a 16-year-old female with primary chondroblastic os-
teosarcoma of the left breast is herein presented.
Case Description: In this report, we describe and report a
16-year-old female with neither a previous history of bone
osteosarcoma nor a family history of breast cancer who was
diagnosed with a primary chondroblastic osteosarcoma
© American Society for Clinical Pathology