Automated Reticulocyte Analysis:
New Parameters for Anemia Diagnosis and
Therapeutic Monitoring
&
Improved Precision & Laboratory Efficiency
Bruce H. Davis, M.D.
William Beaumont Hospital
Royal Oak, Michigan
Automated Hematology:
Desirable New Parameters
• CD4 and CD8 Lymphocyte Subsets
• Reticulocytes with immature reticulocyte maturation
(IRF) - alias RMI
• Neutrophil activation marker, such as quantitative
PMN CD64 expression
• reticulated platelets
• Platelet activation markers (eg. CD62 or CD41
expression)
• Hb F containing RBC enumeration (Kleihauer-Bettke)
• Immune activation profile (cytokine/chemokine Rs)
• CD5+ B Cells or light chain+ B cells (CLL, etc.))
• Stem Cell enumeration (CD34+ cells)
History of Automated Reticulocyte Counting
• 1980-90 Flow cytometric methods
–
–
–
–
Tanke: Pyronin Y
- Jacobberger: DiOC(3)
Ortho: Acridine Orange
- Others: PI, ethidium Br
Metzger, Corash: Thioflavin T
Lee (BDIS), Davis & Bigelow: Thiazole Orange
• 1990: TOA Sysmex R instruments: Auramine O
• 1992-96: Hematology instruments light scatter
– Technicon - H3: Oxazine 750
– Coulter Gen-S, STKS & MAXM: NMB
– Abbott Cell Dyn 3500: NMB
• 1996: Hematology instruments - fluorescence
– Abbott Cell Dyn 4000: thiazole-like dye
– Coulter Gen-S: CPO dye
Automated Reticulocyte Counting:
Methods Available - 1997
Fluoresence Methods
Light Scatter Methods
• Thiazole Orange (BD)
by Flow Cytometry
• CPO dye (Coulter) by
flow cytometry
• TOA Sysmex R series
and SE-Avante by
Auramine O
• Abbott Cell-Dyn 4000
by CD4K530
• ABX Vega by thiazole
orange
• Bayer Technicon H3,
Advia by oxazine dye
• Coulter STKS/MAXM
and Gen-S with new
methylene blue (NMB)
• Abbott Cell-Dyn 3500
with NMB
Advantages of Automated
Reticulocyte Analysis
•Amenable to labor efficiencies or robotics
–faster analysis per sample
–allows for batch analysis or random access
•Improved precision of retic counting
–superior to visual microscopic counts
–greater objectivity
•New parameters of erythropoiesis
–Immature Reticulocyte Fraction (IRF)
–Reticulocyte hemoglobin content
New Parameters with Automated
Reticulocyte Analysis
•Immature Reticulocyte Fraction (IRF)
•Reflects rate of erythropoietic activity
•Available on many instruments, methods
•Formerly termed reticulocyte maturity index (RMI)
•Replaces need for “corrected” reticulocyte count
•Reticulocyte MCHC (hypochromic Retics)
•Detects early functional iron deficiency in Epo Studies by Brugnaro, d’Onofrio
•Available only on Technicon H3 to date
Reticulocyte Enumeration with
Immature Reticulocyte Fraction (IRF)
• IRF measured as fraction (0.00 - 1.00 range)
• Sysmex R: IRF = HFR + MFR (Ref Range: 0.05-0.20)
• Thiazole Orange: Cursor at 95% interval (Ref Range: 0.2-0.5)
• Report with reticulocyte % and absolute count
• Graphic display of retic count vs. IRF
• Superimpose refernce ranges for anemia classification
• Plotting sequential samples shows erythroid response
• Report results with other CBC parameters
• Flags for increased reticulocytosis and
hypoproliferative response
• Automated, random access, discrete testing
4
Immature Reticulocyte Fraction (IRF)
Thiazole Orange by Flow Cytometry
Reticulocytes
RBCs
IRF
– exclude nucleated
cells (nRBCs,
PMNS, lymphs)
– exclude platelets
– define IRF region
– define retics
10
2
FSC-Height -->
10
3
10
• IRF =
#HFR/#Retics
• Data Analysis
Nucleated
Cells
10
1
Platelets
10
1
10
2
10
Thiazole Orange -->
3
10
4
Evidence for Pathophysiologic Relevance
of Immature Reticulocyte Fraction (IRF)
• Erythropoietin therapeutic effect: IRF 1- 3 days
– CD71 vs TO studies
– BJH study, Major et al.
• Animal models
– in vivo biotinylation studies
– CD71 vs. TO studies
• BMT recovery
– IRF earliest parameter of engraftment
– various methods with demonstrated efficacy
1
Erythroid
Maturation
0.9
0.8
Normal
Erythopoiesis
0.7
0.6
Nucleated
0.5
Glycophorin
0.4
CD71 (TfR)
0.3
RNA
Hemoglobin
0.2
Erythrocyte
Reticulocyte, late
Reticulocyte, IRF
Normoblast, late
Erythroblast
0
Normoblast,
early
0.1
• Maturational
continuum
• EPO effect
• blood retic
populations
– IRF retics
(CD71+)
– late retics
– stress retics
10 4
10 4
Reticulocyte Maturation:
in vivo biotinylation (K. Ault)
10 3
10 2
10 1
10 2
BIOTIN LEVEL -->
10 3
6 hours
10 1
BIOTIN LEVEL -->
pre-biotinylation
10
1
10
2
10
3
10
1
10
4
10
2
3
10
4
10
4
10 4
10 4
THIAZOLE ORANGE -->
10 2
BIOTIN LEVEL -->
10 1
10 1
10 2
10 3
72 hours
10 3
24 hours
BIOTIN LEVEL -->
10
THIAZOLE ORANGE -->
10
1
10
2
10
3
THIAZOLE ORANGE -->
10
4
10
1
10
2
10
3
THIAZOLE ORANGE -->
Bone Marrow Regeneration Response:
Consistent Pattern
0.5
Late Post Transplant Period
Immature Reticulocyte Fraction
0.4
0.3
Early Period of Bone
Marrow Recovery
0.2
Prior to Transplant
0.1
Ablation Period
0.0
0
20
40
60
80
Absolute Reticulocyte Count (10^9/L)
10 0
12 0
Erythroid Parameters with
Erythropoietic Response
Changes in Blood with Stimulated Erythropoiesis
Hemoglobin
Retic Count
IRF
14
0.6
12
0.5
10
0.4
8
0.3
6
0.2
4
2
0.1
0
0
0
1
2
3
5
7
10
TIME (days)
14
21
30
45
Evaluation of Erythropoiesis:
Bivariate IRF and Retic Count Display
0.8
Immature Reticulocyte Fraction (IRF)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
10 0
20 0
30 0
40 0
50 0
60 0
70 0
Absolute Reticulocyte Count (10^9/L)
80 0
90 0
10 00
Immature Reticulocyte Fraction (IRF):
Clinical Utility in Medical Practice
• Monitor BM or Stem Cell
Regeneration post-BMT or
ChemoRx
• Monitor Renal Transplant
Engraftment (Epo production)
• Monitor Neonatal Transfusion
Needs
• Monitor Anemia Therapy
• Monitor EPO Therapy: Renal
Failure, AIDS, Infants, MDS
• Monitor Bone Marrow Toxic
Insults from drugs (eg. AZT)
• Prognostic in Anemia of AIDS
and Prematurity
• Timing for Stem Cell Harvests
following Growth Factor or
Cytotoxic Drug Therapy
• Detection of Aplastic Crisis in
Hemolytic Anemias
• Diagnosis and monitoring of
aplastic anemia
• Evaluate Normochromic
Anemias of Various Etiologies
• Detection of Occult or
Compensated Hemorrhage or
Hemolysis
• Classification of Anemias
Patterns of IRF and Retic counts in Anemia
•
•
•
•
•
•
•
•
•
•
Clinical Condition
Retic Ct
IRF
Aplastic anemia/crisis
hypoplastic anemia
BM regeneration
Chronic disease
Iron deficiency
Thalassemia
Folate/B12 deficiency
Myelodysplasia
Hemolytic anemia
Blood loss/anoxia
Low
Low
Low
Low/WNL
Low/WNL
WNL/high
Low/WNL
Any level
High
WNL/high
Low
Low
High/WNL
WNL
High
WNL/high
High
WNL/high
High
High
Intermethod Correlation Studies
• Single site studies: multiple published
– improved precision - CVs <15%
– intermethod bias, but “clinically insignificant”
• Davis et al: AJCP 102:468, 1994
– 8 sites, 11 instruments, 310 blood samples
– IRF and Retic counts compared
• College of American Pathologists’
Reticulocyte RT Survey 1994-96
– >2,600 participants
– surrogate blood material
– Retic % only reported
Retic Counts: Inter-method Correlation
Absolute Reticulocyte Count
Absolute Reticulocyte Count
400
400
Sysmex R-3000
350
300
250
200
150
100
50
450
350
300
250
200
150
100
50
0
400
350
300
250
200
150
100
50
0
0
50 100 150 200 250 300 350 400 450
0
0
50 100 150 200 250 300 350 400 450
Abbott Cell Dyn 4000
0
Sysmex R-3000
Reticulocyte Percentage
Reticulocyte Percentage
10
5
0
0
5
10
15
Abbott Cell Dyn 4000
20
20
Becton Dickinson FACScan
Becton Dickinson FACScan
20
15
50 100 150 200 250 300 350 400 450
Abbott Cell Dyn 4000
Reticulocyte Percentage
20
Sysmex R-3000
Absolute Reticulocyte Count
BD FACScan & Coulter STKS
450
BD FACScan &Coulter STKS
450
15
10
5
0
15
10
5
0
0
5
10
Sysm ex R-3000
15
20
0
5 Abbott Cell
10 Dyn 400015
20
IRF: Inter-method Correlation
Immature Reticulocyte Fraction (IRF)
1
0.9
0.8
Sysmex R-3000
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Abbott Cell Dyn 4000
0.8
0.9
1
Reticulocyte Proficiency Testing
College of American Pathologists
1994-96 Program - surrogate blood material
Methodologies Approved for Testing
New Methylene Blue visual microscopy
Sysmex R series (auramine O)
Flow Cytometry - Thiazole Orange
Flow Cytometry - Other dyes
Coulter STKS/MAXM - NMB
Miles Technicon H3 - Oxazine
Flow Cytometry
Sysmex R
Miller Disc
Technicon H-3
Manual
STKS/MAXM
NUMBER OF PARTICIPANTS
CAP Survey 1995 RT-C: Distribution of Methods
12 50
10 00
75 0
50 0
25 0
0
1994-95 CAP RETICULOCYTE SURVEY
9
COULTE
R X-L RE
TI C%
COULTE
R EP
I CSRETIC %
8
FAC
SCANRET
IC%
M ILLERDI S
C RE
TI C%
7
STK
S/ MAXM RET
IC%
SYS
M EXR R
ETI C%
6
TEC
HNI C
O NH- 3RETIC %
5
4
3
2
1
12
95RT-06
95RT-05
95RT-04
95RT-03
10
8
95RT-02
95RT-01
6
94RT-06
94RT-05
4
94RT-04
94RT-03
94RT-02
94RT-01
2
0
0
RETICULOCYTE PERCENT
MA
NUALRET
IC%
Coulter X-L
Coulter Epics
FACScan
Manual
Miller Disc
STKS/MAXM
Sysmex R
Technicon H-3
1994-95 CAP RETICULOCYTE SURVEY
Coulter X-L
Coulter Epics
FACScan
Manual
Miller Disc
STKS/MAXM
Sysmex R
Technicon H-3
COULTE
R EP
I CSC. V.
40
COULTE
R X-L C.V.
FAC
SCANC. V
.
MA
NUALC. V
.
35
M ILLERDI S
C C.V.
COEFFICIENT OF VARIATION
STK
S/ MAXM C. V
.
30
SYS
M EXR C
. V.
TEC
HNI C
O NH- 3C. V.
25
20
15
10
5
0
0
1
2
3
4
5
6
RETICULOCYTE PERCENT
7
8
9
CAP RT Survey Experience: No Bias with TO methods
secondary to FCM instrument
11.0
R
E
T
I
C
%
A
11.0
10.0
10.0
9.0
9.0
8.0
8.0
7.0
5.0
R
E
T
I
C
4.0
%
6.0
7.0
6.0
5.0
4.0
3.0
3.0
2.0
2.0
R-1
1 R-2
2 R-3
3 R-4
4 R-6
5 R-5
6
Specimen
SPECIMEN
Number
B
A
6
6B
KC
KD
Flow Cytometer
INSTRUMENT
PE
PF
Instrument
G
P
Known or Potential Interferents
•Cellular Elements
–Platelet clumps or giant platelets
–nucleated cells or fragments
•RBC Inclusions
–Howell-Jolly bodies
–Heinz or Pappenheimer bodies
–parasites (malaris, babesia)
•Miscellaneous causes
–Autoflourescence (drugs, porphyria)
–RBC aggregation (paraproteins, cold agglutinins)
–coincidence (eg. platelet and RBC)
–abnormal RBCs, hemolysis
Controls for Clinical Practice
•Commericial Preparations
–R&D Systems, Minneapolis, Mn
–Streck Lab, Omaha, Ne
–Instrument manufacturers
•Refrigerated blood samples
–short term QC by carry-over comparison
–least expensive
–will not detect long-term drift
•Veterinary blood samples
–rabbit
–porcine
Reasons for NOT utilizing
automated reticulocyte counting
•
•
•
•
•
Volume does not exceed 3-5/day
Physicians expect “stat” results
Waiting for the “next generation” instrument
“We’ve always done it this way”
Technologists like doing manual counts