Michael Keeney
London, Canada
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Presence of NRBC’s predict for poor outcome
in many disease states:
Hemoglobinopathies
Critical care
MPD’S, Myelofibrosis
Indication of fetal distress in newborn
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Detection of NRBCs was monitored daily in 383
medical intensive care patients.
The incidence of NRBCs in medical intensive care
patients was 17.5% (67/383).
Mortality of NRBC-positive patients was 50.7% (34/67)
the mortality of NRBC-negative patients was 9.8%
(31/316) (p < 0.001)
Mortality increased with increasing NRBC
concentration. 78.6% of patients with NRBCs of more
than 200/μl died.
Nucleated red blood cells in the blood of medical intensive care patients
indicate increased mortality risk: a prospective cohort study
Axel Stachon
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Prematurity
Increased erythropoiesis from chronic hypoxia,
anaemia
Maternal diabetes
Acute stress mediated release from the marrow
stores
postnatal hypoxia
Extreme increases may occasionally be
idiopathic.
Nucleated red blood cells in the fetus and
Newborn. M C Hermansen
pH
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7.40–7.49
7.30–7.39
7.20–7.29
7.10–7.19
7.00–7.09
<7.00
Results are mean (1 SD).
nRBCs/mm
630 (870)
840 (1630)
1240 (2090)
2330 (5130)
3040 (2060)
7780 (13 810)
Nucleated red blood cells in the fetus and
Newborn. M C Hermansen
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Manual count on Wrights/Giemsa stain.
Two technologist count 200 cell differential and
enumerate nucleated RBC as a non-totalling
cell (excluded from differential)
Express as number per 100 WBC e.g.
200 WBC, 4 NRBC
2 NRBC/100 WBC
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Observed WBC × (100 / (100 + NRBC*))
*NRBC ; The count as the number of NRBC
pert 100 WBC.
WBC count correction is not necessary when
less than 5 (10) NRBC/100 WBC are present
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Depends on artefactual differences in distribution
due to the process of spreading as well as random
distribution
Mean coefficient of variation for manual NRBC40%
In a study addressing the performance of the
manual differential, qualified medical
technologists did not identify NRBC in 19% of
samples
Labour intensive, time-consuming, subjective and
imprecise due to small number of NRBC counted
Tsuji, Sakata, Hamaguchi,Wang and
HouwenCytometry 37:291-301 1999
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reagent lyses the RBC and enucleates, shrinks
and slightly stains the nuclei of NRBC.
WBC become permeable maintain shape, intracytoplasmic organelles and nuclei stain
intensely with a polymethine-based dye
staining intensities between NRBC nuclei and
WBCs coupled with differing volumes detected
by a laser using forward-scattered light and
fluorescence intensities.
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argon laser in combination with two
fluorescence channels to measure NRBC.
reagent lyses the membrane of NRBC and
mature erythrocytes,while WBC are left intact.
propidium iodide binds to the nuclear DNA of
the NRBCand WBC.
combination of cell size and fluorescence
intensity permits the separation of NRBC from
the leucocyte population.
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NRBC enumeration is achieved by the
identification of particles in the NRBC
signature position in the differential DataPlot.
This information is generated from VCS analysis of
the cells
 In addition the far left region the WBC histogram is
examined for the presence of cells.
 If cells are present in both locations then the NRBC
count is derived from the WBC histogram, and
NRBCs are reported.
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No NRBC’s in WBC histogram
NRBC signature
position
No NRBC count and no WBC
correction
NRBC location on WBC
histogram (Service Mode)
NRBC signature
position
NRBC enumerated
WBC correction
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Red Blood Cell Diagnostic Testing Using Flow
Cytometry; Draft Guideline—Second Edition
H52-A2 (Draft 1)
Vol. 0 No. 0
Replaces H52-A
Vol. 21 No. 26
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25 uL whole blood
10 uL CD45 FITC
Incubate 15 minutes at room temperature,
protected from the light
Add 250 uL Solution A (hypotonic acid with
100 mg/L PI) incubate exactly 30 seconds
Add 500 uL Solution B (hypertonic alkaline)
Incubate 5 minutes
Keep samples on ice prior to analysis
Tsuji, Sakata, Hamaguchi,Wang and Houwen
Cytometry 37:291-301 1999
Region A =PI+ CD45- (NRBC)
Region B = PI-/+ CD45+ (WBC)
NRBC PER 100 WBC = A/B*100
Flow 3/100 wbc
Manual 4/100
Flow 7/100 wbc
Manual 4/100
Flow 300/100 wbc
Manual 277/100
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Comparison of The DxH 800 to reference flow
Cytometry methods for platelets, wbc and
NRBC’s
B. D. HEDLEY, M. KEENEY, I. CHIN-YEE,
W. BROWN Blackwell Publishing Ltd, Int.
Jnl. Lab. Hem. 2011, 33, 45–56 45
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NRBC counts for all samples collected (n =
201) on the DxH 800 vs. flow cytometry (FCM).
R2 value for DxH 800 vs. FCM was 0.910.
Receiver Operator Curves for DxH 800 vs.
FCM:
Sensitivity for DxH 800 vs. FCM was 73%
Specificity for DxH 800 vs. FCM was 100%.
LH 780
FP rate
3%
FN rate
10%
Sensitivity 56%
Specificity 96%
Bias
0.29
DxH 800
0%
6%
73%
100%
0.60
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Absence of specific marker for NRBC
Absence of CD45 on subset of B-cell precursor
leukemia, childhood T-cell leukemia and in
subsets of myeloma
Lyse resistant RBCs (sickle cells, burr cells)
Clumped platelets
Sensitivity not validated below 1 NRBC/100
WBC
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Flow cytometry provides a novel and
reliable methodology to evaluate the
accuracy of NRBC, a key
hematological parameter
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Bruce H. Davis, MD
Ross Brown, PhD
Andrea J. Illingworth, MS, H(ASCP), CQYM
May-Jean King, PhD
Beth H. Shaz, MD
Belinda Kumpel, PhD
Emily Riehm Meier, MD, FAAP
S. Gerald Sandler, MD, FACP, FCAP
D. Robert Sutherland, MSc
Brent Wood, MD PhD
CLSI Staff Liaison- Jennifer K. Adams, MT(ASCP),
MSHA
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PROCEDURE
1. Add 25 µL well-mixed whole blood to 10 µL CD45 FITC. Vortex to mix.
2. Incubate 15 minutes at room temperature, protected from the light.
3. Add 250 µL of Solution A (acid lysing reagent). Vortex well. Let stand
for 30 seconds at room temperature. Lyses RBC, Stains NRBC and
degenerating WBC) Timing critical
4. Add 500 µL of Solution B (alkaline reagent). Vortex to mix.
5. Incubate for 5 minutes at room temperature, protected from the light.
6. Following incubation, store samples in melting ice bath, protected from
the light.
Stable for at least 30 mins
NOTE: It is imperative that the incubation times described are strictly
adhered to. In particular, lysing for longer than 30 seconds will
adversely effect the light scatter of the white blood cell population.