Flow Cytometry
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London Health Sciences Centre
Flow Cytometry Unit (Westminster Campus)
Flow Cytometric Enumeration of Nucleated Red Blood Cells

CLINICAL SIGNIFICANCE

The presence of nucleated red blood cells in peripheral blood (NRBCs) is mostly physiological in newborns and very young infants, but in all other situations NRBCs indicate severely stressed or disordered erythropoiesis. NRBCs should therefore be correctly identified, even in low numbers, because their presence may indicate significant underlying disease. The presence of NRBCs in peripheral blood is an important marker to detect significant hematological or non-hematological disease.1

PRINCIPLE

Peripheral white blood cells (WBCs) are identified and subsequently separated from NRBCs by staining with a pan leukocyte marker CD45 FITC. All non-nucleated RBCs are lysed and any NRBCs present are permeabilized and stained by the lysing reagent which contains a nucleic acid dye, propidium iodide (PI). In addition, separation of RBC ghosts, debris and lyse-resistant RBCs, reticulocytes and platelets from NRBCs is possible.

SPECIMEN

1. Whole blood samples anticoagulated with either K2EDTA or K3EDTA. Samples stored at either room temperature or 4ºC are stable up to 48 hours.


QUALITY CONTROL

1. Prior to performing this test, the flow cytometer will be properly aligned and calibrated. Refer to appropriate methodology.

Note: Calibration protocol is named _ANRBC FLOWSET

MATERIALS & EQUIPMENT

1. Beckman Coulter EPICS XL-MCL Flow Cytometer


2. Thermolyne Maxi Mix II


3. Adjustable Eppendorf Pipettes (10 µL, 25 µL, 250 µL and 500 µL)


4. Disposable pipette tips


5. Disposable polypropylene test tubes

6. Melting ice bath


REAGENTS

1. CD45 FITC. Immunotech PN# 0782 - Store at 2-8ºC, protected from the light.

2. Solution A: hypotonic, acid reagent (16 mOsm/kg. H2O, pH = 3.0)

a) 2.10 g/L citric acid monohydrate (EM Science PN# CX 1727-1)
b) 0.56 g/L disodium hydrogenphosphate (Sigma Chemical Co. PN# S-0876)
c) 100 mg/L propidium iodide (Sigma Chemical Co. PN# P-4170)

Dissolve in distilled water. Store at 2-8ºC, protected from the light.

Solution B: hypertonic, alkaline reagent (420 mOsm/kg. H2O, pH = 7.5)

a) 0.95 g/L sodium dihydrogenphosphate dihydrate (Fisher Scientific Co. S-381)
b) 6.24 g/L disodium hydrogen phosphate (Sigma Chemical Co. PN# S-0876)
c) 10.2 g/L sodium chloride (Sigma Chemical Co. PN# S-9888)

Dissolve in distilled water. Store at 2-8ºC.

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.

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.

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.


ANALYSIS

The protocol required is called NRBC ENUMERATION


Gating Strategy

Figure 1 represents a properly gated example of the NRBC ENUMERATION protocol.

Histogram 1: (SS vs FS) displays light scatter properties of all events. The discriminator must be set low enough on forward scatter to ensure that NRBCs are not excluded from analysis.

Histogram 2: (CD45 vs PI) displays CD45+ events as well as PI+ events. Debris, RBC ghosts, lyse-resistant RBCs, reticulocytes and platelets will be detected in the low left hand side of the histogram. Region A is a rectilinear region which encompasses PI+CD45- (NRBCs) events. Region B is a rectilinear region which encompasses all CD45+ (WBC) events.

Histogram 3: (SS vs FS) gated on region A displays the light scatter properties of all PI+CD45- (NRBC) events. Ensure that the discriminator is set low enough on forward scatter so that the entire NRBC population is included in the analysis.

Histogram 4: (SS vs FS) gated on region B displays the light scatter properties of all CD45+ (WBC) events.


CALCULATION

The number of NRBCs/100 WBCs is calculated by dividing the number of events in region A (PI+CD45-) by the number of events in region B (CD45+) and multiplying that ratio by 100.

For example:

Region A x 100 = #NRBCs/100 WBCs
Region B

1106 x 100 = 12.7 NRBCs/100 WBCs
8692


LIMITATIONS

1. The presence of large numbers of clumped platelets will falsely elevate the WBC enumeration.

2. The presence of highly immature or malignant CD45 negative white blood cells will falsely decrease the WBC enumeration.

 

REFERENCES


1. Tomohiro Tsuji, Takashi Sakata, Yukio Hamaguchi, Fu-sheng Wang, Berend Houwen. New Rapid Flow Cytometric Method for the Enumeration of Nucleated Red Blood Cells. Cytometry 37: 291-301 (1999).

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August 12, 2008