automated cell counters - WBC

CBC

white blood cell count

red blood cell count

hemoglobin

hematocrit

red cell indices

WBC differential

platelet count and size - 150-450,000/uL

cells differ in quantity, size, and refractory qualities

WBC 3.6-10.6 x103/L
 WBCs contain cellular inclusions
 RBC 4.0-6.0 x106/L
 Hemoglobin is contained in the red cells
 Platelets 150-450 x103/L
 Hemoglobin g/dl

Current technology

Electrical impedance

radiofrequency

optical scatter (flow cytometry)

WBC differential

• 3 part - lymphocytes, granulocytes, mononuclear cells

• 5 part - neutrophils, lymphocytes, monocytes, eosinophils, and basophils

• 6 part, 5 part + immature granulocytes

impedance counting

Impedance counting (Coulter Principle)
 Cells are suspended in an isotonic diluent
 An electric current is introduced
 Dilution is passed through a small aperture
 Cells are nonconductive and cause an electronic
pulse interruption as they pass through
 Number of pulses created equals the number of
cells passing through the electrode system
 The amplitude of the pulse is proportional to the
size of the cell (MCV)

electrical impedance

first introduced by Wallace Coulter

blood cells are poor conductor of electricity

2 chambers filled with a conductive buffered electrolyte solution

separated by a small aperture

DC current between two electrodes

counting chambers

most common chambers using impedance

• RBC/platelet chamber

• WBC chamber/hemoglobin

differential chamber

reticulocyte channel

RBC/platelet chamber

2-20 femtoliters (fL) - platelet

36-330 fL - RBC

typical WBC impedance histogram

bimodal RBC histogram peaks, reticulocytosis, RDW

bimodal peak can be seen in situations such as -

• reticulocytosis

• colg agglutinins (cold antibodies IgM)

• post-transfusion

• post-treatment of IDA

laser based flow cytometry

due to patent of impedance method competitors developed laser light scatter technology

• initially very limited test offerings

eventually surpassed impedance counting

flow cytometry method

measures cells or particles as they move through a sensing area

• sensing may be optical or electronic

optical sensing is done with an intense light source, usually a laser

• Tungsten-halogen lamp or a helium neon laser

• emitting light with a narrow wavelength spectrum

instrument measures light scatter and or fluorescent signals generated as cells pass through a light beam

flow cytometry components

Five main components
 Flow Cell Fluidics (cell transport system)
 Liquid sheath fluid, carries and aligns the cells so that they
pass single file through the light beam for sensing
 Laser Optical System
 Most common mercury, xenon
 High-power water-cooled lasers (argon, krypton, dye laser);
 Low-power air-cooled lasers (argon (488nm), red-HeNe
(633nm), green-HeNe, HeCd (UV));
 Diode lasers (blue, green, red, violet) resulting in light signals
 Photodetectors for signal detection
 Converts light energy into electrical energy
 Generates FSC and SSC signals from light into electrical
signals
 An Amplification System
 Microprocessor /Computer data management system

process flow cytometry

Fluidics are regulated by pressure
 Cells suspended in fluid are transported to a
special flow chamber
 Chamber has small orifice at tip
 Sample is injected into a stream of fast-
moving cell-free liquid (sheath fluid)
 Hydrodynamically-focused stream of fluid
 Sheath fluid and cell suspension move at
different rates and do not intermingle
 Laminar Flow generates a single file alignment of
cells

A single wavelength of light is directed onto a
hydrodynamically-focused stream of fluid
 A number of detectors are aimed at the point
where the stream passes through the light
beam:
 One in line with the light beam (Forward Scatter or
FSC)
 And several perpendicular to it (Side Scatter
(SSC)
 Possibly one or more fluorescent detectors

As the cell enters the laser beam in flow cytometry

Light is scattered through 360 degrees
 This combination of forward scattered and Side Scattered
light picked up by the detectors
 By analyzing fluctuations in brightness at each detector, it is
possible to derive various types of information about the Cell
size, nuclear complexity, and cytoplasmic granulation of
each individual cell
 FSC correlates with cell volume or size
 0-10 degrees
 SCC correlates with the complexity of the cell
 90 degrees
 Shape of the nucleus, amount and type of cytoplasmic gra

Flow Cytometry Histogram

FSC and SCC histogram

analysis technologies

flow cytometry - white blood cell populations

impedance - red blood cells/platelets

spectrophotometry - hemoglobin, uses light emitted diode (LED)

Hgb concentration is proportional to the absorbance of light

analysis process

aspiration

separation

dilution

staging

measurement

data/analysis

analysis process

aspiration

• closed mode - min volume 1.5 mL, aspirated 240 uL

• open mode - aspirated 180 uL

separation

• takes place in the shear valve

• 20 uL for the WBC dilution

• 1.67 uL for the RBC dilution

• 12 uL for the Hgb dilution

dilution

• components - syringes, shear valve, mixing chambers

• reagents added - diluent/sheath, cyanide-free HGB lyse. WBC lyse (nuclear determination)

diluent/sheath

acts as the diluting substance for RBC/PLT and for HGb

it is used to maintain the stable cell volume of the red cells and platelets during counting and sizing

provides acceptable background counts

rinsing agent for the fluidic system

serves as a sheath fluid during the laminar flow process in the optical flow cell

hemoglobin lyse

Hemoglobin is often determined by a colorimetric method
– Imidazole: Non-cyanide reagent with color change and
read at 540nm
– Abbott CELL-DYN Sapphire
– Sodium Lauryl Sulfate:Non-cyanide reagent with color
change and read spectrophotometrically
– Sysmex XT and XE
– Other Lysing agent: converts free hemoglobin to
cyanmethemoglobin and read spectrophotometrically at
540nm
• Advia 120
• Some Beckman Coulters
• Hemoglobin Lyse: Rapidly lyses RBCs
• Converts hemoglobin to stable chromogen complex
• Measured spectrophotometrically

WBC lyse

WBC Lyse reagent - strips the WBC cytoplasm, leaving
the nuclear membrane intact so that the white cell nuclei can be
enumerated
 It maintains the light scattering properties of
the WBCs for the duration of the
measurement period
 It provides sufficient wetting action to prevent
accumulation of air bubbles in the Optical
Flow Cell system

staging and measurement - analysis

Staging
 Peristaltic Pump
 WBC Mixing Chamber
 RBC/PLT Mixing Chamber
 Optical Flow Cell
 Measurement
 Optical Flow Cell
 HGB Flow Cell
 Sample Injection Syringe

what’s measured and what’s calculated

Measured parameters:
 WBC
 RBC
 HGB
 MCV
 Platelets
 Reticulocytes
 All other parameters are calculated:
 Hct = (RBC x MCV) ÷ 10
 MCH = (Hgb/RBC) x 10
 MCHC = (Hgb/Hct) x 100
 RDW= mean distribution width

data anylisis and results

scatterplots, histogram, calculations

data accumulated can be analyzed using software

gating

the data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram

two dimensional dot plots or even in three dimensions

the regions on these plots can be sequentially separated, ased on FSC, SSC and fluorescence intensity

creating a series of subset extractions, termed “gates”

linearity limits

defined by instrument manufacturer

range of test results where answers are considered accurate

when results are outside the linearity, alternate methods must be used

• dilution

• report as “greater than x”

nucleated RBCS

> 10 nRBCs will falsely elevate Total WBC count
 Because they are counted in WBC chamber
 > 10 needs to be a corrected count
 Corrected WBC = WBC x 100 / #nRBC + 100
 Example: if WBC=5,000 and 10 nRBC were counted,
what is the Corrected WBC?
 Corr. WBC = 5,000 x 100/(10 + 100) = 4545.5
 Automated instruments have this ability

nucleated RBC overlap WBC

sysmex nRBC counts explained

total WBC count

the total WBC count is determined in whole blood in which red cells have been lysed

fully automated multichannel instruments perform WBC counting by either

• impedance

• light scattering

• or both

total WBC count

3-part differential usually counts
 Granulocytes or large cells
 Lymphocytes or small cells
 Monocytes(mononuclear cells) or (middle cells)
 5-part differential classify cells to
 Neutrophils
 Eosinophils
 Basophils
 Lymphocytes
 Monocytes
6-Part Diff: 5-Part + Immature Granulocytes

sysmex histogram

cold agglutinant can affect the patient results so that even tho the QC is within range, not all patient results will be correct

causes of erroneous results

Cold agglutinins
decreased RBC , MCV increase
 Lipemic or hemolyzed sample
 increased Hgb, MCH, MCHC erroneous
 High WBC
 RBC increased, HCT increased
 Sample contaminated with IV fluid
 MCV increased, cell counts increased
 Incomplete aspiration of sample
 Clotted sample
 Platelet clumps may falsely increase the WBC
count, while falsely decreasing the PLT count

cold agglutinins

most common cause of MCHC > 37.0

causes a decreased RBC and increased MCV

what to do next?

• warm the specimen in a 37C heat block

• eliminated cold agglutination and resolves CBC interferences

could be HS

• look for spherocytes too

lipemia

increases Hgb, MCH, MCHC

two methods to resolve

• plasma replacement

  • measure and discard plasma

  • replace with equal part saline

  • mix and repeat CBC

• RBC washing

  • wash aliquot of RBCs

  • perform a count on washed aliquot for indices

  • calculate Hgb from indices

IV fluid contamination

sample contaminated with IV fluid

MCV increased, cell counts decreased, Hgb decreased

look for delta check error

redraw sample

interferences

Cold Agglutinins
 RBC too low, MCV too high
 Agglutination Flag
 Warming sample at 37o usually resolves
 Lipemia
 Hemoglobin, MCH, MCHC erroneous
 Plasma Replacement with saline
 Measure and discard plasma, replace with saline
 High WBC
 RBC too high, HCT too high
 Perform a dilution
 IV Contamination
 MCV too high, RBC, Hb, HcT may be decreased
 Look for delta error
 Redraw Sample

instrument flags

Instrument flags are generated when variations
from normal histograms and cytograms are
present.
 Must be reviewed visually

 Flags
 Blasts, IG, band
 RBC morphology
 Platelet clumps
 Linearity limits (WBC/RBC too Hi or too Low)

maintaining accuracy

Maintaining Accuracy
 Controls
 3 levels (alternate between high & low abnormal
control)
 2 levels, every 8 hours
 Background Count/Checks
 Count using reagents only
 High count indicates a problem with analyzer or
reagent
 Calibration
 Daily maintenance
 Weekly maintenance

Maintaining Accuracy

preventive maintenance

regular helps to keep QC in range

• shear valve

• flow cells

• aperture flush

• dilution chambers rinse

• zap

flagging

parameter marks

flagging WBC suspect and abnormal

Flagging RBC/RET abnormal and suspect flags

manual WBC and platelet count

hemocytometer

WBC and platelet count

Prepare a 1:100 dilution of the sample in a
2% acetic acid or 1% (0.1N) HCl
 The diluting fluid will hemolyze RBC's so that
WBC's and platelets are not obscured
 Allow 10 minutes for the RBCs to lyse
 Load both chambers of the hemocytometer
 Allow 3 minutes for the cells to settle
 Perform the counts

WBC calculation

Counts from each side of the chamber should match within
25%. Average the values for the duplicate determinations when
reporting results
 # of Cells Counted X Dilution Factor = WBC per mm3
# of Squares Counted X Depth Factor
 Example: If 45 WBC's were counted in 4 WBC squares.
 45 X 100 X 10 / 4 = 5000 mm3
 Alternate Calculation: Calculate the average
number of WBCs in 1 square and multiply by
1000

sources of error

apparatus - dirt, chipper cover slip, scratches on hemoctyomter

personal technique

inherent error

errors caused by personal technique

Not thoroughly mixing blood/inadequate
shaking
 Failure to discard first 4 drops (Unopette)
 Not loading chamber properly (overfilling,
trapped air bubbles)
 Counting cells inaccurately (skipping cells,
counting cells twice, counting on wrong
borders)
 Calculation error
 Clerical error

inherent errors

Field Errors - relates to the random
distribution of cells on the counting
chamber
 Statistical error - occurs when total
number of cells is too low to give
statistical confidence in result (this error
is reduced when larger numbers of cells
are counted)