Central Philippine University

College of Medical Laboratory Science

Laboratory Activity No. 7

Automation
Learning Outcomes:
At the end of this activity the student must be able to:
1. Perform accurately hematologic examinations using an automated analyzer.
2. Interpret correctly cell histograms and scattergrams.
3. Perform correctly QA/QC on the automated analyzer.
4. Identify properly errors encountered in automated methods and make necessary
corrective action(s)
Contents
01 Introduction
Cell Counting Principles
Cell Histogram and scattergram

02 Procedure
Materials and Equipment
Specimen
Principle
Procedure

03 QA/QC

04 Problems Encountered in Automated Methods

Spurious increase
Spurious decrease
Corrective action(s)
Introduction

Advantages of Hematologic
Automation:
1. For speed of performance
2. Elimination of visual fatigue
3. Improved precision and accuracy
Introduction

Basic Components of Most Hematology

Analyzers
1. Hydraulics
• Aspirating unit, dispensers, dilutors, mixing chambers, aperture baths
and/or flow cells and hemoglobinometer
2. Pneumatics
• Vacuums and pressures for operating valves and moving the sample
through the system
3. Electrical Systems
• Electronic analyzers and computing circuitry for processing data
Introduction

Basic Cell Counting

Principles
1. Electrical Impedance
• Also low-voltage direct current (DC)
resistance
• Most common methodology used
2. Optical Scatter or Detection
• Flow Cytometry
Introduction

Electrical Impedance
• Developed by Coulter in 1950
• Cells pass through an aperture with an
electrical current flowing through
simultaneously
• Cells do not conduct current but rather
they change electrical resistance, which is
then counted as voltage pulses.
a. Number of pulse is proportional to number of
cells
b. Amplitude of pulse generated is proportional
to size of the cell.
c. The bigger the cell, the bigger the impedance
Introduction

In a typical electrical impedance instrument,

aspirated blood is divided into two separate
volumes for measurements

One volume is mixed with diluent

One volume is mixed with diluent
and is delivered to the cell bath,
where erythrocyte and platelet and a cytochemical-lytic reagent
that lyses only the red blood cells.*
counts are performed. *A commercially available
reagent, which both lyses
RBCs and converts
hemoglobin to
Particles 2-20 fL are counted as Particles >35 fL are recorded as cyanmethemoglobin, can be
platelets, >36 fL are counted as leukocytes. used to determine
erythrocytes. hemoglobin and WBCs in
Hb is measured.
one dilution.
Introduction

Optical Scatter
• Light scattering optical method
• Principle
a. A diluted blood specimen passes in a
steady steam through a quartz flow
cell past a focused light source
b. As each cell passes through the
sensing zone of the flow cell, it
scatters the focused light.
c. Scattered light is detected by a
photodetector and converted to an
electrical pulse
d. No. of pulse generated = No. of cells
passing through the sensing zone in a
specific period
Introduction

Optical Scatter
• Forward angle light scatter measures
cell size.
• Side angle light scatter provides
information on cell granularity and
lobularity.
• RBC = no nucleus = no light scatter
• WBC = with nucleus = light scatter
• Number of pulses generated is
proportional to the number of cells
present.
Light Sources in Optical Scatter
1. Tungsten – halogen lamp
2. Helium-neon laser (monochromatic light)

• Laser light is the most common light source used in flow cytometers
because of the properties of intensity, stability and monochromatism.
• Laser = "light amplification by stimulated emission of radiation."
Introduction

Flow Cytometry
1. Cells in a suspension of buffered solution are labelled with one to
several fluorescent compounds*.
2. Cells are run under high pressure and in a single, narrow stream
through a laser, causing excitation of the fluorescent compound(s)
resulting in the emission of light energy.
3. This energy is detected by a photomultiplier tube and is
subsequently converted into computerized data, which upon
analysis provides information regarding number, size, and cellular
components of a flow cytometer.
*Example of fluorescent dyes used: allophycocyanin (APC), acridine orange, chromomycin A3,
cyanine dye, fluorescein isothiocyanate, peridinin chlorophyll protein (PerCP), etc.
Introduction

Major Components of FC
1. Fluidics
• flow chamber for single cell separation, sheath fluid, and hydrodynamic
focusing
2. Optics
• excitation light source includes lasers (argon, krypton, helium-neon, helium-
cadmium, diode) or lamps (mercury, xenon-mercury). Light is separated by
dichroic mirror and filters.
3. Electronics
• photomultiplier tube detects light energy, then converts this to voltage pulses;
computers translate pulses into data files.
 Instruments
Beckman Coulter UniCel Siemens ADVIA
Parameter DxH 800 Sysmex XN Series Abbott CELL-DYN Sapphire 2120i
WBC Impedance volume and con- Fluorescent staining; forward Light scatter (primary count), Light scatter and
ductivity and five-angle light light scatter and side fluores- impedance (secondary count) absorption
scatter measurement cent light detection
RBC Impedance Impedance Impedance Low-angle and high-
angle laser light scatter
HGB Modified cyanmethemoglobin Sodium lauryl sulfate-HGB Modified cyanmethemoglobin Modified cyanmethemo-
(525 nm) (555 nm) (540 nm) globin (546 nm)
HCT (RBC ! MCV)/10 Cumulative RBC pulse height (RBC ! MCV)/10 (RBC ! MCV)/10
Methods for detection

Hemogram,
MCV Mean of RBC volume (HCT/RBC) ! 10 Mean of RBC volume distribution Mean of RBC volume
distribution histogram histogram distribution histogram

Reticulocyte, MCH
MCHC
(HGB/RBC) ! 10
(HGB/HCT) ! 100
(HGB/RBC) ! 10
(HGB/HCT) ! 100
(HGB/RBC) ! 10
(HGB/HCT) ! 100
(HGB/RBC) ! 10
(HGB/HCT) ! 100
Nucleated RBC, Platelet count Impedance volume and con- Impedance; light scatter; fluores- Dual angle light scatter analysis; Low-angle and high-

and WBC ductivity and five-angle light

scatter measurement
cent staining, forward light
scatter, and side fluorescent
impedance count for verification;
optional CD61 monoclonal
angle light scatter;
refractive index
Differential light detection antibody count

Counts on Four RDW RDW as CV (%) of RBC histo-

gram or RDW-SD (fL)
RDW-SD (fL) or RDW-CV (%) Relative value, equivalent to CV CV (%) of RBC histogram

Major Reticulocyte
count
Supravital staining;
impedance volume and
Fluorescent staining; forward light Fluorescent staining; low-angle
scatter and side fluorescent scatter, and fluorescent light
Supravital staining
(oxazine 750); low-
Hematology conductivity and light scatter light detection detection angle and high-angle

Instruments measurement light scatter and

absorbance
NRBC* Impedance volume and con- Fluorescent staining; forward light Red fluorescent dye staining; Multi-angle light scatter
ductivity and five-angle light scatter and side fluorescent forward light scatter and fluores- measurements in the
scatter measurement light detection cent light detection two WBC differential
channels
WBC differential Impedance volume and con- Fluorescent staining; forward and Multi-angle polarized scatter sepa- Peroxidase staining, light
ductivity and five-angle light side light scatter, and side ration (MAPSS) and three-color scatter and absorp-
scatter measurement fluorescent light detection fluorescence detection tion; for basos, differ-
ential lysis, low-angle
and high-angle laser
light scatter

CV, Coefficient of variation; DC, direct current; HCT, hematocrit; HGB, hemoglobin; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell
Introduction

BLOOD CELL
HISTOGRAMS
• Utilizes impedance technology y-axis =
• Representation of cell number no. of
versus one measured property, cells
usually cell size.
• Volume (cumm or fL) is plotted
against the relative frequency
for platelets, WBCs and RBCs x-axis = cell size
Introduction

WBC
Histogram
• 45 – 450 fL is the
reference size
range to WBCs
• Normal WBC First Peak (35-90 fL) = small mononuclear population of cells
Histograms have (lymphocytes)
Second Peak (90 – 160 fL) = minor population of large
three (3) distribution mononuclear cells (monocytes, reactive lymphocytes, immature
peaks WBCs)
Third Peak (160 – 450 fL) = normal mature types of granulocytes
Introduction

Abnormal WBC Histogram

• The “R” stands for the region:
• R1: Population before 35 fL may indicate nucleated RBCs (nRBCs), giant or
clumped platelets.
• R2: Peak overlap at 90 fL may indicate reactive lymphocytes or blast cells.
• R3: Peak overlap at 160 fL may indicate an increase in bands, immature
neutrophils, eosinophils, or basophils.
• R4: Population after 450 fL may indicate a high granulocyte count.
• RM – is the error code for multiple region overlap
• Other flags:
• H – when a parameter value is higher than the set normal limit
• L – when a parameter value is lower than the set normal limit
Introduction

RBC Histogram
• 36 – 360 fL or >36 fL is the
size range for RBCs
• Will show a single peak at
between 70 and 110 fL

a. 24 – 36 fL: rejected (RBC histogram can measure cells as small as 24 fL)

b. Shift to the right: macrocytic
c. Shift to the left: microcytic
d. Bimodal: cold agglutinin disease, hemolytic anemia with schistocytes present, or anemias with
different cell size; when a patient received a blood transfusion; dimorphic eythrocyte population
e. Increased curved width: anisocytosis (correlate with an increase RDW)
Introduction

Platelet Histogram
• 2-20 fL is the reference
size range for platelets

a. Lower region interference (<2 fL): electrical

interference
b. Upper region interference (>20 fL): microcytic
RBCs, schistocytes, giant or clumped platelets
Introduction

SCATTERGRAM
• Also Cytogram or Scatterplot
• A 2-dimensional representation of
a two or more cell properties or y-axis =
characteristics plotted against size
each other
• Scatterplots for WBCs are displayed
on a monitor and are color coded for
different subpopulations
• Methodologies include
radiofrequency, fluorescence and
cytochemistry x-axis = granularity
Introduction

Optical Scatter/Detection
• Forward light scatter (0 degres) – cell
volume
• Orthogonal light scatter (90 degrees) or
side scatter – internal complexity
• Forward low-angle scatter (2 to 3 degrees)
and forward high-angle scatter (5 to 15
degrees) – cell volume and refractive
index or with internal complexity
• Differential scatter is the combination of
this low-angle and high-angle forward light
scatter and is primarily used on Siemens
systems for cellular analysis
ADVIA 2120i.
The ADVIA 2120i printout
displays the CBC, DIFF, and
reticulocyte data for the same
patient in Figures 15-6, 15-7,
and 15-9. A, CBC data; B, Six-
part differential, including large
unstained cells (LUCs);
nucleated RBCs; C,
Reticulocyte information
includes the CHr (cellular
hemoglobin reticulocyte). D,
Cytograms for the differential,
both the perox and baso
channels, on the right; E,
Scattergram for the
reticulocyte and RBC counts;
F, Morphology flags and
Sample/System Flags where
flags are displayed.
Procedure

Reagents and Equipment

1. Automated Hematology Analyzer
2. Phlebotomy kit
3. EDTA tube 3

Specimen
Whole blood anticoagulated with EDTA 1 2

Principle
In electrical impedance, sizing and counting particles uses measurable changes in electrical resistance produced by
nonconductive particles suspended in an electrolyte. Cells pass through an aperture with an electrical current flowing
through simultaneously. Cells do not conduct current but rather they change electrical resistance, which is then counted
as voltage pulses. the number of pulses is proportional to number of cells Amplitude of pulse generated is proportional to
size of the cell. The bigger the cell, the bigger the impedance.
Optical scatter systems (flow cytometers) use detection of interference in a laser beam or light source (at aperture) to
differentiate and enumerate cell types based on scattering properties of cells. Forward angle light scatter measures cell
size while side angle light scatter indicates cell granularity and lobularity.
Procedure

1 Check the status of the analyzer and confirm

if the analyzer is ready. Verify if the host
connections are working properly. Perform
visual inspections of the reagents also.

2 Check specimen identification. Ensure that

the EDTA tube and patient request match. A
request form with pertinent history details
should be submitted concurrently with the
sample.
Procedure

3 Check acceptability of the specimen. A

properly filled EDTA tube should be
submitted. Partially filled EDTA tubes affect
the cells because EDTA is hypertonic (e.g.
echinocytes will form in underfilled EDTA
tubes and red cells shrink, decreasing the
mean cell volume and increasing the mean
cell corpuscular hemoglobin concentration).

4 Scan the barcode on the tube or sample ID

can be entered manually in the hematology
analyzer.
Procedure

5 Ensure that the blood is mixed promptly prior to

aspiration. This should be done by rolling the
tube between your palms or gentle inversion
several times. Do not shake the tube.

6 Aspirate the sample through the sample needle

of the analyzer.

Or properly mix the specimen and place in tube

holder. Press the switch on the analyzer. The
tube holder will slide in and the sample will be
aspirated. When the analysis is complete, the
tube holder slides out.
Procedure

7 Check test results for flagging and

correlate with patient’s diagnosis.
Discrepancies in the results
should be investigated
immediately and resolved.
QA/QC

Quality Control
1. Before turn-over of analyzers to laboratory users, all automated instruments
must be standardized by manufacturers against reference methods (e.g., Hb
cyanide or against a single channel instrument that has been calibrated).

2. Once instrument has been standardized/calibrated, commercially prepared

control materials may be used as day to day controls.
• Commercial control material consists of blood cells (human, avian, porcine, etc.) that
have been fixed, buffered, stabilized, or preserved.
• Nucleated avian red cells are frequently used as “white” cells in control material
• Controls must be run at least once a day
• At least two levels of control materials (i.e., normal, abnormal) must be utilized
QA/QC

Validating Results
1. Correlate hematology results with previous results and other
laboratory data
• High Hb may be due to high glucose
• Increased platelets may be due to many microcytes, leukemic cell fragments, red cell
inclusions, hemolysis, and presence of cryoglobulins
• Decreased platelets in clotted specimens, presence of giant platelets, use of heparin,
platelet clumping, and platelet sattelitosis
2. Follow the International Society for Laboratory Hematology
(ISLH) suggested criteria for action following automated CBC
QA/QC

Additional Action After Doing an Automated CBC

RULE # PARAMETER CRITERION ACTION
1 Neonate 0-30 days old Smear review
2 Hb, RBC, WBC, PLT Exceeds linearity Dilute specimen and retest
3 WBC, PLT Lower than instrument Follow laboratory SOP
linearity
4 Hb, RBC, WBC, PLT No result Check specimen for clots; retest specimen,
perform an alternate count method
5 WBC (× 109/L) <4 or >30 Smear review
6 PLT (× 109/L) <100 or >1000 Smear review; re-draw sample if necessary
7 PLT (× 109/L) Any value AND delta fail* Smear review; re-draw sample if necessary
QA/QC

Additional Action After Doing an Automated CBC

RULE # PARAMETER CRITERION ACTION
8 Hb (g/L) <70 for any age; >20 above Smear review; verify sample integrity;
upper reference for age correlate with RBC indices
9 MCV (fL) <75 or >105 (Adult) Smear review
10 MCV (fL) Follow-up specimen differs by Verify sample identity
2 fL, and specimen is <24
hours old
11 MCHC ≥2 units above upper limit of Check for lipemia, hemolysis, RBC
reference range agglutination, spherocytes
12 RDW >22 Smear review
13 Differential count Incomplete or no differential Smear review with manual differential
count count
QA/QC

Additional Action After Doing an Automated CBC

RULE # PARAMETER CRITERION ACTION
14 Neutrophil absolute count (× 109/L) <1 or >20 Smear review
15 Lymphocyte absolute count (× >5 (adult) or >7 (children Smear review
109/L) <12 yrs old)
16 Monocyte absolute count (× 109/L) >1.5 (adult) or >3 Smear review
(children <12 yrs old)
17 Eosinophil absolute count (× 109/L) >2 Smear review
18 Basophil absolute count (× 109/L) >0.5 Smear review
19 Nucleated red blood cell Any number Compute for corrected WBC
count
20 Machine flag Any Smear review; check
histograms
Problems Encountered in Automated Methods

TYPES OF ERROR

Instrumental Errors

Nature of the
Specimen
Problems Encountered in Automated Methods

Instrumental Errors
1. Positive (+) Error
• Aperture plugs – MOST COMMON problem in cell counting
• Bubbles in the samples (caused by vigorous mixing)
• Extraneous electrical pulses
2. Negative (-) Error
• Excessive lysing of RBCs

• Improper settings of aperture current/threshold will cause EITHER

positive (+) or negative (-) error
Problems Encountered in Automated Methods

Nature of the Specimen

1. Giant platelets – may be counted as RBCs or WBCs
2. Fragments of WBC cytoplasm (may be seen in leukemia
therapy) – may be counted as platelets or RBCs
3. Some abnormal RBCs (for example: sickle cells, extremely
hypochromic cells, target cells) resist lysis – may cause
falsely elevated WBC count
Hematocrit

Spurious Increase Spurious Decrease

• Cryoglobulin • Autoagglutination
• Cryofibrinogen • Clotting
• Giant platelets • In-vitro hemolysis
• High WBC • Microcytic RBCs
• Hyperglycemia • Excess EDTA
(especially K3
EDTA)
www.beaumontlaboratory.com
 Hemoglobin

Spurious Increase Spurious Decrease

• Carboxyhemoglobin • Clotting
(>10%)
• Cryo-globin/fibrinogen
• Monoclonal proteins
• Hyperlipemia
• Hyperbilirubinemia
• Heparin
• In-vitro hemolysis
• High WBC
biochemistryquestions.wordpress.com
 RBC Count

Spurious Spurious
Increase Decrease
• Cryoglobulin • Autoagglutination
• Cryofibrinogen • Clotting
• Giant platelets • In-vitro
• High WBC hemolysis
(>50,000/uL) • Microcytic RBCs
imagebank.hematology.org
WBC Count

Spurious Increase Spurious Decrease

• Cyoglobulin • Clotting
• Cryofibrinogen • Smudge cells
• Heparin • Uremia plus
• Monoclonal proteins immunosuppressants
• nRBCs
• Platelet clumping
• Unlysed RBCs
www.medical-labs.net
 Platelet count

Spurious Increase Spurious Decrease

• Turbid plasma • Incipient clotting

(some systems) • EDTA-induced
• Precipitated platelet satellitism
cryoglobulin and agglutinatination
• RBC or WBC • Giant platelets
cytoplasmic
fragments
• Very microcytic www.sciencedirect.com
RBCs
www.researchgate.net
 MCV

Spurious Spurious
Increase Decrease
• Autoagglutination • Cryofibrinogen
• High WBC and globulin
• Hyperglycemia • Giant platelets
• Rigid RBC • In-vitro hemolysis

MCH and MCHC: spurious levels usually

www.researchgate.net
secondary to factors affecting Hb and Hct
Problems Encountered in Automated Methods

Common Automated Cell Count Errors

1. WBC counts exceeding instrument linearity limits result in
increased cell turbidity and may falsely increase the
hemoglobin, MCH, and MCHC.
2. Glucose over 600 mg/dL (hyperosmolarity) may increase the
MCV and hematocrit and decrease the MCHC.
3. Cold agglutinins increase the MCV, MCH, and MCHC and
decrease the RBC count and hematocrit.
4. Lipemia increases the hemoglobin, MCH, and MCHC.
 TABLE 15-2 Conditions That Cause Interference on Most Hematology Analyzers
Parameters
Condition Affected* Rationale Instrument Indicators Corrective Action
Cold agglutinins RBC g, MCV h, MCHC Agglutination of RBCs Dual RBC population on RBC Warm specimen to 37° C and
h, grainy appearance map, or right shift on RBC rerun
histogram
Lipemia, icterus HGB h, MCH h h Turbidity affects spectropho- HGB ! 3 HCT " 3, abnormal Plasma replacement‡
tometric reading for HGB histogram/cytogram†
Hemolysis RBC g, HCT g RBCs lysed and not counted HGB ! 3 HCT " 3, may Request new specimen
show lipemia pattern on his-
togram/cytogram†
Lysis-resistant RBCs with WBC h, HGB h RBCs with hemoglobin S, C, Interference at noise-WBC inter- Perform manual dilutions,
abnormal hemoglobins or F may fail to lyse; will be face on histogram/cytogram allow incubation time for
counted as WBCs lysis
Conditions Microcytes or schistocytes RBC g, PLT h Volume of RBCs or RBC frag- Left shift on RBC histogram,
ments less than lower RBC MCV flagged if below limits;
Review blood film

That Cause threshold, and/or within

PLT threshold
abnormal PLT histogram may
be flagged
Interference Nucleated RBCs, mega-
karyocyte fragments, or
WBC h
(older instruments)
Nucleated RBCs or micro-
megakaryoblasts counted
Nucleated RBC flag resulting
from interference at noise-
Newer instruments eliminate
this error and count nucleated

on Most micromegakaryoblasts as WBCs lymphocyte interface on

histogram/cytogram
RBCs and correct the WBC
count; count micromega-

Hematology karyoblasts per 100 WBCs

and correct

Analyzers Platelet clumps PLT g, WBC h Large clumps counted as

WBCs and not platelets
Platelet clumps/N flag, interfer-
ence at noise-lymphocyte
Redraw specimen in sodium
citrate, multiply result
(Rodak, page 226) interface on histogram/ by 1.1
cytogram
WBC # 100,000/$L HGB h, RBC h, HCT h Turbidity affects spectropho- HGB ! 3 HCT " 3, WBC Manual HCT; perform manual
incorrect tometric reading for HGB, count may be above linearity HGB (spin/read superna-
WBCs counted with RBC tant),‡ correct RBC count,
count recalculate indices; if
above linearity, dilute for
correct WBC count
Leukemia, especially with WBC g, PLT h Fragile WBCs, fragments Platelet count inconsistent with Review film, perform phase
chemotherapy counted as platelets previous results platelet count or CD61 count
Old specimen MCV h, MPV h, PLT g, RBCs swell as specimen ages, Abnormal clustering on WBC Establish stability and
automated differential platelets swell and degen- histogram/cytogram specimen rejection criteria
may be incorrect erate, WBCs affected by
prolonged exposure to
EDTA

h, Increased; g, decreased; EDTA, ethylenediaminetetraacetic acid; HGB, hemoglobin; HCT, hematocrit; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration;
Problems Encountered in Automated Methods

Repeat the analysis if…

1. Rule of three failure on a normocytic
sample (especially MCHC >37 g/dL) RULE OF THREE
2. Any result outside linearity limits a)RBC × 3 = Hb
established by manufacturer (dilute
into linearity range) b)RBC × 9 = Hct
3. Unexplained delta check failures
(e.g., results do not correlate with c) Hb × 3 = Hct
recent previous results, especially
MCV)
References

• Brown, B. A. (1993). Hematology: Principles and procedures (6th ed.). Estados

Unidos: Lea & Febiger.
• Keohane, E. M., Smith, L. J., Walenga, J. M., Rodak, B. F. (2016). Rodak's
hematology: Clinical principles and applications (5th ed.). St. Louis, MO: Elsevier.
• Lo, R. W., Liu, E. B., Orillaza, M. A., Faundo, A. C., & J., D. C. (2012). PCQACL'S
Standardization and harmonization of complete blood count in the Philippines
(1st ed.). Quezon City: C & E Publishing.
• McPherson, R. A., & Pincus, M. R. (2011). Henry's Clinical Diagnosis and
Management by Laboratory Methods E-Book. Saunders.
• Stiene-Martin, E. A., Lotspeich-Steininger, C. A., & Koepke, J. A. (1998). Clinical
Hematology: Principles, procedures, correlations. Philadelphia: Lippincott-Raven.