Chapter 33
Blood Routine Examination
Xiong Lifan
BLOOD CELL COUNTING
The process of performing a basic hematologic
analysis of peripheral blood involves four primary steps:
1.collection and processing of the peripheral blood
sample
2.determination of the CBC
3.determination of the differential WBC
4.blood film examination
Specimen Collection & Processing
Some of the preanalytic and analytic errors that
can affect hematologic results (see Table 33-1).
THE CBC: SOURCES OF
SPURIOUS RESULTS
CBC
Component
Causes of
Spurious Increase
Causes of
Spurious Decrease
WBC
Cryoglobulin
Clotting
Heparin
Smudge cells
Monoclonal Proteins
Nucleated red cells
Platelet clumping
Unlysed red cells
Table 33-1(continued)
THE CBC: SOURCES OF SPURIOUS RESULTS
CBC
Causes of
Causes of
Component Spurious Increase Spurious Decrease
Red blood
Cryoglobulin
Autoagglutination
cells
Giant platelets
Clotting
High WBC
Hemolysis
(＞50×109/L)
(in vitro)
Microcytic red
cells
Table 33-1(continued)
THE CBC: SOURCES OF SPURIOUS RESULTS
CBC
Component
Causes of
Spurious Increase
Hemoglobin Carboxyhemoglobin (
＞10%)
Cryoglobulin
Hemolysis (in vivo)
Heparin
Hyperbilirubinemia
Lipemia
Monoclonal proteins
Causes of
Spurious
Decrease
Clotting
Table 33-1(continued)
THE CBC: SOURCES OF SPURIOUS RESULTS
CBC
Component
Causes of
Spurious Increase
Causes of
Spurious
Decrease
Hematocrit
(automated)
Cryoglobulin
Autoagglutination
Giant platelets
Clotting
High WBC
(＞50×109/L)
Hemolysis (in vitro)
Hyperglycemia (＞6g/L)
Microcytic red cells
Hyponatremia
Excess EDTA
Plasma trapping
Hemolysis (in vitro)
Hematocrit
(microhematocrit)
Hypernatremia
Table 33-1(continued)
THE CBC: SOURCES OF SPURIOUS RESULTS
CBC
Component
Causes of
Spurious Increase
Causes of
Spurious
Decrease
Mean corpuscular
volume
Autoagglutination
Cryoglobulin
High WBC (＞50×109/L)
Giant platelets
Hyperglycemia
Hemolysis (in vitro)
Microcytic red cells
Swollen RBC
Mean corpuscular
hemoglobin
High WBC (＞50×109/L)
Spuriously low Hb
Spuriously high Hb
Spuriously high RBC
Spuriously high RBC
Table 33-1(continued)
THE CBC: SOURCES OF SPURIOUS RESULTS
CBC Component
Mean corpuscular
hemoglobin concentration
Causes of
Spurious
Increase
Causes of
Spurious
Decrease
Autoagglutination
High WBC (＞
50×109/L)
Clotting
Spuriously low Hb
Hemolysis (in vitro)
Spuriously high Hct
Hemolysis (in vivo)
Spuriously high Hb
Spuriously low Hct
Table 33-1(continued)
THE CBC: SOURCES OF SPURIOUS RESULTS
CBC
Component
Platelets
Causes of
Spurious
Increase
Causes of
Spurious
Decrease
Cryoglobulin
Clotting
Hemolysis (in vitro and
in vivo)
Giant platelets
Microcytic red cells
Heparin
Red cell inclusions
Platelet clumping
White cell fragments
Platelet satellitosis
Venipuncture specimen
•Venipuncture specimen:
The blood should be collected into a tube containing
an anticoagulant and thoroughly mixed.
Anticoagulants
• The choice of anticoagulants for hematologic
studies:
-EDTA
-trisodium citrate
-heparin
Complete Blood Count
•The CBC includes a determination of
-red blood cell data: RBC,Hb,Ht,
MCV, MCHC, MCH,RDW
-white blood cell data
-platelet count, MPV
Hematology
Analyzers/Instrumentation
•The evolution of robotic techniques :
specimens handling
•Technologist interaction: needed at the point of
-troubleshooting ;
-manual slide making
•Computers’ power: to store and analyze large clinical
databases
.
Total RBC
•Methods:
-electrical impedance methods
-laser light-scatter
•Clinical significance:
-The RBC is the basis for calculating the
hematocrit(HCT), MCH, and MCHC.
-In iron deficiency: the RBC diminishes in proportion
with Hb.
-In thalassemia: the RBC may be normal to increased
relative to the degree of anemia
Total RBC
•The electrical impedance or light-scattering
techniques : allow both the counting of total cells and
determining the cell size (MCV) of the red blood cells.
•Spurious decrease in total RBC come from:
red blood cell autoagglutination
extreme red blood cell microcytosis
•False elevations in total RBC come from:
very high WBCs(＞ 100.0×109cells/L）
cryoglobinemia
Hemoglobin
•Method: spectrophotometry using a cyanomethemoglobin
procedure
-The formation of cyanated methemoglobin
-Falsely elevated hemoglobin can occur owing to
hyperlipemia, fat droplets,hypergammaglobulinemia,
cryoglobulemia, leukocytosis,improperly collected blood
specimens
•Clinical significance:Hemoglobin concentrations vary
according to age and gender
(Table 33-2A, 33-2B).
Table 33-2 A COMPLETE BLOOD COUNT:
NORMAL VALUES
Age
Hb
(g/L)
Hct
(%)
RBC(
×1012/
L)
MCV
(fl)
MCH
(pg)
MCHC
(g/L)
RDW
(%)
At birth
135～
195
42～60
3.9～
5.4
98～
118
31～37
300～
360
－
Adult
female
117～
157
34.9～
46.9
3.8～
5.2
80.8～
100
26.5～
34.0
314～
358
＜15
Adult
male
135～
175
39.8～
52.2
4.4～
5.9
80.5～
99.7
26.6～
33.8
315～
363
＜15
Table 33-2B WBC AND DCs:NORMAL VALUES
Age
WBC
Lymph
Neutrophils (×l09/L)
ocytes
(×l09
/L )
Total
Band
Segmen
ted
At
18.1
birth (9.0～
30.0)
Adult
11.0
(6.0～
26.0),
61%
7.4 4.4 (I .8
(4.5～ ～7.7),
I 1.0)
59%
1.61,
9.4,
9.1%
52%
0.22 (0
～0.7),
3.0%
4.2 (I .8
～7.0),
56%
5.5 (2.0
～11.0)
,
31%
Mono
cytes
Eosino Baso
phils
phils
1.05
(0.40
～3.1)
,
5.8%
0.40
(0.02
～
0.85),
2.2%
0.10
(0～
0.64),
0.6%
2.5 (I .0 0.30 (0 0.20 (0 0.04
～4.8), ～0.8),
(0～
～
34%
4.0% 0.45), 0.20),
2.7% 0.5%
Hematocrit
•HCT: is the ratio of the volume of the red blood
cells to the volume of the whole blood.
•Determined directly by centrifugation
•Calculated directly from the RBC and MCV:
= RBC (cells/L)×MCV (liter/cell)
Red Blood Cell Indices
•MCV: is important in classifying anemias with
parameter RDW. (Fig.33-1)
•MCH and MCHC：are useful tools primarily for
quality-control purposes.
Red Cell Distribution Width
•RDW: provides quantification into the variation in
red cell size, or anisocytosis.
•It may be a more sensitive indicator of a change in
cell size than purely the MCV
• The elevated RDW has been associated with
anemias.
•The normal RDW classically characterizes the
microcytic anemias seen in thalassemia.
WBC
•WBC: detemined by either electrical impedance
methods or light-scatter techniques.
•Hemacytometers may be used if the automated counters
fail to provide accurate results
•Clinical sinificance: primary hematologic disease or
acute / chronic infectious processes,trauma, surgery ,
hemorrhage, delivery, tissue necrosis, corticosteroids,
other medications
WBC
•Heparinized blood: should not be used for
determining the WBC
•Nucleated red blood cells (NRBC), cryoglobulin,
platelet clumps, large platelets, and unlysed red
blood cells may all lead to false elevations.
•The corrected WBC:
= (measured WBC × 100) / [100 + (n red cells / 100
white cells)]
Differential Count
•DC(or differential leukocyte count) (Fig.33-2)
•In addition to the DC, it is important to give
morphologic evaluation of all components of the
peripheral blood morphologically, including red
blood cells, white blood cells, and platelets.
Differential Count
•The manual DC ：a time-consuming, laborintensive, and relatively expensive procedure.
The manual DC has other medical and scientific
limitations： poor sensitivity, specificity, and
predictive value；it is imprecise.
•The manual DC: has remained the gold standard
of differential WBCs..
Differential Count
•Two basic methodologies as automated DC：
-digital image analysis systems
-flow cell-related techniques
• Automated DC by modern hematology analyzer：
more accurate, more precise, more economical, faster,
and safer
But in some cases AHA fails to provide important
morphologic detail that only the manual differential /
review can provide.
Differential Count
•The key to successful implementation of DC by
AHA is based on the ability of the instrument ：
•(Fig.33-3)
-to recognize both quantitative and qualitative
abnormalities
-to flag particular cases for further review（a manual
differential count or a manual review of the stained
blood smear）
Differential Count
•The newer analyzers: uses technologies including
electrical impedance, cytochemistry, and optical
absorbance or uses a complex multiangle, lightscattering to categorize white cells (Fig.33-4)
•The differential WBC: In the outpatient group, a
differential WBC should be performed only in
patients in whom the information may provide
important diagnostic, prognostic, or therapeutic
decisions.( Fig.33-5, Fig.33-6, Fig.33-7 )
Differential Count
-In hospitalized patients, there are many clinical
situations in which an abnormal differential count
will correlate with a particular clinically important
disease.
-An unexpected leukocytosis or leukopenia found on
a CBC may be more specifically elucidated if a
leukocyte differential count is obtained.
Platelet Count
•A platelet count(PLT) : provides the starting point
in the functional evaluation of the hemostatic
system.
-A diminished platelet count may be the result of
either a marrow production problem or a peripheral
destructive process.
-Evaluation of the bone marrow may reveal an
infiltrative malignant process
Platelet Count
-Various drugs and some viral infections may lead
to a reduction in platelet production.
-In patients receiving chemotherapeutic regimens,
platelets are commonly diminished
-These are primarily immunebased
thrombocytopenias but may occasionally involve
splenic sequestration of platelets.
Platelet Count
-In individuals with EDTA-dependent platelet
agglutinin, citrate is the preferred alternative
anticoagulant.
-Today‘s PLT is routinely measured with AHA.
However, manual hemacytometer counts are still
essential in patients with low platelet counts(＜
50.0×109/L)
-Various red and white blood cells, platelet, and
instrument artifacts may interfere with PLT
Platelet Count
•Artifacts: that can interfere with PLT in the AHA
include:
-red/white blood cell fragments/debris
-electronic noise
-microcytic RBC
-giant platelets
-platelet clumping
•Phase microscopy: is necessary to obtain an
accurate platelet count．
MPV
•MPV: The high MPV is suggestive of younger
platelets found in peripheral destructive processes
such as immune thrombocytopenias.( Fig.33-8, 33-9 )
-The MPV may falsely increase or decrease with
EDTA anticoagulation.
-The patients with thrombocytopenia owing to
marrow suppression typically have decreased MPV
values
MPV
Low MPV
NormaI to High MPV
High MPV
Marrow suppression
Hyperdestruction with
marrow compensation
Hereditary disorders
Chemotherapy
Immune-related(ITP,druginduced)
Bernard-Soulier syndrome
Megaloblastic anemia
Mechanical(consumptive
coagulopathies，vasculitis)
May-Hegglin anomaly
Aplastic anemia
Miscellaneous
Marrow infiltration
Hemorrhage(major)
α-and β-thalassemia
trait(unknown cause)
Sepsis
Sepsis(without marrow
suppression)
Myelodysplastic syndrome
Hypersplenism(variable)
Hereditary disorders
Wiskott-Aldrich syndrome
Myeloproliferative
disorders(in some cases)
ReticuIocyte Count
•Reticulocytes :may take on various morphologic
appearances depending on the amount of residual
ribosomes and organelles，or reticulum.
•Clinically，the reticulocyte percentage can be used
as an indicator of erythropoiesis and is often utilized
for evaluating patients with anemia(as in iron，
folate，or vitamin B12 deficiency，or as a result of
a bone marrow infiltrative process)
ReticuIocyte Count
•An increased RET generally reflects a rapid
erythroid turnover(as in acute blood loss or acute
or chronic hemolysis).
In other words，the RET level can be used as
a general indicator of bone marrow erythropoiesis
and release．
ReticuIocyte Count
•Laboratory microscopic methods: make the
reticulocyte visible by precipitating the residual
ribosomal RNA material with a dye such as new
methylene blue or brilliant creosol blue
•The manual determination of reticulocyte
counts: is a very imprecise method with CV over
25%
ReticuIocyte Count
•Automated counting methods:
image analysis and flow cytometry (FCM) (Fig. 33-10)
-Both these procedures remove much of the subjective
interpretation ，allow evaluation of large numbers of
red blood cells ，and provide a standard and uniform
analysis
-FCM procedures depend on the binding of a suitable
fluorescent dye to residual erythrocyte RNA (auramine
O , thiazole orange)
ERYTHROCYTE
SEDIMENTATION RATE
•The ESR: measures the distance a red blood cell falls
in a vertical tube over a given period of time．
-The Westergren method: the standard procedure ;
The modified Westergren procedure uses EDTA as the
anticoagulant
-Various factors: a clotted blood sample, prolonged
delay analysis ,etc. may all lead to a false decrease in
ESR values．
ERYTHROCYTE
SEDIMENTATION RATE
-Clinical significance : Anemia，
hypercholesterolemia,chronic renal
failure,inflammatory disease may all produce an
elevated ESR．
-fragmented
red blood cells(e．g．，sickle cell
anemia，burn patients)，spherocytes，
microcytic red blood cells，steroids，
hypofibrinogenemia may all lead to a decrease in
the ESR value．
ERYTHROCYTE
SEDIMENTATION RATE
-An elevated ESR has been used as evidence for
an inflammatory process．
The only consistent diagnostic use for the ESR is
in the diagnosis and monitoring of temporal arteritis
and polymyalgia rheumatica．
- The
ESR should not be used as a screening device in
the healthy，asymptomatic population．
ERYTHROCYTE
SEDIMENTATION RATE
-No study has shown a significant contribution of
an elevated ESR in detecting unsuspected disease in
the asymptomatic patient．
-Patients with a markedly elevated ESR greater than
100 mm/h usually have underlying malignancy，
acute infection，or some type of connective tissue
disease．
END