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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

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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
Specimen Collection & Processing

Some of the preanalytic and analytic errors that can affect hematologic results (see Table 33-1).

venipuncture specimen
Venipuncture specimen

Venipuncture specimen:

The blood should be collected into a tube containing an anticoagulant and thoroughly mixed.


The choice of anticoagulants for hematologic studies:


-trisodium citrate


complete blood count
Complete Blood Count

The CBC includes a determination of

-red blood cell data: RBC,Hb,Ht,


-white blood cell data

-platelet count, MPV

hematology analyzers instrumentation
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
Total RBC


-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 rbc1
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)



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).


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
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
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: 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


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
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 count1
Differential Count

The manual DC :a time-consuming, labor-intensive, 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 count2
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 count3
Differential Count

The key to successful implementation of DC by AHA is based on the ability of the instrument :


-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 count4
Differential Count

The newer analyzers: uses technologies including

electrical impedance, cytochemistry, and optical

absorbance or uses a complex multiangle, light-

scattering 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 count5
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
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 count1
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 count2
Platelet Count

-In individuals with EDTA-dependent platelet

agglutinin, citrate is the preferred alternative


-Today‘s PLT is routinely measured with AHA.

However, manual hemacytometer counts are still

essential in patients with low platelet counts(<


-Various red and white blood cells, platelet, and instrument artifacts may interfere with PLT

platelet count3
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: 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

reticuiocyte count
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 count1
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 count2
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 count3
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 rate1

-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 rate2

-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 rate3

-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.