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
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
Some of the preanalytic and analytic errors that can affect hematologic results (see Table 33-1).
The blood should be collected into a tube containing an anticoagulant and thoroughly mixed.
The choice of anticoagulants for hematologic studies:
The CBC includes a determination of
-red blood cell data: RBC,Hb,Ht,
MCV, MCHC, MCH,RDW
-white blood cell data
-platelet count, MPV
The evolution of robotic techniques :
Technologist interaction:needed at the point of
-manual slide making
Computers’ power: to store and analyze large clinical databases
-electrical impedance methods
-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
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)
MCV: is important in classifying anemias with parameter RDW. (Fig.33-1)
MCH and MCHC：are useful tools primarily for quality-control purposes.
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)]
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.
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..
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.
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）
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 )
-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.
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
-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.
-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
Artifacts: that can interfere with PLT in the AHA include:
-red/white blood cell fragments/debris
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
-The patients with thrombocytopenia owing to marrow suppression typically have decreased MPV values
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)
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．
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%
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)
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．
-Clinical significance : Anemia，hypercholesterolemia,chronic renal failure,inflammatory disease may all produce an elevated ESR．
-fragmented red blood cells(e．g．，sickle cell
microcytic red blood cells，steroids，
hypofibrinogenemia may all lead to a decrease in
the ESR value．
-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．
-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．