chapter 33 blood routine examination xiong lifan

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 Component



Red blood cells

CryoglobulinAutoagglutination

Giant platelets Clotting

High WBC

( ＞ 50×109/L)

Hemolysis

(in vitro)

Microcytic red cells


CBC Component



Hemoglobin Carboxyhemoglobin (＞ 10%)

Clotting

Cryoglobulin

Hemolysis (in vivo)

Heparin

Hyperbilirubinemia

Lipemia

Monoclonal proteins


CBC Component



Hematocrit

(automated)

Cryoglobulin Autoagglutination

Giant platelets Clotting

High WBC

( ＞ 50×109/L)

Hemolysis (in vitro)

Hyperglycemia ( ＞ 6g/L) Microcytic red cells

Hematocrit (microhematocrit)

Hyponatremia Excess EDTA

Plasma trapping Hemolysis (in vitro)

Hypernatremia


CBC Component



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


CBC Component Causes of Spurious Increase


Mean corpuscular hemoglobin concentration

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


CBC Component



Platelets 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 (×l09/L )

Neutrophils (×l09/L) Lymphocytes

Monocytes

Eosinophils

BasophilsTotal Band Segmen

ted

At birth

18.1 (9.0～

30.0)

11.0 (6.0 ～26.0), 61%

1.61,

9.1%

9.4,

52%

5.5 (2.0～ 11.0)

,

31%

1.05 (0.40～ 3.1)

,

5.8%

0.40 (0.02～

0.85), 2.2%

0.10 (0 ～0.64), 0.6%

Adult 7.4 (4.5～ I 1.0)

4.4 (I .8～ 7.7),

59%

0.22 (0～ 0.7),

3.0%

4.2 (I .8～ 7.0),

56%

2.5 (I .0～ 4.8),

34%

0.30 (0～

0.8),

4.0%

0.20 (0～

0.45),

2.7%

0.04 (0 ～0.20),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, 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 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, 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 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,drug-induced)

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) Myeloproliferative disorders(in some cases)

Hereditary disorders Wiskott-Aldrich syndrome

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 ．


-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 ．


-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 ．

END

chapter 33 blood routine examination xiong lifan

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