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).
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).
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
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