Difference between impedance and optical platelet count methods ...

ISLHLaboratory Hematology 5:22–27© 1999 Carden Jennings Publishing Co., Ltd.Official PublicationDifference between impedance and opticalplatelet count methods in patientswith microcytosis of red blood cellsR. PIŃKOWSKIDepartment of Laboratory Diagnostics, Military University of Medicine, Clinical Hospital WAM, Lódź, PolandABSTRACTPlatelet counts (PLT) were conducted in 30 patients diagnosedwith microcytosis due to iron deficiency anemia and 30 normalsubjects using optical and impedance methods (PLTo and PLTi) inan Abbott Cell-Dyn 4000 analyzer. To achieve the best estimationof the platelet and red blood cell quantity and quality (includingplatelet aggregation, platelet satellitism, microcytes, and schistocytes),a stained preparation was estimated and platelet count wascarried out with the microscopic chamber method (PLTh). Whenboth automated methods were used, result reproducibility was betterthan with the manual method. Correlation of the plateletcounts between the methods was high (r > 0.98), and highest forPLTo and PLTh (r = 0.997). When the impedance method wasused, platelet counts were higher on average than when the opticalmethod was used and differed, in absolute values, by 34.110 9 /L(9.7%) (p < 0.001). In some cases, the differences between PLTiand PLTo were bigger, even exceeding 15010 9 /L (>30%). Thedifference between PLTi and PLTo had an inverse correlation withmean corpuscular volume (MCV) (r = –0.635). Samples from normalsubjects did not demonstrate the above discrepancies or a similarrelationship with MCV. The platelet volume histogram ishelpful for evaluating the interference of microcytes and plateletsestimated using the impedance method. The optical methodyields more reliable results in the patients with microcytosis. PLTiestimation in cases of microcytosis is insufficient, and its resultsare not always reliable. Lab Hematol 5:22–27, 1999KEY WORDS: Hematology analyzer · Impedanceplatelet count · Microcytosis ·Optical platelet countAddress correspondence to Roman Pińkowski, Zak ⁄lad Diagnostyki LaboratoryjnejWojskowej Akademii Medycznej, Szpital Kliniczny WAM, ul.Żeromskiego 113, 90-549 Lódź, Polska; e-mail: rpin@skwam.wam.lodz.pl.Received 15 February 1999; accepted 22 June 1999INTRODUCTIONPlatelet count estimation is an important element of the diagnosticand treatment process in many disorders [1]. In patientswith thrombocytopenia, especially in the case of platelet transfusion,the reliability of the PLT estimation is highly desired andnecessary to provide appropriate treatment [2,3].The impedance method (PLTi), used in the majority of automatedhematology analyzers, is the most common method of PLTestimation. Applied less frequently are the microscopy methodusing a hemocytometer, the immunophenotypic method usingmonoclonal antibodies and a flow cytometer, and the opticalmethod. Both methods, optical and impedance, are employed inthe Abbott Cell-Dyn 4000 hematology analyzer [1,4–6]. Animportant complementary element of laboratory tests is plateletsize, shape, and grain structure in a stained microscopic preparation,a preliminary estimation of platelet quantity, and analysis ofphenomena such as aggregation or platelet satellitism.The impedance method, commonly used, is characterized byhigh precision and accuracy, better than manual microscopy andcomparable to the optical method. It should be remembered, however,that several factors interfere with the PLTi estimation and cansignificantly affect the result of a laboratory examination: fragmentsof red or white blood cells, reagent contamination or outside contamination,air bubbles, blood parasites, microclots, and other factors(Table 1).In patients with anemia due to iron deficiency, the populationof the smallest microcytes can lead to overestimated PLT. It wasshown in our earlier studies [1] that thrombocytosis, estimated onthe basis of PLTi, occurred relatively frequently in patients withiron deficiency. In turn, the analysis of the PLT estimations carriedout at the same time by both methods, PLTi and PLTo, showedthat PLTi was generally higher than PLTo in patients with microcytosis.This observation led us to a hypothesis that interference ofvarious factors with PLTo estimation is smaller than with PLTi estimationin these patients. The aim of this study was to confirm thishypothesis.22

Impedance and Optical Platelet Count Methods and Microcytosis 23TABLE 1. Some reasons for interference with platelet count estimationusing automated methodsFalsely high resultsCryoglobulinHemolysisSchistocytesMicrocytosisWhite blood cell fragmentsArtifactsMATERIALS AND METHODSFalsely low resultsMacrothrombocytesMicroclotsClotsUse of heparinPlatelet satellitismPlatelet degranulationPatient SamplesThirty patients with anemia due to iron deficiency and 30 normalsubjects were examined. Blood samples were stored using theBecton-Dickinson Vacutainer system (4 mL K 3 EDTA).PLTo and PLTi together with complete blood count (CBC) anddifferential were estimated using the Cell-Dyn 4000 hematologyanalyzer. For control purposes, each blood sample also underwent amicroscopy examination (stained blood smear) to confirm microcytosis,hypochromia, or presence of interfering elements.Microscopy Examination of Blood SmearRoutine microscopy of the stained peripheral blood smear wasused to evaluate the platelet count. This method was also applied toestimate the occurrence of aggregates or platelet satellitism,macrothrombocytes, microcytes, schistocytes, or white blood cellfragments, which could interfere with PLT estimation.Direct Microscopy Platelet CountsA phase contrast microscope, a hemocytometer (Burker’s chamber),and appropriate liquid (ammonium oxalate) were used to estimateplatelet counts in both groups. The final result was the averageof the two estimations. In the case of significant divergence ofplatelet counts estimated with automated methods, the hemocytometerplatelet count (PLTh) was used.Impedance Platelet CountsThe Cell-Dyn 4000 uses the impedance method (PLTi) to estimateplatelet count. The method draws on a hydrodynamic focusingflow in an impedance chamber. On the basis of impedance,platelet size is estimated and a histogram of platelet volume is prepared.Platelet number and mean platelet volume are estimated onthe basis of the PLT histogram surface, limited by the lower andupper volume discriminator (Fig. 1).Optical Platelet CountsThe Cell-Dyn 4000, apart from the routine PLTi estimation,also uses the optical method (PLTo) to measure platelet count,applying multiangle polarized light scatter separation (MAPSS). Atwo-dimensional analysis estimates the complexity and optical densityof the platelets, represented as a cytogram of the light intensityat 7- and 90-degree angles. Three moving discrimination lines createa window separating PLTo from other elements (Fig. 2).RESULTSCompared with manual microscopy, automated methods showbetter reproducibility of platelet counts (Table 2).If interference of nonplatelet elements (e.g., microcytes, schistocytes)occurs, then PLTi counts may be biased in their entirety(falsely high results). In such instances, the interference phenomenoncan be evaluated on the basis of the platelet volume histogram(Fig. 1). Such a procedure is insufficient, however, and should onlybe used for general information.Tables 3 and 4 contain detailed PLT counts estimated with thethree methods, summary of the quantitative results, and anaccounting of mean corpuscular cells in blood samples frompatients with microcytosis and normal subjects.In blood samples from patients with microcytosis, PLT valuesestimated using the impedance method were on the average higherby 9.7% than the optical method. The difference between PLTi andPLTo was statistically significant (p < 0.001). In individual cases,these differences were higher, as high as 30% (16710 9 /L as absolutevalues). In blood samples of normal subjects, PLT values estimatedusing the impedance method were on average higher by 1.7% thanthe optical method (p > 0.05). In individual cases, these differencesranged from –7.8% to 8.4% ( 0.98).The differences between PLTi and PLTo had an inverse correlationwith mean corpuscular volume (MCV) patients with microcy-ABFIGURE 1. Platelet volume histograms: normal (A) and with hindered separation due to presence of microcytes (B).

24 R PińkowskiTABLE 2. Platelet count reproducibility (coefficient of variation)depending on the blood examination method in patients withmicrocytosisAutomated methodsPlatelet count (10 9 /L) Manual (hemocytometer) PLTi PLTo130–400 9.9 4.7 3.8>400 8.7 3.4 2.9FIGURE 2. Cytogram outlining PLT, red blood cell (RBC),and microcyte (MICRO) separation in the optical measurementconducted with a Cell-Dyn 4000 analyzer. 7°, complexity;90°, optical density.tosis (r = –0.635) (Fig. 6). There was no such relationship in samplesfrom normal subjects (Fig. 7).On the basis of the PLT reference limit (150–40010 9 /L) acceptedin our laboratory, thrombocytosis and thrombocytopenia were detectedwith varying frequencies depending on the method (Table 5).The frequency of platelet changes in patients with microcytosiswas identical whether the hemocytometer or the opticalTABLE 3. Platelet count and microcytosis in patients with iron deficiency anemiaPatient PLTh (10 9 /L) PLTo (10 9 /L) PLTi (10 9 /L) PLTi – PLTo (10 9 /L) PLTi – PLTo (%) MCV (fL) Microcytosis*1 567 564 561 –3 –0.5 80 +2 134 139 133 –6 –4.3 76 ++3 254 257 259 2 0.8 78 +4 230 239 238 –1 –0.4 80 +5 211 222 248 26 11.7 79 ++6 146 148 152 4 2.7 78 +7 135 128 150 22 17.2 78 +++8 193 191 207 16 8.4 80 +9 230 231 247 16 6.9 73 ++10 191 188 200 12 6.4 77 ++11 194 191 207 16 8.4 71 ++12 241 245 260 15 6.1 76 +13 281 283 309 26 9.2 72 ++14 380 381 416 35 9.2 71 ++15 267 262 288 26 9.9 75 +++16 432 412 463 51 12.4 73 ++17 457 444 506 62 14.0 65 ++18 391 383 401 18 4.7 77 +19 273 261 308 47 18.0 64 +++20 297 304 341 37 12.2 72 +++21 417 428 496 68 15.9 72 +++22 368 364 386 22 6.0 73 +23 270 263 300 37 14.1 71 ++24 226 230 250 20 8.7 73 +25 381 386 432 46 11.9 79 ++26 539 543 617 74 13.6 77 +++27 568 575 668 93 16.2 53 +++28 536 503 552 49 9.7 62 +++29 271 262 288 26 9.9 67 ++30 548 538 705 167 31.0 64 +++Average 321 319 353 34.1 9.7 73Standard deviation 135 133 158 34.5 6.8 5.9Minimum 134 128 133 – 6 – 4.3 57Maximum 568 575 705 167 31.0 80*Severity of microcytosis was estimated by microscopic evaluation.

Impedance and Optical Platelet Count Methods and Microcytosis 25TABLE 4. Platelet count and mean corpuscular volume in normal subjectsPatient PLTh (10 9 /L) PLTo (10 9 /L) PLTi (10 9 /L) PLTi – PLTo (10 9 /L) PLTi – PLTo (%) MCV (fL)1 157 148 156 8 5.7 982 145 153 157 4 2.7 973 152 157 149 –8 –5.1 954 164 158 171 13 8.4 945 163 159 155 –4 –2.7 876 159 163 155 –8 –5.1 867 161 163 155 –8 –5.1 908 158 166 153 –13 –7.8 899 168 169 166 –3 –1.8 8710 182 178 193 15 8.4 9611 189 180 192 12 6.4 9012 187 193 209 16 8.3 9113 189 196 192 –4 –1.8 8414 197 200 213 13 6.3 9815 204 200 215 15 7.3 8816 211 206 203 –3 –1.4 8717 229 239 238 –1 –0.4 8618 263 254 271 17 6.7 8219 254 260 259 –1 –0.4 9420 296 301 316 15 5.0 9221 317 303 300 –3 –0.9 8922 323 312 316 4 1.2 9023 324 317 330 13 4.2 9524 331 321 323 2 0.6 9225 337 322 340 18 5.7 8926 348 356 353 –3 –0.9 9827 362 365 358 –7 –1.9 9428 372 371 388 17 4.5 8329 387 376 389 13 3.4 8630 391 383 386 3 0.7 82Average 244 242 247 4.4 1.7 90Standard deviation 84 81 84 9.6 4.6 4.8Minimum 145 148 149 –13 –7.8 82Maximum 391 383 389 18 8.4 98FIGURE 3. Correlation of the results of PLT counts using the optical method (PLTo) and the impedance method (PLTi) in bloodsamples from patients with microcytosis.

26 R PińkowskiFIGURE 4. Correlation of the results of PLT counts using the optical method (PLTo) and the manual method in blood samples frompatients with microcytosis.FIGURE 5. Correlation of the results of PLT counts using the impedance method (PLTi) and the manual method in blood samplesfrom patients with microcytosis.method was used. When the impedance method was used, five of30 cases had excessive PLT values, including two false-negativeresults for thrombocytopenia and three false-positive results forthrombocytosis.DISCUSSIONImpedance platelet counts, based only on the estimation of volume,prove imperfect in the presence of nonplatelet elements similarin size to platelets. Microcytes, which are present in blood frompatients with anemia due to iron deficiency, interfere with PLTicounts to various degrees. Excessive results in the 150–40010 9 /Larea of the scope or slight thrombocytosis do not constitute clinicalproblems. False-negative results in the case of thrombocytopeniamay jeopardize life. The new optical method of platelet count estimationin hematology analyzers significantly increases the reliabilityof the data. Optical methods are less susceptible to interference ofnonplatelet elements such as microcytes.As other authors have observed [3], the impedance method providesbiased results when macrothrombocytes or red blood cell frag-TABLE 5. Frequency of thrombocytopenia (PLT 40010 9 /L) in blood samples from 30patients with iron deficiency anemiaPLTh PLTo PLTiPLT 40010 9 /L 8/30 8/30 11/30

Impedance and Optical Platelet Count Methods and Microcytosis 27FIGURE 6. Correlation of the impedance and optical platelet count difference (PLTi and PLTo) and the MCV in blood samplesfrom patients with microcytosis.FIGURE 7. Relationship of impedance and optical platelet count difference (PLTi and PLTo) and MCV in normal blood samples.ments are present. In the former case, PLTi is falsely low; if schistocytesare present (as in microcytosis), results are falsely high.Currently, the application of both PLT estimation methods, opticaland impedance, constitutes the best solution, especially in clinicallydifficult cases in which only a reliable result ensures the successof treatment (such as in cytoreductive treatment, after bone marrowtransplant, marrow aplasia, or in severe cases of thrombocytopenia).REFERENCES1 PINKOWSKI R: Blood platelets: Current indices and some selected problemsrelated to the use of hematology analyzers. Abbott Laboratories Poland:educational materials, ISBN 83-907097-0-8, 1997 [in Polish]2 AULT KA: Platelet counting: Is there room for improvement? Lab Hematol2:139, 19963 KCKLER TS, ROTHE M, BLOSSER L, SCHISANO T, VAN HOVE L: Improvingplatelet transfusion therapy using the ImmunoPLT method on the CELL-DYN 4000. Lab Hematol 4:80, 19984 GOOSSENS W, S COTT CS, WALSH A, VANDEKERKHOVE PH, VANORSHOVEN A, VAN HOVE L: First basic performance evaluation of theCELL-DYN 4000. Lab Hematol 2:151, 19965 TATSUMI N, TSUDA I, SHIMIZU A, WATANABE K, MATSUNO K: Basic performanceevaluation of the new hematology analyzer, CELL-DYN 4000:Japanese hematology analyzer study group. Lab Hematol 2:157, 19966 VAN HOVE L, JANIK J, BECKER R, SCHISANO T, KIM YR, CHUPP V: CELL-DYN 4000, the next hematology analyzer. Int J Hematol 64 (Suppl1):164, 19967 PENNINCK D, CONTEMPRE B, WIJNS W, CAPEL P: Evaluation of AbbottCell-Dyn 4000, with special emphasis on platelet and nucleated red bloodcell. Lab Hematol 2:181, 1997