Accurate Results in the Clinical Laboratory || Sources of Errors in Hematology and Coagulation Testing

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<ul><li><p>C H A P T E R</p><p>19</p><p>Sources of Errors in Hematology andCoagulation TestingAndy Nguyen, Amer Wahed</p><p>University of Texas Health Sciences Center at Houston, Houston, Texas</p><p>INTRODUCTION</p><p>This chapter is divided into two parts: The first partdiscusses sources of errors in hematology testing, andthe second part addresses challenges in coagulationtesting. To understand sources of errors in hematology,it is important to understand the steps involved inproviding blood count values as well as interpretationof peripheral blood smears [1].</p><p>Blood for complete blood counts (CBC) is typicallycollected in vacuum tubes that contain the anticoagulantethylenediaminetetraacetic acid (EDTA). The blood col-lected in the vacuum tube is analyzed on automatedhematology analyzers for CBC results. These automatedinstruments have various channels. Different channelsare used to obtain different counts. One channel is usedfor red blood cell (RBC) count and platelet count.Another channel is used to obtain the total white bloodcell (WBC) count and hemoglobin level. In this channel,the red cells are lysed. Some instruments have a sepa-rate channel for hemoglobin. Other channels are forWBC differential count, reticulocyte count, and nucle-ated red cell count. Different methodologies exist toobtain the actual counts, including impedance (basedon the measurement of changes of electrical resistanceproduced by a particle suspended in a conductivemedium as it passes through an aperture of knowndimension), conductivity measurement with high-frequency electromagnetic current, light scatter, andflorescence-based (flow cytometric) methods. For themeasurement of hemoglobin concentration, the red cellsare lysed and hemoglobin (and also methemoglobinand carboxyhemoglobin) is converted to cyanmethemo-globin. The absorbance of light at 540 nm is measuredto provide a hemoglobin level [2]. Each time a cell</p><p>passes through the aperture, a pulse is produced. Thepulse height is proportional to the cell volume. The dis-tribution curves for the volume are separated from eachother with a moving discriminator. Cells with a volumebetween 2 and 30 fL are counted as platelets. Cells witha volume of 40250 fL are counted as red cells. In addi-tion to the actual counts, RBC and platelet histogramsare also provided. Each cell volume is measureddirectly, and the mean corpuscular volume (MCV) iscalculated by averaging the volume of all the cells or bydrawing a perpendicular line from the peak of the RBChistogram to the baseline. The red cell distributionwidth (RDW), which is a measure of anisocytosis, is cal-culated from the RBC histogram at 20% of peak height.</p><p>The lysing reagent causes WBCs to lose cytoplasm,and the cell membrane collapses around the nucleus.This allows differentiating the cells as the nuclear sizedifferences are accentuated. WBCs are countedbetween the range of 30 and 300 fL. Typically, threepeaks are seen in the WBC histograms. The first peakrepresents the lymphocytes, and the third peak repre-sents the neutrophils. All other white cells are repre-sented as the second peak.</p><p>As discussed previously, the automated instrumentsare actually measuring red cell counts, volumes, andhemoglobin levels. The RDW and MCV are calculatedby the instrument from the red cell volume histogram.Values for hematocrit, mean corpuscular hemoglobin(MCH), and mean corpuscular hemoglobin concen-tration (MCHC) are calculated by the instrument asfollowing:</p><p>Hematocrit5MCV3RBC countMCH5hemoglobin=RBC countMCHC5hemoglobin=hematocrit</p><p>305Accurate Results in the Clinical Laboratory.</p><p>DOI: 2013 Elsevier Inc. All rights reserved.</p></li><li><p>ERRORS IN HEMOGLOBINMEASUREMENTAND RBC COUNT</p><p>Hemoglobin measurement is based on absorption oflight at 540 nm. If the sample is turbid, this will pro-duce higher hemoglobin levels. Examples of such stateinclude hyperlipidemia [3], patients on parenteralnutrition [4], hypergammaglobulinemia, and cryoglo-bulinemia. Turbidity from very high WBC count canalso falsely elevate hemoglobin levels. Smokers havehigh carboxyhemoglobin, which may falsely elevatethe measured hemoglobin level.</p><p>Large platelets may be counted by some instrumentsas red cells. Also, red cell fragments greater than 40 fLwill be counted as whole red cells. In both situations, theRBC count will be falsely high. Cold agglutinins willcause red cell agglutination in vitro and result in lowRBC counts. If cold agglutinins are suspected, the sam-ple should be warmed to obtain an accurate RBC count.</p><p>ERRORS IN MCVAND RELATEDMEASUREMENTS</p><p>If there is red cell agglutination, then red cellclumps will be counted as single red cells but the vol-ume of the estimated cell will be much higher. Thiswill result in falsely high MCV values. If large plateletsare counted as red cells, then these platelets typicallyhave less volume than a normal red cell. This willresult in falsely low MCV values.</p><p>If the patient is in a state of high osmolarity, thecytoplasms of the red cells are also hyperosmolar.When diluents are added to the blood in the analyzer,water will move into the red cells, causing them toswell in size. MCV values will be higher than that inthe in vivo state. Examples of hyperosmolar states areuncontrolled diabetes mellitus, hypernatremia, anddehydration [5]. The converse will occur in hypo-osmolar states.</p><p>Values for hematocrit, MCH, and MCHC are obtainedby calculation using hemoglobin levels, RBC counts, andMCV values. If there is an error in any of these values,the calculated values will also be inaccurate.</p><p>ERRORS IN WBC COUNTS AND WBCDIFFERENTIAL COUNTS</p><p>Falsely high WBC counts are more common thanfalsely low WBC counts. There are several situations inwhich the WBC count may be falsely elevated. One ofthe most frequent situations is high WBC count in thepresence of a significant number of nucleated red blood</p><p>cells (NRBCs). If an accurate WBC count is required,then a corrected WBC count needs to be performed.This can be done by some hematology analyzers byrunning the sample again in the NRBC mode or per-forming a manual count. Platelet aggregates and nonly-sis of red cells are other causes of spuriously high WBCcounts. If the high WBC count is due to nonlysis of redcells, this may be a tip-off for hemoglobinopathies.Target cells seen in hemoglobinopathies are typicallyresistant to lysis. Platelet aggregates may be due toEDTA, and redrawing blood in a citrate tube may bethe solution in such cases. Erroneous WBC counts withspurious leukocytosis can be seen with the presence ofcryoglobulins and microorganisms. Spurious leukope-nia can be seen in cold agglutinins and EDTA-dependent leukoagglutination [6].</p><p>As discussed previously, WBC histograms havethree peaks. The first peak represents lymphocytes,and it is during this peak that WBCs have the lowestcell volume. It is easy to understand that when thereare giant platelets or nucleated red cells or red cellsresistant to lysis, these may be counted as lymphocytesin some instruments, giving rise to a falsely high lym-phocyte count. Hemoglobinopathies and target cellsare important causes of nonlysis of red cells. The pres-ence of malarial parasites in red cells has also beenknown to increase the lymphocyte count.</p><p>In myelodysplastic syndrome, if the myeloid seriesis affected, then hypolobated and hypogranular neutro-phils can be present. Automated analyzers may no lon-ger count these dysplastic neutrophils as such; instead,these neutrophils may be counted as lymphocytes.</p><p>Basophilia is typically seen in chronic myelogenousleukemia. Basophils are cells with coarse granules thatmay even obscure the nucleus. If the analyzer falselyrecognizes all the dense granules of basophils as onesingle nucleus, then these cells could be counted aslymphocytes.</p><p>It is thus apparent that there can be multiple situa-tions in which the lymphocyte count is inappropriatelyelevated. Whereas falsely low lymphocyte count israre, falsely low neutrophils can be encountered morefrequently. If there is an error in the neutrophil count,it is more likely to be a falsely low count than a highcount. Neutrophil aggregation is a documented phe-nomenon and can result in low neutrophil count.Neutrophils have fine granules, whereas the granulesof eosinophils are larger. Basophils have quite largegranules. If neutrophils have hemosiderin granules,they may be counted as eosinophils. If eosinophils arehypogranular, they may be counted as neutrophils.Red cells infected by malarial parasites may containmalarial pigments. Malaria-infected red cells are resis-tant to lysis. These red cells with malarial pigmentsmay be counted as eosinophils.</p><p>306 19. SOURCES OF ERRORS IN HEMATOLOGY AND COAGULATION TESTING</p><p>ACCURATE RESULTS IN THE CLINICAL LABORATORY</p></li><li><p>A key difference between lymphocytes and mono-cytes is that monocytes are significantly larger.Reactive (activated) lymphocytes typically have moreabundant cytoplasm compared to nonreactive lympho-cytes. Their size approaches that of a monocyte. Theselymphocytes may thus be counted as monocytes.</p><p>Also, it has been reported that abnormal lympho-cytes such as those seen in chronic lymphocytic leuke-mia, lymphoblasts, and leukemic or lymphoma cellscan be miscounted as monocytes. When there is leftshift in the WBC series, there is a tendency for slightlymore immature cells such as bands and metamyelo-cytes to be seen. Cells that are more immature are nat-urally larger and may also be counted as monocytes.Storage of blood at room temperature and delay inrunning the sample on the analyzer may also contrib-ute to inaccurate WBC differential values.</p><p>When differential counts obtained by automatedanalyzers are compared to differential counts per-formed manually, differences in results are relativelyfrequent. Most often, they are clinically inconsequen-tial. However, it is important to correlate significantlyabnormal results with morphological review of theperipheral smear.</p><p>ERRORS IN PLATELET COUNT</p><p>In certain situations, hematology analyzers areknown to provide a falsely low platelet count whenthe true platelet count is adequate. This may give riseto suboptimal clinical management.</p><p>Partial clotting of specimen or platelet activationduring venipuncture may cause platelet aggregation.Both mechanisms may lead to low platelet counts.Checking the specimen for clots, analyzing the histo-grams, as well as reviewing the smear are all impor-tant steps to avoid misleading low platelet counts.There are various other mechanisms to explain falselylow platelet counts, otherwise referred to as pseudo-thrombocytopenia: anticoagulant-induced pseudo-thrombocytopenia, platelet satellitism, giant platelets,and cold agglutinin-induced platelet agglutination.</p><p>Falsely elevated platelet counts are much less com-mon than falsely low counts. Fragmented red cells orwhite cell fragments may be counted as platelets, giv-ing rise to high platelet counts. Fragmented red cellscan be seen in states of microangiopathic hemolysissuch as disseminated intravascular coagulation (DIC).White cell fragments can be seen in leukemic or lym-phoma states. Patients with leukemia, especially acuteleukemia, need supportive therapy in the form ofblood component transfusions. If the platelet count isfalsely elevated in a patient with acute leukemia, thedecision to transfuse platelets may be delayed, with</p><p>undesirable clinical consequences. Falsely high plateletcounts may also be seen in the presence of cryoglobu-lins and microorganisms present in blood [7].</p><p>ERRORS IN SPECIFIC HEMATOLOGYTESTING</p><p>In the following sections, specific selected test errorsoften seen in the hematology laboratory are discussedin more detail.</p><p>Cold Agglutinins</p><p>Cold agglutinins are polyclonal or monoclonalautoantibodies directed against RBC i or I antigensand preferentially binding erythrocytes at cold tem-peratures [8]. These autoantibodies are typicallyimmunoglobulin M subtype, which may be associatedwith malignant disorder (e.g., B cell neoplasm) orbenign disorders (e.g., postinfection and collagen vas-cular disease) and can be manifested clinically asautoimmune hemolytic anemia [9]. In the hematologylaboratory with automated analyzers, cold agglutininstypically present as a discrepancy between the RBCindexes [9,10]. The agglutinated erythrocytes may berecognized as single cells or may be too large to becounted as erythrocytes; subsequently measuredmean corpuscular volume is falsely elevated and theRBC count is disproportionately low. Although themeasured hemoglobin is correct due to its indepen-dence of cell count, the calculated indexes are incor-rect: The hematocrit (red cell count3MCV) is low,whereas the MCH (hemoglobin/red cell count) andthe MCHC (hemoglobin/hematocrit) are elevated.Hemagglutination may be grossly visible to theunaided eye [8], and microscopic examination of theperipheral blood smear would show erythrocyteclumping [9]. By rewarming the blood sample to 37C,the erythrocyte agglutination is alleviated and correctvalues may be obtained [9]. More severe cases of coldagglutinin may require saline replacement techniqueif rewarming the sample fails to resolve the RBC indexdiscrepancy.</p><p>Spurious leukopenia due to cold agglutinin is alsooccasionally encountered with automated hematologyanalyzers. The mechanism is postulated to be an IgMautoantibody directed against components of the gran-ulocyte membranes [11]. Cold agglutinin-induced leu-kopenia should be recognized as a potential cause ofpseudogranulocytopenia so that WBC counts can beaccurately reported and unnecessary evaluation ofpatients for leukopenia can be avoided.</p><p>307ERRORS IN SPECIFIC HEMATOLOGY TESTING</p><p>ACCURATE RESULTS IN THE CLINICAL LABORATORY</p></li><li><p>Cryoglobulins</p><p>Cryoglobulins are typically IgM immunoglobulinsthat precipitate at temperatures below 37C, producingaggregates of high molecular weight [8]. The first clueto a diagnosis of cryoglobulinemia is laboratory arti-facts detected in the automated blood cell counts [12].The precipitated cryoglobulins of various sizes mayfalsely be identified as leukocytes or platelets causingpseudoleukocytosis and pseudothrombocytosis. At thesame time, the RBC indexes are generally unaffected.Correction of the artifacts for automated counts can beobtained by warming the blood to 37C or by keepingthe blood at 37C from the time of collection to thetime of testing. Peripheral blood smear typically showsslightly basophilic extracellular material, and leukocytecytoplasmic inclusions are occasionally found.</p><p>Pseudothrombocytopenia</p><p>Pseudothrombocytopenia is caused by various etiolo-gies, including giant platelets [13], anticoagulant-inducedpseudothrombocytopenia [14], platelet satellitism [15,16],and cold agglutinin-induced platelet agglutination [17].Regarding giant platelets, due to their large size, they areexcluded from electronic platelet counting causing pseu-dothrombocytopenia [13]. This scenario is of particularclinical importance in patients with rapid consumptionof platelets in the peripheral circulation, such as i...</p></li></ul>


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