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Sources of Errors in Hematology andCoagulation TestingAndy Nguyen, Amer Wahed

University of Texas Health Sciences Center at Houston, Houston, Texas

INTRODUCTION

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].

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

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 40�250 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.

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.

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:

Hematocrit5MCV3RBC countMCH5hemoglobin=RBC countMCHC5hemoglobin=hematocrit

305Accurate Results in the Clinical Laboratory.

DOI: http://dx.doi.org/10.1016/B978-0-12-415783-5.00019-0 © 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-415783-5.00019-0

ERRORS IN HEMOGLOBINMEASUREMENTAND RBC COUNT

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.

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.

ERRORS IN MCVAND RELATEDMEASUREMENTS

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.

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.

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.

ERRORS IN WBC COUNTS AND WBCDIFFERENTIAL COUNTS

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

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].

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.

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.

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.

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.

306 19. SOURCES OF ERRORS IN HEMATOLOGY AND COAGULATION TESTING

ACCURATE RESULTS IN THE CLINICAL LABORATORY

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.

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.

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.

ERRORS IN PLATELET COUNT

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.

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.

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

undesirable clinical consequences. Falsely high plateletcounts may also be seen in the presence of cryoglobu-lins and microorganisms present in blood [7].

ERRORS IN SPECIFIC HEMATOLOGYTESTING

In the following sections, specific selected test errorsoften seen in the hematology laboratory are discussedin more detail.

Cold Agglutinins

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 37�C,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.

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.

307ERRORS IN SPECIFIC HEMATOLOGY TESTING


Cryoglobulins

Cryoglobulins are typically IgM immunoglobulinsthat precipitate at temperatures below 37�C, 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 37�C or by keepingthe blood at 37�C from the time of collection to thetime of testing. Peripheral blood smear typically showsslightly basophilic extracellular material, and leukocytecytoplasmic inclusions are occasionally found.

Pseudothrombocytopenia

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 in DIC,acute immune thrombocytopenic purpura, or thromboticthrombocytopenic purpura. Effective platelet productionby bone marrow in these cases will present with manylarge platelets in peripheral blood, many of which maynot be identified by automated analyzers. An accurateplatelet count can be obtained with a manual count usingphase contrast microscopy.

Anticoagulant-induced pseudothrombocytopenia isan in vitro platelet agglutination phenomenon generallyseen in specimens collected into EDTA [14]. It has beenreported both in healthy subjects and in patients withvarious diseases (including collagen vascular diseaseand neoplasm) [12], and it has an overall incidence ofapproximately 0.1% [13,18]. Although the agglutinationis most pronounced with EDTA, it may occasionallyoccur with other anticoagulants, such as heparin, cit-rate, and oxalate [14]. Because the platelet aggregatesare large, the automated hematology counters do notrecognize them as platelets, leading to lower plateletcounts [14]. In some cases, the aggregates are largeenough to be counted as leukocytes by automatedinstruments, causing a concomitant pseudoleukocytosis[14]. The platelet aggregation in pseudothrombocytope-nia is usually temperature-sensitive [14], with maximalactivity at room temperature. The EDTA-induced pseu-dothrombocytopenia is mediated by autoantibodies of

IgG, IgM, and IgA subclasses [19] directed at an epi-tope on glycoprotein IIb [20]. This epitope is normallyhidden in the membrane GP IIb/IIIa due to ionized cal-cium maintaining the heterodimeric structure of the GPIIb/IIIa complex [20]. Through its calcium chelatingeffect, EDTA dissociates the GP IIb/IIIa complex withGP IIb epitope exposure [20]. It has been noted that inGlanzmann’s thrombasthenia, a disorder characterizedby the quantitative and/or qualitative abnormality ofglycoprotein IIb/IIIa, pseudothrombocytopenia doesnot occur [20]. Interestingly, in recent years, Abciximab(a GP IIb/IIIa antagonist) has been found to be associ-ated with pseudothrombocytopenia [21]. Ifanticoagulant-induced pseudothrombocytopenia is sus-pected, a peripheral blood smear should be examinedfor platelet clumping [14].

Platelet satellitism has features similar toanticoagulant-induced pseudothrombocytopenia. Inthe presence of EDTA, platelets bind to leukocytes andform rosettes [15,16]. The binding is usually to neutro-phils [15], but binding to other leukocytes has alsobeen reported [16]. The automated analyzers do notidentify platelets that bind to leukocytes, resulting inpseudothrombocytopenia. Platelet satellitism is medi-ated by autoantibodies of IgG type directed at GP IIb/IIIa on the platelet membrane and to an Fcγ receptorIII on the neutrophil membrane [22].

Platelet agglutination due to cold agglutinins caus-ing pseudothrombocytopenia is a rare condition. Theplatelet agglutination is anticoagulant-independent,usually occurs at 4�C, and is mediated by IgM autoan-tibodies directed against GP IIb/IIIa [17]. Becausethese autoantibodies have little activity at temperaturesabove 30�C, they are not associated with any clinicalsignificance [1].

Spurious Leukocytosis

The presence of microorganisms in the peripheralblood can result in spuriously high WBC counts or dif-ferentials by automated analyzers. Organisms thathave been shown to be associated with this artifactinclude Histoplasma capsulatum, Candida sp., Plasmodiumsp., and Staphylococcus sp. [23]. Spurious leukopeniadue to EDTA is sometimes encountered [24,25].Leukoagglutination has been reported as a transientphenomenon in neoplasia (especially lymphoma),infections (infectious mononucleosis, acute bacterialinfection, etc.), alcoholic liver diseases, and autoim-mune diseases (rheumatoid arthritis, etc.). It can alsooccur in the absence of any obvious underlying disease,even though an inflammatory condition is often found.Other well-known EDTA-dependent counting errorsare platelet clumps and platelet-to-neutrophil



satellitism. Association of neutrophil clumping andplatelet satellitism has also been observed [26].

False-Positive Osmotic Fragility Test

The osmotic fragility test is useful for diagnosis ofhereditary spherocytic hemolytic anemia [27].Spherocytes are osmotically fragile cells that rupturemore easily in a hypotonic solution than do normalRBCs. Because they have a low surface area:volumeratio, they lyse at a higher solution osmolarity than donormal RBCs with discoid morphology. After incuba-tion in a hypotonic solution, a further increase inhemolysis is typically seen in hereditary spherocytosis.Cells that have a larger surface area:volume ratio, suchas target cells or hypochromic cells, are more resistantto lysing in a hypotonic solution.

Conditions associated with immunologically medi-ated hemolytic anemias may present with many micro-spherocytes in peripheral blood. Consequently, thefragility test can be positive in immunologically medi-ated hemolytic anemias other than hereditary sphero-cytosis, but the former would have a positive directCoombs test and the latter would not.

Errors Related to Sample Collection, Transport,and Storage

EDTA is the typical anticoagulant used in blood col-lection tubes. It can be in a dry format or as a solution.The amount and concentration of EDTA require thatblood should be collected up to a specific mark on thetube. If too little blood is collected, dilution of the sam-ple can become an issue with alteration of parameters.Relative excess EDTA in such cases also affects themorphology of blood cells. Transport of specimenshould ensure that high temperatures are avoided. Redcell fragmentation is a feature of excess heat [28].

Prolonged storage will result in degenerativechanges in WBCs. This is best illustrated in neutrophils,in which WBCs have a round pyknotic nucleus. To thecasual observer, these cells may appear as nucleatedred cells. Abnormal lobulation of the lymphocyte nucleiis another established phenomenon with prolongedstorage of blood [29]. These cells may be considered asatypical lymphocytes, with an incorrect implication ofan underlying lymphoproliferative disorder.

Table 19.1 summarizes sources of laboratory errorsin hematology testings.

COAGULATION TESTING

Patients with coagulation disorders may eitherbleed or form thromboses. Hemostasis involves

TABLE 19.1 Sources of Laboratory Errors in Hematology

Falsely high hemoglobin

Turbid sample (hyperlipidemia, parenteral nutrition,hypergammaglobulinemia, cryoglobulinemia, marked leukocytosis)Smokers (high caboxyhemoglobin)

Falsely low hemoglobin

Rare

Falsely high RBC count

Large plateletsRed cell fragments

Falsely low RBC count

Cold agglutinin

Falsely high MCV

Cold agglutininHyperosmolar state (uncontrolled diabetes mellitus)

Falsely low hemoglobin

Large plateletsHypoosmolar state

Falsely high WBC count

Nucleated red cellsNonlysis of red cells (due to target cells in hemoglobinopathy)Giant platelets or platelet clumps (due to EDTA)CryoglobulinsMicroorganisms

Falsely low WBC count

Leukoagglutination (due to EDTA)Cold agglutinin

Falsely increased lymphocyte count

Nucleated red cellsNonlysis of red cells (due to target cells in hemoglobinopathy)Giant platelets or platelet clumps (due to EDTA)Malarial parasitesDysplastic neutrophils (hypolobated neutrophils)Basophilia

Falsely decreased lymphocyte count

Rare

Falsely high neutrophil count

Rare

Falsely low neutrophil count

Neutrophil aggregationNeutrophil with hemosiderin granules (counted as eosinophils)

Falsely increased eosinophil count

Neutrophils with hemosiderin granules (counted as eosinophils)Red cells with malarial pigments

Falsely low eosinophil count

Hypogranular eosinophils

(Continued)

309COAGULATION TESTING


activation of the clotting factors and platelets [30].Evaluation of platelet events may include a CBC,examination of the peripheral smear, bleeding time,and platelet aggregation test. Evaluation of the clottingfactors is typically done by partial prothrombin time(PT) and activated partial thromboplastin time (aPTT)measurements. Abnormal PT or aPTT will usually leadto mixing studies to determine whether the abnormalresult is due to factor deficiency or inhibitors. If thereis correction of the prolonged clotting time in the mix-ing study, then factor assays will be performed toidentify the deficient factor(s). Inhibitors includespecific clotting factor inhibitors as well as lupus anti-coagulants. Inhibitor screen and inhibitor assays orconfirmatory tests for lupus anticoagulants will follow.Blood samples for coagulation tests are typicallyobtained in tubes with sodium citrate buffer. Thereare various sources of erroneous test results in coagu-lation testing, and these are addressed in the followingsections.

ERRORS IN PTAND aPTTMEASUREMENTS

PT measures the time required for a fibrin clot toform after addition of tissue thromboplastin and cal-cium to platelet-poor plasma collected in a citrated

tube. PT measures the activity of VII, X, V, II, andfibrinogen. If aPTT is normal, then a prolonged PT isdue to factor VII deficiency. PT is relatively insensitiveto minor reductions in the clotting factors. aPTT is pro-longed with deficiencies of XII, XI, X, IX, VIII, V, II,and fibrinogen. Just like PT, aPTT can be normal inminor deficiencies. In general, the deficient factor hasto be approximately 20�40% to cause a prolongedaPTT. Most laboratories use automated methods forPT and aPTT measurements. Either optical or mechani-cal methods are employed to monitor clot formation. Ifmeasured with optical methods, shortened times maybe seen with turbid plasma (e.g., hyperlipidemia andhyperbilirubinemia). aPTT is a test conventionallyused to monitor heparin therapy. It is important toproperly separate plasma from platelets as soon aspossible. Platelet factor 4 can neutralize heparin, thusspuriously reducing aPTT values. Factor VIII levels arereflected in aPTT measurements. Factor VIII is an acutephase reactant. Again, if aPTT is being used to monitorheparin therapy in a patient who has an underlyingcause for acute phase reactants to be elevated, aPTTvalues may be falsely lower than expected.

ERRORS IN THROMBIN TIMEMEASUREMENT

Thrombin time (TT) measures the time to convertfibrinogen to fibrin. Dysfibrinogenemia, elevated levelsof fibrin degradation product, and paraproteins caninterfere with fibrin polymerization, thus falselyprolonging TT. Amyloidosis can inhibit the conversionof fibrinogen to fibrin, also prolonging TT. In certainmalignancies, heparin-like anticoagulants have beenknown to be the cause of prolonged TT. Next, specificselected test errors that are often seen in the coagula-tion laboratory are discussed in more detail.

Incorrectly Filled Tubes

Citrate tubes for coagulation tests are designed for a9:1 ratio of blood to citrate buffer. Both underfillingand overfilling of the citrate tube result in imbalancesin this blood-to-buffer ratio and produce artificiallyprolonged or shortened clotting times, respectively[31]. Both underfilling and overfilling result in too littleor too much blood sample for fixed amount of antico-agulant in the tube, respectively. The amount of bloodthat fills the citrated tubes is controlled by vacuum,which maintains the proper 9:1 ratio of blood to antico-agulant [32]. Underfilling may be caused by air bub-bles in the tube, vacuum loss, or not allowing the tubeto completely fill during the blood collection process.

TABLE 19.1 (Continued)

Falsely increased monocyte count

Large reactive lymphocytesLymphoblastsLymphoma cellsImmature granulocytes

Falsely low monocyte count

Rare

Falsely high platelet count

Fragmented red cells (in microangiopathic hemolysis)Fragmented white cells (in leukemia)MicroorganismsCryoglobulin

Falsely low platelet counts

Partial clotting or platelet activationGiant plateletsPlatelet clumps and platelet satellitism (due to EDTA)GP IIb/IIIa antagonistsPlatelet agglutination (due to cold agglutinin)

False-positive fragility test

Immunologically mediated hemolytic anemias

MCV, mean corpuscular volume.



If the tube stopper is removed, it also becomes difficultto obtain the correct amount of blood and attain theproper 9:1 ratio. If the patient’s hematocrit is known inadvance of blood collection to be greater than 55%(e.g., in patients with polycythemia) or below 21% (e.g.,in patients with severe anemia), the amount of sodiumcitrate must be adjusted using the following formula:

C5 0:001853 100 Hð Þ3V

where C is the volume of 3.2% sodium citrate inmilliliters, H is the hematocrit in percentage, and V isthe volume of blood in milliliters.

Errors in clotting tests due to hematocrit changeswithout adjustment in the citrate volume are most sig-nificant with elevated hematocrits because even severeanemia does not significantly change PT or aPTT.

Dilution or Contamination with Anticoagulants

Blood collection from indwelling lines or catheterscould be a potential source of testing error. Sampledilution from incomplete flushing or hemolysis causedby improper catheter insertion can alter coagulationtest results. Heparinized lines should be avoided ifblood must be drawn from an indwelling catheter. Ifusing a heparinized line is absolutely necessary, ade-quate line flushing must be achieved before blood col-lection. The National Committee for ClinicalLaboratory Standards recommends flushing lines with5 mL of saline [33]. At least 5 mL or six times the deadspace volume of the catheter should be discardedbefore blood collection. The Intravenous NursingStandards of Practice recommends that manufacturers’instructions should always be followed regarding theappropriate discard volume. These guidelines alsostate that blood should not be acquired from varioustypes of indwelling cannula, venous administrationsets, and indwelling cardiovascular or umbilical lines[34]. Even when the initial volume drawn is discardedbefore blood collection according to these guidelines,specimens drawn from a heparinized line are still eas-ily contaminated with heparin. Consequently, bloodfor coagulation tests should be drawn directly from aperipheral vein, avoiding the arm in which heparin,hirudin, or argatroban is being infused for therapy[30]. Before coagulation testing, heparin may beremoved or neutralized with polybrene in the coagula-tion laboratory; however, residual heparin may con-tinue to cause testing interferences [35].

By enhancing antithrombin activity, heparin inhibitsactivated factors II (thrombin), X, IX, XI, XII, and kalli-krein. In contrast, lepirudin, danaparoid, and argatro-ban inhibit only activated factor II [30]. Theseanticoagulants (heparin, lepirudin, and argatroban)

prolong aPTT and interfere with coagulation tests suchas factor assays and lupus anticoagulant assays. Factorassays may yield falsely low levels, whereas lupusanticoagulant may be falsely positive.

Traumatic Phlebotomy

Traumatic phlebotomy can result in artificiallyshortened coagulation results such as PT and aPTT.This is due to excessive activation of coagulation fac-tors and platelets by release of tissue thromboplastinfrom endothelial cells [36]. A proper free-flowingpuncture technique will avoid this release of tissuethromboplastin and avoid this artifact.

Fibrinolysis Products and Rheumatoid Factor

Fibrinolysis is mediated by plasmin, whichdegrades fibrin clots into D-dimers and fibrin degrada-tion products. Plasmin also degrades intact fibrinogen,generating fibrinogen degradation products. Fibrindegradation products and fibrinogen degradation pro-ducts are collectively known as fibrin/fibrinogen deg-radation products (FDPs) or fibrin/fibrinogen splitproducts (FSPs). Assays for D-dimer and FDPs aresemiquantitative or quantitative immunoassays.

Latex Agglutination

Patient plasma is mixed with latex particles that arecoated with monoclonal anti-FDP antibodies [37]. IfFDP is present in the patient plasma, the latex particlesagglutinate as FDP binds to the antibodies on the latexparticles. These agglutinated clumps are detected visu-ally. Various dilutions of patient plasma can be testedto provide a semiquantitative result known as FDPtiter. Latex agglutination assays are also available forD-dimers. Various automated and quantitative ver-sions of this assay are commercially available forD-dimers in which the agglutination is detected turbi-dimetrically by a coagulation analyzer rather thanvisually by a technologist [38,39].

Enzyme-Linked Immunosorbent Assays

Quantitative enzyme-linked immunosorbent assays(ELISAs) are also available for FDPs and D-dimers.The traditional ELISA method is accurate but is notuseful due to long analytical time. An automated,rapid ELISA for D-dimers is also available (VIDAS,bioMerieux) [40�42].

One of the most important limitations of D-dimerand FDP assays is interference by high rheumatoid fac-tor levels. This may cause false-positive results withalmost all available assays. The most useful clue todetect this interference is evaluation of the DIC panel.

311ERRORS IN THROMBIN TIME MEASUREMENT


If all values in this panel (PT, aPTT, TT, and fibrino-gen) are normal except for FDP or D-dimer, the pres-ence of rheumatoid factor is most likely the cause.

PLATELET AGGREGATION TESTINGWITH LIPEMIC, HEMOLYZED, ORTHROMBOCYTOPENIC SAMPLES

Platelet aggregation measures the ability of plateletsto adhere to one another and form the hemostaticplug, which is the key component of primary hemosta-sis [30]. It can be performed using either platelet-richplasma or whole blood. Substances such as collagen,ristocetin, arachidonic acid, adenosine 50-diphosphate,epinephrine, and thrombin can stimulate platelets andhence induce aggregation. Response to these aggregat-ing agents (known as agonists) provides a diagnosticpattern for different disorders of platelet function.Measurement of aggregation response is typicallybased on changes in the optical density of the sample.

Platelet aggregation is affected by a number of con-founding variables. Lipemic and hemolyzed samplescomplicate aggregation measurements because theyobscure spectral changes due to platelet aggregation.Thrombocytopenia also makes platelet aggregationevaluations difficult to interpret because a low plateletcount by itself may yield an abnormal aggregationpattern.

CHALLENGES IN ANTICOAGULANTSAND LUPUS ANTICOAGULANT TESTS

The International Society on Thrombosis andHaemostasis Scientific Subcommittee on LupusAnticoagulant recommended two sensitive screeningtests for lupus anticoagulants that assess different com-ponents of the coagulation pathway: clotting time-based assays, such as the dilute Russell viper venomtime (DRVVT), and aPTT-based assays, such as kaolinclotting time and dilute prothrombin time (tissuethromboplastin inhibition test) [43]. Lupus anticoagu-lants prolong various phospholipid-dependent clottingtimes in the laboratory because they bind to phospho-lipid and thereby interfere with the ability of phospho-lipid to serve its essential co-factor function in thecoagulation cascade. Lupus anticoagulant screeningassays usually have a low concentration of phospho-lipid to enhance sensitivity. Any abnormal (prolonged)screening result typically requires a 1:1 mixing study inwhich the patient plasma is mixed with one equal vol-ume of normal plasma to demonstrate that the clottingtime remains prolonged upon mixing. Confirmatoryassays are performed if the screening assay remains

abnormal after the 1:1 mixing. Confirmatory assaystypically demonstrate that upon addition of excessphospholipid, the clotting time shortens toward nor-mal. The platelet neutralization procedure is a confir-matory assay in which the source of the excessphospholipid is freeze�thawed platelets. The hexago-nal phospholipid neutralization procedure is also basedon the same principle—that is, the clotting timebecomes corrected after addition of phospholipid inhexagonal phase. Note that aPTT may or may not beprolonged, depending on the amount of phospholipidin the reagent.

In many lupus anticoagulant assays, heparin(including subcutaneous low-dose heparin) may causefalse-positive lupus anticoagulant results. By enhanc-ing antithrombin activity, heparin inhibits activatedfactors II (thrombin), X, IX, XI, XII, and kallikrein.Subsequently, clotting times such as PT and aPTT areprolonged and interfere with lupus anticoagulantassays. Lepirudin, danaparoid, and argatroban inhibitactivated factor II and can also prolong clotting times.Before coagulation testing, heparin may be removed orneutralized with polybrene in the coagulation labora-tory; however, residual heparin may continue to causetesting interferences [44]. Results for lupus anticoagu-lant assays can be interpreted correctly in patients onCoumadin. Table 19.2 summarizes important labora-tory coagulation errors due to various entities.

CASE STUDIES

Two case studies highlight the issues discussed inthis chapter.

Case Study 1

A cardiac surgeon was following up on his patientduring the first week of surgery. A CBC was done toassess current hematologic parameters. The hemoglo-bin level was acceptable at 12.5 g/dL. However, theRBC count was low at 2.9 million/mm3 of blood. Thevalues for MCV and MCH were also high. The sur-geon was naturally concerned with the low RBC countand found the values for hemoglobin and RBC countdiscrepant. He called the pathologist to discuss thefindings. The pathologist reviewed the smear andfound red cell agglutination. Red cell agglutination canbe seen in cold hemagglutinin disease. Because hemo-globin levels are measured after lysing red cells,whether the red cells are agglutinated or not does notmatter. However, with red cell agglutination, the totalRBC count would be reduced. The MCV and MCHvalues would be falsely high. The high MCH value



should have been an indication for the lab technologistto preview the smear and to warm the blood prior to arepeat CBC on the hematology analyzer.

Case Study 2

A 52-year-old female who has long-standing rheu-matoid arthritis is under the care of an oncologist dueto a recently diagnosed soft tissue sarcoma. Theoncologist is concerned about chronic DIC and decidesto evaluate her. Her CBC results show thrombocytope-nia. Her PT and aPTT results are within normal limits.However, her D-dimer values are elevated. The

oncologist calls the pathologist to discuss the findingsin this case. The pathologist reviews her peripheralsmear and observes platelet clumping. The pathologistexplains that this phenomenon may be seen especiallywith samples collected in EDTA tubes. Recollectionin heparin or sodium citrate tubes should result inan accurate and higher platelet count. Rheumatoidfactor is an example of a false-positive D-dimer test.Ultimately, it was proven that this patient does nothave DIC.

CONCLUSIONS

In this chapter, common tests performed in thehematology and coagulation section of the laboratorywere discussed. Sources of errors can potentiallyinclude all steps in testing—collection of samples,transportation, storage, and methodology used—aswell as intercurrent issues of the patients. It is impera-tive to follow procedures and protocols for all con-cerned to attempt to obtain meaningful, accuratevalues. Laboratory technologists and pathologists needto be aware of situations in which erroneous resultsmay be obtained. Correlation with clinical informationprovided or from the medical records is required incertain situations. If aware of issues related to possibleerroneous results, clinicians will also contribute to pro-viding appropriate interpretation of laboratory results.In essence, it is a team effort of laboratory personneland clinicians to provide an accurate interpretation oflaboratory tests for better clinical decisions and patientmanagement.

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TABLE 19.2 Sources of Laboratory Errors in Coagulation

Falsely prolonged clotting times

Underfilling of citrate tubePolycythemiaSample from indwelling catheters (dilution or contamination withanticoagulant)

Falsely shortened clotting times

Overfilling of citrate tubeTraumatic phlebotomyTurbid plasma (e.g., hyperlipidemia, hyperbilirubinemia) in opticalinstrument

Falsely shortened aPTT in patients on heparin

Delay in separation of plasma from plateletsElevated factor VIII (acute phase reactant)

Falsely prolonged TT

DysfibrinogenemiaElevated levels of FDPs and paraproteinsAmyloidosisHeparin-like anticoagulants (in malignancy)

Falsely high FDPs and D-dimer

Rheumatoid factor

Falsely abnormal platelet function

LipidemiaHemolysisThrombocytopenia

Falsely low factor levels

HeparinLepirudinDanaparoidArgatroban

False-positive results of lupus anticoagulant tests

HeparinLepirudinDanaparoidArgatroban

aPTT, activated partial thromboplastin time; FDPs, fibrin/fibrinogen

degradation products; TT, thrombin time.

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