accurate results in the clinical laboratory || sources of errors in hematology and coagulation...
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C H A P T E R
Sources of Errors in Hematology andCoagulation TestingAndy Nguyen, Amer Wahed
University of Texas Health Sciences Center at Houston, Houston, Texas
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 .
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 . 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 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.
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.
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 , patients on parenteralnutrition , 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 . 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 .
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 cl