令人吃惊的血常规结果之红细胞与白细胞

M.ZANDECKI,F.GENEVIEVE,J.GERARD,A.GODON

Haematology Laboratory, University Hospital of Angers, Angers,France Correspondence:

M.Zandecki,Haematology Labor-atory,University Hospital of Angers, 4,rue Larrey,49000Angers, France.Tel.:+33241355353;Fax: +33241355599;E-mail: mazandecki@chu-angers.fr

doi:10.1111/j.1365-2257.2006.00871.x Received30January2006; accepted for publication30July 2006

Keywords

Haematology analysers,automated count,cell blood count,spurious count,white blood cells,haemo-globin,red blood cells,mean cell volume SUMMARY

Haematology analysers provide quick and accurate results in most situations.However,spurious results,related either to platelets(part I of this report)or to other parameters from the cell blood count(CBC) may be observed in several instances.Spuriously low white blood cell (WBC)counts may be observed because of agglutination in the presence of ethylenediamine tetra-acetic acid(EDTA).Cryoglobulins, lipids,insufficiently lysed red blood cells(RBC),erythroblasts and platelet aggregates are common situations increasing WBC counts.In most of these instancesflagging and/or an abnormal WBC differential scattergram will alert the operator.Several situations lead to abnormal haemoglobin measurement or to abnormal RBC count,including lipids,agglutinins,cryoglobulins and elevated WBC counts.Mean(red) cell volume(MCV)may be also subject to spurious determination, because of agglutinins,excess of glucose or salts and technological considerations.In turn,abnormality related to one measured param-eter will lead to abnormal calculated RBC indices:mean cell haemo-globin content(MCHC)is certainly the most important RBC indices to consider,as it is as important asflags generated by the haematology analysers(HA)in alerting the user to a spurious result.In many circumstances,several of the measured parameters from CBC may be altered,and the discovery of a spurious change on one parameter frequently means that the validity of other parameters should be considered.Sensitiveflags now allow the identification of several spurious counts,but only the most sophisticated HA have optimal flagging and more simple HA,especially those without a WBC differential scattergram,do not possess the same sensitivity for detecting anomalous results.Reticulocytes are integrated now into the CBC in many HA,and several situations may lead to abnormal counts.

INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

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Journal compilationÓ2007Blackwell Publishing Ltd,Int.Jnl.Lab.Hem.2007,29,21–4121INTRODUCTION

Many haematology analysers(HA)that enumerate WBC generate a WBC differential scattergram,which is the basis for the automated WBC differential.Such WBC scattergrams are also pivotal for the generation offlags or alarms,detecting that the WBC count(and the WBC differential)is possibly erroneous and giving some information about the possible origin of the anomaly.As mentioned in part I of this report,the WBC differential scattergram is also of crucial import-ance to identify anomalies of platelet(PLT)counting, PLT clumps being the most obvious example.

Red blood cell(RBC)counts,haemoglobin(Hb), and mean(Red)cell volume(MCV)are other parameters measured by HA,which are also subject to the spurious values in several situations.Red cell (Wintrobe)indices other than MCV and packed cell volume(haematocrit;Hct)are usually calculated from these measured parameters,and in most cir-cumstances erroneous directly measured RBC param-eters will subsequently cause in turn erroneous calculated RBC indices.These indices can be used as aflag for abnormal counts.Reticulocytes may be considered now as a part of the CBC,and many HA have integrated reticulocyte counts under a fully automatized method.

WHITE BLOOD CELLS

For blood cell analysis,each large sized particle (greater than the size of a PLT)that is not destroyed by haemolytic agents will be identified as a WBC on most HA.After enumeration,and according to the type,impedance with low-and high-frequency elec-tromagnetic or direct current,laser light scattering(at one or at various angles),or peroxidase staining intensity are used,either individually or together,to generate afive-,six-,or even seven-part differential (for review,see Bain&Bates,2001).It is not in the scope of this report to study how the various WBC are classified but it must be kept in mind that scatter-grams generated by the HA to display the WBC differ-ential must be fully understood by operators(Bain& Bates,2001).In many instances,WBC scattergrams allow the detection of abnormalities related to spur-ious counts and/or help to explain them.As a rule, instruments that do not generate WBC differential scattergrams will be most prone to overlook several factitious counts.

Spuriously low WBC counts Polymorphonuclear neutrophil aggregates

Polymorphonuclear neutrophil(PMN)aggregates may be observed on blood samples drawn into EDTA.The incidence is low but certainly underestimated,corre-sponding to2/65000full blood counts in USA (Epstein&Kruskall,1988),1/9500in Italy(Bizzaro, 1993),1/7500in France(Lesesve et al.,2000).Both male and female show the anomaly.Although no pathology or no specific disease is associated with clustering of PMN,an acute or chronic inflammatory context(Luke,Koepke&Siegel,1971;Savage,19; Kahlil,1991;Robbins,Conly&Oettinger,1991; Imbing et al.,1996;Lesesve et al.,2000),liver diseases (Epstein&Kruskall,1988;Savage,19;Kobayashi et al.,1991;Vinatier et al.,1994;Imbing et al.,1996), or circumstances associated with the generation of cold agglutinins(Guibaud,Plumet-Leger&Frobert, 1983;Epstein&Kruskall,1988;Robbins,Conly& Oettinger,1991;Deol,Hernandez&Pierre,1995; Imbing et al.,1996;Lesesve et al.,2000)have been reported in many instances.The phenomenon may or may not be transitory(Antonsen&Beyer,19;Bizz-aro,1993;Schinella,Kojikara&Curci,1995).The decrease may be either moderate or clinically signifi-cant,leading at times to suspect agranulocytosis and to generate unneeded investigations(bone marrow aspiration or biopsy)or therapy(antibiotics;Epstein& Kruskall,1988;Vinatier et al.,1994;Schinella,Kojikara &Curci,1995).There is no relationship between the phenomenon described here,which is a pure in vitro anomaly related to sampling in EDTA anticoagulant, and the in vivo anomaly occurring in diseases such as adult respiratory distress syndrome or leukostasis,in which PMN tend to aggregate as a consequence of membrane interactions with complement(Jacob et al., 1980).

The mechanism leading to agglutination is not fully elucidated(Carr et al.,1996).It is always an in vitro phenomenon,mainly EDTA dependent(Epstein &Kruskall,1988),although agglutination after the use of sodium citrate or heparin as anticoagulants has also been reported in some instances(Rohr&

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Journal compilationÓ2007Blackwell Publishing Ltd,Int.Jnl.Lab.Hem.2007,29,21–41 22M.ZANDECKI ET AL.SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERSRivers,1990;Robbins,Conly&Oettinger,1991;Vin-atier et al.,1994;Deol,Hernandez&Pierre,1995; Carr et al.,1996).Using transfer experiments,either plasma from the affected patients in contact with WBC from normal patients or normal plasma in con-tact with PMN from the affected patients,the anom-aly has proved to be related to a plasma component, and incubation of plasma with an anti-IgM antibody or with dithiothreitol abolished or quantitatively decreased PMN aggregation(Bizzaro,1993).Using flow cytometry,IgM was found on the PMN surface in a patient(Carr et al.,1996).Some authors have reported that the size of the aggregates was larger at low temperature and that they disappeared at37°C (Bizzaro,1993;Carr et al.,1996).Several other authors reported that WBC agglutination in EDTA-anticoagulated blood was not corrected by warming

the EDTA blood sample,at least in some cases,min-imizing the implication of a cold agglutinin in the genesis of the clusters(Robbins,Conly&Oettinger, 1991;Vinatier et al.,1994;Deol,Hernandez&Pierre, 1996).A high level of integrin expression(CD11b–CD18)on PMN membrane was proposed to be rela-ted to the generation of PMN clumps(Galifiet al., 1993).

White blood cell differential scattergrams(using either size and complexity or size and peroxidase con-tent)may demonstrate anomalies,but it must be kept in mind that the largest aggregates are overlooked, and only the smallest will triggerflagging.On imped-ance-type HA,the anomaly is suspected when events are present above the area of PMN(top of the WBC graph),or when it is difficult to separate WBC classes (Galifiet al.,1993),and the most frequentflags gener-ated using such HA are‘immature granulocytes’or ‘band cells’(Lippi et al.,1994).On Bayer-Technicon HA,the smallest clusters may appear as a band of dots in the upper right of the WBC differential scattergram: as these aggregates contain many PMN they are reported as peroxidase-rich particles,and an alarm corresponding to high peroxidase content is gener-ated.On some HA,analysis of WBC nuclei is per-formed on a dedicated channel after the use of a drastic solution ensuring lysis of WBC membranes: number of free nuclei is used as a control for WBC count.Discrepancy between WBC count obtained from that channel and that from thefluorescence or peroxidase channel is observed(Bayer,Tarrytown,NY,USA;Abbott,Abbott Park,IL,USA).White blood cell aggregates are destroyed after drastic lysis of WBC membranes(Galifiet al.,1993).Bloodfilms may show small(up tofive cells),moderate(up to50cells),or large(>100cells)clusters of PMN(Figure1).Careful morphological examination of these aggregates shows that a few lymphocytes or monocytes may be at times entrapped within the aggregates(Hillyer,Knopf& Berkman,1990).Immature granulocytes(myelocytes, metamyelocytes)and band cells are not infrequently reported as a part of the clustered cells,and it was proposed that aggregates might develop around myelocytes,whereas PMN alone failed to cluster together(Deol,Hernandez&Pierre,1995).Aggregates of PMN are devoid of PLT,in contrast to PLT–PMN aggregates(discussed in part I).Eventually,as aggre-gation of PMN in the presence of EDTA leads to a reduction in their number,HA may generateflags corresponding to spuriously abnormal WBC differen-tials,e.g.spurious agranulocytosis or spurious lympho-cytosis(Epstein&Kruskall,1988;Vinatier et al.,1994; Schinella,Kojikara&Curci,1995).

Home-made anticoagulants were proposed to over-come the agglutination(Schinella,Kojikara&Curci, 1995).As mentioned above,warming the sample at 37°C may reduce both size and number of clusters in some instances,but full disappearance is far from being a consistentfinding,and that method cannot be proposed to overcome the anomaly.Finger prick and immediate dilution of the blood sample prevents the

agglutination.

Figure1.Aggregate of polymorphonuclear neutroph-ils observed in an infected patient.

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M.ZANDECKI ET AL.SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS23Aggregation of WBC other than polymorphs

in the presence of EDTA

Clusters of normal(nonmalignant)lymphocytes were reported in a patient with urinary tract infection,in a patient with a B-cell lymphoma without bloodstream involvement,in a patient with escarres,and in another one with chronic myelomonocytic leukaemia (Deol,Hernandez&Pierre,1995;Lesesve et al.,2001). Lymphocyte aggregation was also reported to occur in chronic lymphocytic leukaemia patients,spontane-ously when lymphocyte counts are extremely high (>400·109/l;O’Flaherty,Kreutzer&Ward,1978),or not(Bizzaro&Piazza,1991).Aggregates of three to 50lymphoma cells were observed in two cases of Splenic Lymphoma with Villous Lymphocytes(SLVL; Juneja et al.,1992;Imbing et al.,1995)and in one case to non-Hodgkin lymphoma mimicking SLVL (Shelton&Frank,2000).In these situations,the lar-gest aggregates are overlooked by the HA and WBC count is spuriously but variably low.The smallest aggregates may disturb WBC differential and may or may not generate aflag,depending on the type of HA used(Shelton&Frank,2000).In all cases reported so far EDTA was implicated,although small clusters of cells were also observed on heparinized samples drawn as controls(Deol,Hernandez&Pierre,1996) and sodium citrate did not appreciably change the clustering tendency of the lymphocytes in one case (Shelton&Frank,2000).Fingerpricking and immedi-ate dilution of blood seems the best way to avoid aggregates(Lesesve et al.,2001).Heating(37°C)was reported to reduce clumping either partly(Shelton& Frank,2000)or had no significant effect on the size of clumps(Lesesve et al.,2001).As the number of cases reported is low,only hypotheses on the mech-anism(s)leading to lymphocyte agglutination have been proposed,implicating various molecules such as adrenalin(epinephrin),arachidonic acid,or leukotrien B4(Villa et al.,1984;Shelton&Frank,2000).In one instance aggregates involving all WBC classes was reported,in a patient with a long-standing history of alcohol abuse and alcoholic cirrhosis(Savage,19).

Nature and amount of anticoagulant

A decrease in WBC count not related to agglutination was reported in situations corresponding to samples containing excess of K3-EDTA(but not K2-EDTA) anticoagulant,resulting from insufficient blood drawn after vein puncture(Goossens,Van Duppen&Verwil-ghen,1991).

Spuriously high WBC counts

PLT aggregates and large platelets

Pseudoleukocytosis may be secondary to PLT clumps large enough to mimic WBC size(Solanki&Blackburn, 1983;Cornbleet,1983;Savage,1984;Payne&Pierre, 1984;Lombarts&de Kieviet,1988;Schrezenmeier et al.,1995).All modern HA that analyse WBC sub-populations detect this anomaly:PLT clumps are localized as a rocket shaped area of dots at the lower left hand corner of the WBC differential scattergram, and aflag corresponding to inability to discriminate among the WBC categories,namely lymphocytes, is generated(see Figure2,part I).Several authors have reported that HA without WBC differential scattergrams are unable to detect such anomaly (Payne&Pierre,1984;Lombarts&de Kieviet,1988).

Some very large PLT may be encountered in myelo-proliferative and in myelodysplastic disorders,whose size and volume may reach those of WBC and which may be enumerated as WBC:flags are usually gener-ated,that mention the presence of pathological parti-cles that cannot be classified as WBC(usually‘giant PLT’,or‘PLT aggregates’,depending on the HA).

Nucleated red blood cells(NRBC)

They may be found in the blood stream in physiologi-cal(newborns)and in pathological circumstances,and at times they may be much more numerous than WBC.Using HA,these NRBC are in contact with lysis agents that destroy their membrane,leaving nuclei free,the latter being responsible for anomalies.Free NRBC nuclei are usually particles<40–50fl,a size that is superimposable to that of PLT clumps or large PLT.Unsurprisingly,NRBC and PLT clumps disturb WBC counts identically and both artifacts generate equally the correspondingflags(Figure2).According to the HA,NRBC are either included within the WBC count(instruments that do not generate any WBC dif-ferential scattergram,but also some that do),or not enumerated as WBC,or counted separately.On

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several recent and sophisticated HA,NRBC are specif-ically identified and may be enumerated using fluor-escence technology (Kim et al.,1998;Wang et al.,2003).

Red blood cells resistant to lysis

Red blood cell may not be destroyed by lysis reagents in various circumstances,either physiological (neo-nates)or pathological (abnormal haemoglobins,liver disease,uraemia,chemotherapy):they are considered as WBC and falsely increase the WBC count (Luke,Koepke &Siegel,1971;Cornbleet,1983;Griswold &Champagne,1985;Vinatier et al.,1994;Mellors &McArdle,1995;Elghetany &Hudnall,1996).White blood cell differential scattergrams demonstrate dots of small size located near or within the lymphocyte win-dow,and inability to perform a WBC differential leads to the generation of a flag.The picture is more or less superimposable to that observed with the presence of NRBC.This phenomenon seems to be a consistent finding in haemoglobin C –containing RBC (either CC,C b thal,or CS)but not in b thalassemic patients (either trait or major)or in haemoglobin S disease (Booth &Mead,1983).HA without WBC differential

scattergrams may fail to detect the anomaly,and in patients undergoing chemotherapy it may be of cru-cial importance,as it leads to normal or even elevated WBC counts in otherwise leukopenic patients:com-parison with previous results (delta check)and exami-nation of blood smear are necessary in such instances.Some HA generate a message «RBC resistant to lysis »and give access to an extended lysis mode which lengthens contact of blood cells with the lysis reagent (Abbott CellDyn 4000).On HA using a dedi-cated channel to enumerate WBC nuclei,discrepancy between full WBC count and WBC nuclei count is obvious:the channel analysing WBC nuclei uses lysis agents strong enough to destroy all membranes (WBC and RBC)and gives the accurate WBC count.

Cryoglobulins

The presence of cryoglobulin was first reported as causing erroneous WBC counts (Emori,Bluestone &Goldsberg,1973;Taft et al.,1973;Haeney,1976;Gul-liani,Hyun &Gabaldon,1977),but it also leads in many instances to spuriously elevated PLT counts,and at times to altered RBC counts or Hb measure-ments (cf.,see Fohlen-Walter et al.,2002).

Abnormal-

Figure 2.Nucleated red blood cells (NRBC ¼85·109/l)in a neonate (haemolytic anaemia):the WBC scattergram (right)shows a large number of dots,corresponding to NRBC nuclei,as compared to a normal WBC scattergram (left;Bayer Advia 120).

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M.ZANDECKI ET AL.SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 25

ities that occur on HA are neither consistent nor rela-ted to the amount of cryoglobulin or to its nature (each type may be involved in spurious counts).How-ever,anomalies generated must be considered because they have some peculiar characteristics,and their recognition may be the first clue leading to the diag-nosis of cryoglobulinaemia (Zandecki et al.,19;Foh-len-Walter et al.,2002).Cryoprecipitates may be observed in some instances on stained blood smears (Figure 3),and in nearly all cases after examination of fresh blood sample using a phase contrast microscope (more obvious if performed at low temperature).Var-ious morphological aspects were reported,including dense amorphous clusters or flake-like particles,nee-dle-shaped crystals,and pinkish globules (cf.,see Zan-decki et al.,19).As cryoglobulins are immunoglobulins that precipitate at temperature lower than 37°C,abnormal counts are observed mainly on HA that both use reagents and perform analysis at room temperature.However,HA that use prewarmed reagents,even with low pH reagents that normally avoid precipitation of cryoglobulins,are not free of anomalies and spurious counts are also observed (Fohlen-Walter et al.,2002).According to their size,cryoprecipitates may only affect PLT counts,leading to spuriously elevated ones,up to eight times (Fohlen-Walter et al.,2002):histograms of PLT vol-umes show large number of small-sized particles,and

an alarm is usually generated (see Figure 8,part I).If cryoglobulin particles cluster together or if their size is larger,reaching that of leukocytes,WBC count is spu-riously increased:a cloud of small dots may be observed on the WBC differential scattergram,near the lymphocytes or above the threshold for PLT,or at the top of the PMN cloud,depending on the HA used (Fohlen-Walter et al.,2002).When samples are warmed to 37°C for at least 30min and reanalysed promptly anomalies disappear,but it is not a fully consistent finding and not infrequently a new sample must be taken and kept at 37°C until analysis to obtain accurate CBC counts (Zandecki et al.,19;Infanti et al.,1998;Figure 4).Hb determination and RBC counts may at times be altered in the presence of cryoglobulins (see later).In some situations cryoglob-ulins may form a gel,leading to inaccurate aspiration,and an alarm may be generated on some HA (insuffi-cient sampling,or related).

Cryofibrinogen or related

Particles related to cryofibrinogen or to fibrin have been reported to elevate WBC counts and at times PLT counts and,according to the reports,PLT and WBC counts may be found up to twice and up to 16times the accurate value,respectively (Gulliani,Hyun &Gabaldon,1977;Griswold &Champagne,

1985;

Figure 3.Several morphological aspects of cryoglobulins may be observed on peripheral blood samples,and two among them are shown here:small lucent precipitates scattered around RBC (left),or invisible precipitates that change morphology of RBC into a ‘moth-eaten’aspect (right).

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26M.ZANDECKI ET AL.SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

Corberand et al.,1991).Careful examination of the corresponding blood smears shows thin fibrils,related to fibrin strands after electron microscopic study (Figure 5).These fibrils might correspond either to cryofibrinogen,although biochemical research may be negative in some instances (Griswold &Champagne,1985),or to a mixture of cryoglobulin and cryofibri-nogen (Gulliani,Hyun &Gabaldon,1977).However,analysis of new EDTA blood samples from the patients frequently fail to reproduce the phenomenon,hypothesizing that such particles might be the result of fibrin polymerization after a difficult venepuncture,initiating coagulation in vitro before contact of the blood with EDTA (Corberand et al.,1991).After warming the samples at 37°C abnormal counts are either less pronounced or fully disappear.

Lipids

As previously discussed (part 1),lipids may generate droplets large enough to disturb PLT counts,but also WBC counts,together with or independently of PLT count.Abnormal WBC differential scattergrams are generated,displaying various changes,at times more or less superimposable to those outing from the presence of cryoglobulins (Figure 6).Comments on lipids disturbing Hb determination will be discussed

later.

Figure 5.In some instances (see text),fibrin strands may be observed on peripheral blood smears (MGG staining;left);according to their size they may be enumerated as PLT or/and as WBC.Electron microscopic analysis (right;a)shows such strands intermixed with PLT and WBC (arrows),and inset (right;b)shows peculiar periodic ultra-structure corresponding to fibrin

strands.

Figure 4.Low grade B-cell lymphoma and cryoglobu-lin type I (Ig M Kappa).Analysis at room tempera-ture (20°C;left)shows that WBC count was not performed and that PLT count was inaccurate,and several flags were generated.After warming the

same sample at 37°C and prompt analysis all abnor-malities disappear (37°C;right;STKS II Coulter).

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Microorganisms

Bacteria or fungi may be observed on peripheral blood smears and,as previously discussed (part I),may lead to spuriously elevated PLT counts.They were also reported as disturbing WBC differentials if they clumped together,and in vitro studies adding large number of such microorganisms were shown to increase WBC count (Marshall et al.,1990).

Haemozoin contained in PMN and monocytes in malarial cases may cause depolarization of the laser light and subsequently misclassification of WBC types on some HA (Sysmex,Kobe,Japan),although full WBC count does not clearly change (Huh et al.,2005)

Adipose tissue

Total WBC count was reported as artefactually eleva-ted,caused by contamination with subcutaneous adi-pose tissue of the blood sample obtained by traumatic femoral vein puncture:peculiar scatter plots also dem-onstrated an abnormal WBC differential (Whiteway &Bain,1999).

Overfilling blood collection vacuum tubes

As previously mentioned together with spurious PLT counts,overfilling the collection tube leads to inade-quate sample mixing and setting of cellular contents:all the parameters are altered (Pewarchuk,Vander-Boom &Blajchman,1992).

Concluding remarks on spurious WBC counts

As for spurious PLT counts,it is not always possible to find or to identify the phenomenon that leads to spur-ious WBC counts,even if something is visible on per-ipheral blood smears (Savage,19).The WBC scattergram from HA is useful and must be interpreted carefully;examination of stained films must also be considered.

HAEMOGLOBIN,RBC COUNT AND RBC PARAMETERS

HA measure several RBC parameters,including RBC count and MCV both determined on the same

chan-Figure 6.Lipids in a patient with liver disease.When compared with a normal scattergram (left),lipids generated here a strand of dots elongating PLT region (arrow;Sysmex XE2100).

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Haemoglobin

Hb concentration is measured using a colorimetric (spectrophotometric)method,either by a modification of the manual cyanmethaemoglobin method or by addition of various reagents,such as sodium lauryl sulphate or imidazole.According to the manufactur-ers,various nonionic detergents are included to insure rapid RBC lysis and to reduce turbidity because of cell membranes and plasma lipids.

Lipids and hyperchylomicronemia

Lipemia may cause erroneous PLT(see part I)and WBC counts(see above),but may also induce inter-ferences by turbidity(Creer&Ladenson,1983;Sand-berg,Sonstabo&Christensen,19).An abnormally high MCHC(>36g/dl)corresponding to an errone-ously high Hb has been reported for patients with severe constitutional or acquired hypertryglyceridemia (Gagne et al.,1977;Mayan et al.,1996),and for patients receiving intravenous administration of fat emulsions(Nosanchuck,Roark&Wanser,1974; Shah,Patel&Rao,1975;Nicholls,1975,1977;Creer &Ladenson,1983;Artiss&Zak,1987;Sandberg, Sonstabo&Christensen,19;Cantero,Conejo&Jimenez,1996).After the study of various types of hyperlipoproteinemias,it was observed that erroneous Hb and MCHC>36g/dl were observed in patients with at least20g/l of triglycerids,corresponding to type I and part of type V hyperlipoproteinemias(rich in chylomicrons)but not to type IV hyperlipoprote-inemias(rich in very low density lipoproteins;Gagne et al.,1977).Samples taken after a meal may at times demonstrate superimposable spurious Hb measure-ment.Even the most recent HA are sensitive to hyperlipemia,although to a variable extent,as for example Abbott Cell Dyn4000that is defined as giv-ing true Hb values for up to13and9g/l of triglycerid and cholesterol levels,respectively(M.C.Chrieten, personal communication).Although excess of lipids usually spuriously increases Hb,a spurious fall of Hb was also reported once(Savage,19).Similar to the presence of cold agglutinins,anomaly related to lipids is suspected when MCHC is>36g/dl,or when WBC scattergrams demonstrate high number of particles of low to moderate size(Figure6).For laser-beam HA that measure Hb level within each RBC and generate the so-called«measured MCHC»or CHCM,a differ-ence between the CHCM and the calculated MCHC is observed.Various methods have been proposed to obviate the abnormality on the relevant sample, including isovolumetrical replacement of hyperlipemic plasma with isoosmotic diluent,or ether extraction of lipids.As previously mentioned,such methods may in turn lead to erroneous PLT and WBC counts.

High WBC counts

White blood cell may induce excessive turbidity and disturb Hb measurement if their number is sufficiently high.There is no clear-cut threshold for WBC counts associated with spuriously elevated Hb,but one must be careful with all samples with WBC count over50 or100·109/l(Cornbleet,1983;Sandberg,Sonstabo &Christensen,19),although some HA define threshold at250·109/l or even seem to be fully insensitive to WBC count because of entire WBC lysis performed before Hb measurement(Sysmex).As for lipid disturbance,CHCM determined on some HA may help tofind the true Hb value(McVeigh,Faim& van der Weyden,19).However,RBC count may be also disturbed by high WBC counts(see later),and one must pay attention to the limits in recalculating

Ó2007The AuthorsRBC indices,and a packed cell volume(centrifuged Hct)should be considered.

Immunoglobulins

They have been reported to interfere with a number of clinical laboratory tests(cf.,see Roberts,Fontenot &Lehman,2000).False elevation of Hb measurement by automated methods was observed for several patients with Waldenstro¨m’s macroglobulinemia and monoclonal IgM,or with multiple myeloma and monoclonal IgA or IgG(Wallis&Ford,1987;McMul-lin,Wilkin&Elder,1995;Roberts et al.,2000).This anomaly is related to high levels of Ig that interact with reagents of the lysis solution.For IgM,this phe-nomenon has been related to the amount of mono-meric component within the circulating paraprotein (Goodrick et al.,1993).HA employing Hb conversion to cyanmethaemoglobin seem to be more affected than others,because of addition of surfactants for cyanide-free methods.Red blood cell count and MCV are unaffected in this situation but,as Hb is overesti-mated,MCHC usually exceeds36g/dl.In order to obtain more accurate results in this instance,it was proposed to determine‘plasma Hb’after centrifugation of the sample(which gives turbidity because of the paraprotein)and to withdraw it from Hb of the whole blood(Roberts et al.,2000).For Sysmex instruments, it seems that if the sample is half diluted by the oper-ator before analysis on the HA,the phenomenon does not occur(Roberts et al.,2000).For laser-beam HA that determine the measured MCHC(CHCM)a clear-cut difference with the calculated MCHC generates an alarm,and accurate Hb from the sample may be obtained from measured MCHC.

Cryoglobulins

Spurious Hb values have been reported in some instances,and several mechanisms are proposed to explain these abnormalfindings.In some cases spuri-ously high Hb values were related to a mechanism similar to that described above for immunoglobulins (Taft et al.,1973;Cornbleet,1983)or to the distur-bance of light transmittance,whereas in other cases a slight decrease of both Hb measurement and RBC count was related to aflow anomaly(Taft et al.,1973; Bremmelgaard&Nygard,1991;Fohlen-Walter et al.,2002).However,spurious Hb measurement is far from being a consistentfinding in the presence of cryoglob-ulins(Fohlen-Walter et al.,2002).

Haemolysis

Hb free within plasma is measured together with that from the RBC,but its amount ranges from10to 40mg/l in normal conditions and does not affect total Hb measurement.However,in situations related to major intravascular haemolysis,including chemicals, mechanical haemolysis associated with heart valves, and haemolytic anaemias associated with blood trans-fusion,free plasma Hb may be elevated enough to affect total Hb measurement.MCHC may be>36g/dl. Centrifuged Hct shows a pink or red plasma tinge, namely if free plasma Hb is>200mg/l,and is,in some instances,the only reliable RBC parameter.Some laser-beam HA directly determine Hb within each RBC,the Cellular Haemoglobin Concentration Mean (CHCM),which allows the accurate Hb value to be calculated.A short time lapse between venepuncture and analysis is of crucial importance because haemo-lysis may continue in vitro,leading to a spurious decrease of RBC count and total Hb with a spurious increase of free plasma Hb.

Chemical structure of haemoglobin and bilirubin

In physiological situations,Hb is more or less coupled to oxygen or to carbon-di-oxide and,according to which molecule is coupled to Hb,the peak of optimal light absorbance differs slightly.The addition of several reagents leave Hb free from coupled molecules and changes it into one stable molecule(cyanmethae-moglobin is an example),the latter demonstrating a narrow peak of light absorbance,which allows accu-rate determination of Hb concentration.However, high amounts of carbon monoxide coupled to Hb may not be fully transformed,and in such situations a spuriously high Hb concentration is reported (Cornbleet,1983;Vinatier&Flandrin,1993).In con-trast,sulfhaemoglobin in high amount has been reported as lowering Hb measurement(Cornbleet, 1983).Bilirubin:although one must pay attention to very high amounts of bilirubin within the plasma, most HA do not presently demonstrate any interfer-ence with bilirubin,at least for concentrations up to

Ó2007The Authors250mg/l.Above theses values,however,attention is needed.

Spurious RBC counts and red cell parameters

According to the technology used,spurious results may or may not be observed using some HA,and some knowledge about the methods analysing blood parameters is necessary.On impedance–type HA an aliquot of the blood sample is diluted isoosmotically and the number and height of electric pulses gener-ated by the electrical resistance of RBC that pass through a small orifice allow the determination of both the RBC count and the MCV.Using laser-beam methods,scattering at least at two angles allows the determination of RBC count and MCV.In order to improve accuracy,pretreatment of RBC with a specific reagent that changes them isovolumetrically from dis-coid to a sphere is performed on some HA.Setting dis-crimination thresholds is an important consideration: discriminating the smallest RBC from the largest PLT is at times a challenge,already discussed(part I).On the contrary,even in extreme pathological situations, MCV does not exceed150–160fland,as there is no particle above that size in the blood stream in health or in disease,HA do not analyse any particle above 200–300flin volume.

Spuriously elevated RBC counts

High WBC counts

Most HA enumerate RBC and WBC together within the same channel(s),and the RBC count reported is the sum of both RBC and WBC counts.In physiologi-cal conditions,it is not of any importance,as it corres-ponds to overestimate RBC count by0.1%(if we consider a WBC count of5·109/l and a RBC count of5·1012/l).However,high WBC counts (>100·109/l)may lead to a significant change in the RBC count,particularly if the patient is also anaemic (Bain&Bates,2001).Moreover,in the latter instances the MCV reported corresponds to the mean volume of RBC and of WBC from the sample and may be spurious,variably according to the nature and the number of WBC from the relevant sample.So, high WBC counts may induce several abnormalfind-ings,including Hb(discussed above),RBC count,MCV,and subsequently lead to abnormal calculated MCHC(discussed later).

Giant platelets

High number of giant PLT may lead to spuriously low PLT count(see part1)but,as they are enumerated as RBC,they may also affect RBC count in a way similar to that for WBC;however,RBC count is usually only slightly affected in this instance(Cornbleet,1983; Bain&Bates,2001).

Spuriously decreased RBC counts

Cold agglutinins

Cold agglutinins aggregate RBC when the temperature is lower than37°C.Unsurprisingly,peculiar anomal-ies of RBC parameters in the presence of cold aggluti-nins were reportedfirst on counters acting at room temperature(Hattersley et al.,1971;Petrucci,Duanne &Chapman,1971;Bessman&Banks,1980).Accord-ing to the HA,the upper threshold that may consider particles as RBC into the RBC channel(s)is located between200and300fl.So,only particles correspond-ing either to isolated RBC or to small RBC clumps (two or three RBC),are analysed,whereas large RBC clumps are fully neglected by the HA.This leads to spuriously low RBC counts and to abnormally high MCV(each small RBC clump is considered as one sin-gle particle).Haematocrit(RBC·MCV)is erroneous and spuriously low,contrasting with Hb that is meas-ured after RBC lysis and is unaffected by agglutinins. As a rule,the MCHC is spurious,usually>36g/dl.The peculiar association of low RBC count,high MCV and MCHC>36g/dl is almost pathognomonic of the cold induced artifact on instruments that work at laborat-ory temperature,and not infrequently helps to diag-nose the cold agglutinin in the patient analysed (Petrucci,Duanne&Chapman,1971).HA working with reagents at temperatures near37°C are not fully insensitive to cold agglutinins,however,but changes are less obvious and may remain undiscovered in some instances(Solanki&Blackburn,1985).HA that mea-sure directly the amount of Hb within each RBC(mea-sured MCHC,or CHCM)usually show discrepancy between the measured MCHC and the calculated one. Whatever the HA used,the commonfinding is that Hb

Ó2007The Authorsvalue is unaffected and that anomalies disappear when the sample is warmed at37°C and analysed promptly afterwards.Amplification of the anomalies after the sample has been cooled at4°C for1or2h reinforces the diagnosis.As viscosity of the sample may be high, leading to inaccurate aspiration,an alarm may be generated on some HA(insufficient sampling,or related).Co-existence of RBC agglutination with EDTA-dependant thrombocytopenia was reported but antibodies directed against RBC and those directed against PLT differed(Bizzaro&Fiorin,1999).

Warm autoimmune haemolytic anaemia

In some instances warm autoantibodies were also reported as inducing RBC agglutination,leading to spurious MCV and RBC count,in a situation superim-posable to that observed for cold agglutinins,but not reversible by warming(Weiss&Bessman,1984).

Very small RBC and discrimination with PLT

As discussed in part1,PLT counts may be disturbed by microcytic cells,namely if RBC volume is<36fl: some HA consider particles over this threshold as RBC,and in severe microcytic anaemias PLT count may be altered.Underestimation of RBC counts may be observed in turn,because of the elimination of the smallest erythrocytes from the RBC count.In such instances up to6%lowered RBC counts were repor-ted(Savage et al.,1985).

Cryoglobulins

As previously mentioned,a slight decrease of both Hb measurement and RBC count was reported in a few instances,related to aflow anomaly(Taft et al.,1973; Cornbleet,1983;Bremmelgaard&Nygard,1991;Foh-len-Walter et al.,2002).

Considerations related to the quality of the sample.

Clotting,or abnormal mixing secondary to overfilling of the sample(Pewarchuk et al.,1992),may lead to an aspirated aliquot unrepresentative of the blood sample.Cryoglobulins and cold agglutinins may lead to high viscosity of the sample,leading to inadequate aspiration.In vitro haemolysis

As mentioned above,in some situations,namely hae-molysis related to chemicals or blood transfusion,RBC may continue to lyse within the sample,leading to spuriously low RBC count,together with abnormal Hb measurement and abnormal red cell indices(see Hb measurement).

Mean cell volume and haematocrit

Some situations leading to spurious MCV(see Table1) are already discussed above[high WBC counts,cold (warm)agglutinins].Spurious Hct is a frequentfind-ing,related to abnormal MCV or/and to abnormal RBC count as it is a parameter that is usually calcula-ted by the HA:situations leading to abnormal Hct, either increased or decreased,will not be discussed in details as they may be deducted from MCV changes (Table1).

MCV and the technology used for measurement

Using impedance-type HA,disc-shaped RBC become elongated into a cigar shape as they pass through the aperture.Elongation is more pronounced for particles flowing at the periphery of the orifice than at the cen-tral part,and also when Hb content is low within the RBC.Mean(red)cell volume is spuriously lowered and MCH is spuriously elevated,mainly in hypochro-mic disorders,and for MCV of55fla spurious decrease of up to7flmay be observed(Mohandas et al.,1980;Paterakis et al.,1994;Bain&Bates,2001). Hydrodynamic focusing,a technical change that directs theflow of particles at the centre of the aper-ture,improves MCV measurement.For laser-beam HA,the discoid shape of RBC was a major drawback for peer analysis of RBC until the isovolumetric spher-isation was applied,allowing current instruments to generate accurate MCV(Mohandas et al.,1986).Some impedance-type HA use both technical improvements to analyse RBC(Abbott),whereas others measure the Hct and calculate MCV using the HCT and RBC(Sys-mex;ABX,Kyoto,Japan;some Beckman instruments, Miami,FL,USA).In that latter instance however, the presence of cold agglutinins,which disturbs RBC count(see above)leads to erroneous MCV(Sysmex XE-2100operator’s manual–revised April2004).

Ó2007The Authors

Table1.Situations leading to erroneous results on haematology analysers

Other parameters altered

WBC:spurious decrease

Agglutination of PMN(EDTA-related)

Agglutination of WBC other than PMN

(lymphocytes,lymphoma cells,leukaemic blasts)

Excess amount of K3-EDTA anticoagulant Shrinkage of RBC:MCV and HctflCoagulation within the sample All parameters

WBC:spurious increase

PLT aggregates PLTfl

(Very)large PLT PLTfl

Nucleated red blood cells

RBC resistant to lysis(newborns,abnormal Hb,

chemotherapy,uraemia,liver disease…)

Cryoglobulin,cryofibrinogen,immunoglobulins PLT›

Lipids PLT›,Hb›,MCH›Microorganisms(bacterial aggregates)PLT›

Others(adipose tissue,overfilling vacuum tubes)All parameters

RBC:spurious decrease

Cold agglutinins,warm agglutinins MCV›,MCH›

Very small RBC PLT›

Cryoglobulins(flflow,inadequate aspiration)Hbfl,WBCfl,PLTfl

In vitro haemolysis Hb›,MCH›

Coagulation All parameters

RBC:spurious increase

High WBC counts MCV›,Hct›,MCH›

Giant PLT PLTfl

Haemoglobin:spurious increase

Lipids WBC›,Hb›,MCH›

High WBC counts MCH›,RBC›

Immunoglobulins(and cryglobulins)WBC›,PLT›,MCH›,

In vitro haemolysis MCH›

Carboxyhaemoglobin(high amount)

Bilirubin(>250–300mg/l)MCH›

Haemoglobin:spurious decrease

Coagulation within the sample All parameters

Overfilling vaccum tube All parameters

Veinipuncture near a drip MCV›(glucose drip) Sulfhaemoglobin

MCV

Cold agglutinins,warm agglutinins:›MCH›,RBCfl,PLTfl

High WBC counts:›RBC›

Hyperglycemia:›MCH›

K2EDTA in excess:›MCHfl

Hyper-or hyponatremia:›orflMCHflor›

Technology:impedance without hydrodynamic focusing

MCH›

(MCVflin hypochromic anaemias)

MCH>36g/dl(not related to spurious counts in some disorders:spherocytosis,xerocytosis,abnormal Hb:see text) Cold agglutinins,warm agglutinins RBC,MCV›

Lipids WBC›,Hb›

Immunoglobulins Hb›

In vivo and in vitro haemolysis Hb altered,Hct Carboxyhaemoglobin(>10–20%)

Bilirubin(>300mg/l,at times less)

Immunosuppressive drugs(see also spuriously high Hb,spuriously low MCV)

MCH<32g/dl

Hyperglycemia(see also spuriously high MCV,spuriously low Hb)MCV›

Ó2007The AuthorsMCV and high WBC counts

As mentioned above,WBC are counted together with RBC,and MCV reported is the mean of both RBC and WBC volumes.In situations related to high WBC counts and low RBC counts(e.g.acute leukaemias), MCV is altered.Up to a15–20flincrease may be observed in some instances.HA which calculate MCV using the HCT and RBC(Sysmex,ABX,some Beck-man instruments)are also sensitive to leukocytosis over100·109/l(Sysmex XE-2100operator’s manual –revised april2004).

Severe hyperglycemia

Mean(red)cell volume is reported as spuriously high in blood samples containing high levels of glucose, either related to severe hyperglycemia in the relevant patient or to a sample,which has been drawn near an intravenous glucose infusion(Morse et al.,1981; Strauchen et al.,1981;Holt,DeWandler&Arvan, 1982;Evan-Wong&Davidson,1983;Savage&Hoff-man,1983;Planas,Van Voolen&Kelly,1985;Van Duijnhoven&Treskes,1996).In the latter situation, the sample is diluted leading at times to a severe spur-ious macrocytic and hypochromic anaemia(Figure7). The calculated Hct is spuriously high and the MCH is spuriously low:RBC count and Hb are unaffected in diabetic patients and variably decreased if samples are diluted because of glucose infusion(Figure7).This phenomenon is related to plasma glucose concentra-tion,which balances that of intraerythrocytic,either in the body or in the sample if an excess of glucose is present(blood drawn near a glucose drip).Studies performed on RBC at various concentrations of glu-cose have shown that within a few seconds after dilu-tion into the HA(diluents from the manufacturers) water penetrates within RBC rich in glucose,inducing swelling:return to a normal size is progressively observed and is complete after up to5min.According to the time spent between dilution and MCV meas-urement,the anomaly will be more or less conspicu-ous(Holt,DeWandler&Arvan,1982).The anomaly is more obvious if RBC are isovolumetrically sphered, because stabilization of RBC volume occurs within a few seconds after the dilution of the sample(Van Duijnhoven&Treskes,1996).For the above-men-tioned reasons HA are more or less sensitive to this phenomenon,but glucose concentrations over 20mmol/l may begin to increase MCV,and blood glucose>35mmol/l may overestimate MCV up to 50fl(Holt,DeWandler&Arvan,1982;Planas,Van Voolen&Kelly,1985;Van Duijnhoven&Treskes, 1996;Figure7).Increased MCV leads to an increase in the calculated Hct and to a spuriously low MCHC. CHCM measured on some HA is also altered.CBC demonstrating macrocytic hypochromia may be con-sideredfirst as related to hyperglycemic sample.

Considerations related to the anticoagulant

The use of either K2-or K3-EDTA salt does not induce any difference in the CBC,in optimal condi-tions of sampling.However,when concentration of anticoagulant is increased,because of insufficient volume of blood drawn after venepuncture(or in

RBC RBC

50100200300fL

Ó2007The Authorsneonates),some changes may be observed.Decrease of centrifuged Hct resulting from shrinking of RBC was reported in conditions related mainly to excess of K3salt(Goossens,Van Duppen&Verwilghen,1991; Hinchliffe,Bellamy&Lilleyman,1992),in contrast with data obtained on HA:no influence of K3-EDTA concentration was observed on MCV,while K2-EDTA at high concentrations resulted in a slight increase in MCV,and such phenomenon could be observed using several different HA(Goossens,Van Duppen& Verwilghen,1991).

Hyper-and hypo-natremia

Macrocytic and hypochromic RBC changes were observed on blood samples in conditions related to hypernatremia,whereas hyponatremia generated a tendency towards microcytic and hyperchromic RBC (Cornbleet,1983).Such situations were also reported in animals(Boisvert,Tvedten&Scott,1999).Hyper-and hypo-natremia are both situations also reported as leading to spurious centrifuged Hct(Cornbleet, 1983).

Storage of the sample and MCV

EDTA used as anticoagulant allows the accurate deter-mination of the CBC up to24h after the sample has been drawn.However,after that time,MCV may increase,namely if the sample is stored at room tem-perature.This coupled with low haemoglobin could cause the operator to suspect that a slightly microcytic anaemia is normocytic and that a normocytic could be mistaken for a macrocytic anaemia(Cohle,Saleem& Makkaoui,1981).

Mean cell haemoglobin content(MCHC)

Measured parameters allow the calculation of mean cell haemoglobin of individual RBC[MCH as expressed in pg¼Hb(g/l)/RBC(1012/l)]and MCHC [MCHC in g/dl¼Hb(g/l)·100/MCV(fl)·RBC (1012/l)].Some HA measure Hb concentration directly within RBC,named cellular Hb concentration mean (CHCM):discordant values(usually difference over 1.5g/dl)between MCHC and CHCM allow in many instances the detection of anomalies related to one of the measured RBC parameters.

MCHC>36g/dl is infrequent on most impedance-type HA,whereas it may be occasionally observed on laser-beam HA,in several constitutive disorders in which RBC are‘dehydrated’,including hereditary spherocytosis,various haemoglobin disorders(CC,SC, C b thal),and some rare RBC disorders(xerocytosis). Some acquired conditions mimic constitutive ones, namely acquired immune haemolytic anaemias caused by warm agglutinins,in which RBC coated with warm antibodies may transform gradually into spheres in vivo or in vitro after venepuncture,leading to more or less dehydrated and spherised cells.Many situations corresponding to abnormally high Hb val-ues,and/or abnormally low RBC,and/or spuriously low MCV,also lead to increase MCHC(see the corres-ponding paragraphs and Table1).Although the mech-anism is unknown,some immunosuppressive drugs may slightly increase MCHC,usually not above 37.5g/dl(Cornbleet1983).

MCHC<32g/dl.As mentioned above,hyperglyce-mia leads to artefactual hypochromic macrocytosis. Storage of blood samples has been reported to cause a facticious increase in percentage of hypochromic cells, and become conspicuous after24h of storage (Murphy,Spaven&Casey2002).Although changes are not major,the number of hypochromic cells may be an important element to consider,as for example in iron deficiency anaemias or during erythropoietin therapy(Richardson,Bartlett&Will,2001).

RETICULOCYTES

Enumeration of peripheral blood reticulocytes is essential in the diagnosis and management of anaemic patients and may be considered now as a part of the CBC.If manual counting by light microscopy remains the standard of reticulocyte enumeration,automated methods developed during the past two decades are now more accurate,precise,and cost-effective than manual counting and,in addition,provide a variety of reticulocyte-related parameters,such as volume,hae-moglobin concentration,and maturity,which are unavailable with light microscopy(Cavill,1993;Van Petegem et al.,1993;Davis et al.,1994;Koepke,1999; Brugnara,2000;Siekmeier,Bierlich&Jaross,2000; Riley et al.,2001;Bain&Bates,2001;Pierre,2002; Riley et al.,2002).Automated reticulocyte counts can be performed using general purposeflow cytometers,

Ó2007The Authorsdedicatedflow cytometers,or integrated with the other parameters from the CBC under a fully automa-tized method.RNA from reticulocytes is stained using the classical new methylene blue(part of Abbott and Beckman Coulter instruments),oxazine(Bayer),or one among variousfluorescent dyes,including thiaz-ole orange(Horiba ABX),auramine O(Sysmex), CD4K530(Abbott),or coriphosphine O(Beckman Coulter).According to the HA and to the reagent used,flow cytometric analysis is performed,using either the measurement of volume,conductivity,and light scatter(VCS)or thefluorescence detection(for review see Riley et al.,2002).

Careful gating of reticulocytes is crucial in deter-mining accurate counts,because dyes stain also RNA from PLT and WBC,and may also combine with the DNA of nucleated cells.Red blood cells and reticulo-cytes being larger than PLTs and smaller than WBC, both the former are discriminated from both the latter according to the size.Unsurprisingly,gating may be difficult in the presence of blood components of abnormal size and containing stained RNA/DNA. Giant PLT,PLT clumps,abnormal WBC,abnormal number of WBC,and WBC fragments,are situations reported as a potential source of interference with automated methods of reticulocyte analysis (Oyamatsu et al.,19;Pappas,Owens&Flick,1992; Riley et al.,2001).NRBC have been reported as dis-turbing reticulocyte counts(Pappas,Owens&Flick, 1992;Riley et al.,2001),but,as NRBC themselves are not counted as reticulocytes in the HA,it has been hypothesized that abnormal reticulocyte count observed in that instance could be due to increased number of very young reticulocytes which frequently appeared with NRBC(Oyamatsu et al.,19).

After gating of RBCs,reticulocytes are identified within the RBC population according to the coloured particles orfluorescent material they contain.Cyto-plasmic particles other than RNA that can be stained by supravital dyes,including Howell–Jolly bodies, Pappenheimer bodies,or basophilic stippling,may be confused with reticulum granules using automated techniques in HA,as well as they interfere in manual techniques(Pappas,Owens&Flick,1992;Ghevaert et al.,1997;Riley et al.,2001),although such interfer-ences have been found to be more or less conspicuous according to the machines used(Oyamatsu et al., 19;Lofsness,Kohnke&Geier,1994).Heinz bodies observed in severe haemolytic anaemias,thalassaemia major,congenital Heinz-body anaemia,or post-sple-nectomy disorders,are also situations reported as dis-turbing automated reticulocyte counts(Hinchliffe, 1993;Espanol,Pedro&Remacha,1999;listed in Riley et al.,2001),as well as sickle cells(Pappas,Owens& Flick,1992;Ghevaert et al.,1997),spherocytes (Ghevaert et al.,1997),and haemoglobin H inclusion bodies(Riley et al.,2001).

Although they are only faintly stained usingfluor-escent dyes,intraerythrocytic parasites(malaria,babe-sia)may interfere with automated reticulocyte count, and value six times higher than the manual count was reported in a patient demonstrating70%of RBC infected with plasmodium falciparum(Laurencet, Martinez&Beris,1997).

Intensity of intracellular staining orfluorescence of reticulocytes is assessed in most HA,the brightest reti-culocytes being the youngest,leading to peculiar indices such as the immature reticulocyte fraction (IRF;for review see Brugnara,2000).White blood cells displaying intense staining were reported as lead-ing to an erroneous estimation of reticulocyte matur-ation index,the error being directly correlated with the WBC count(Villamor et al.,1996).Very mature reticulocytes contain only a few coloured dots and may be insufficiently detected in neonates usingflow cytometry,possibly due to the low concentration of the colouring matter used in some HA(Bayer;Wieg-and et al.,2004).Although reticulocyte counts are s after storing blood samples for72h at4°C and24–48h at room temperature(Brugnara et al.,1994; Rudensky,1997;Lacombe et al.,1999;Bain&Bates, 2001),immature reticulocyte fraction was observed as stable for only8h at4°C and6h at room tempera-ture(Lacombe et al.,1999).

Various other situations are mentioned as gener-ating occasionally spurious reticulocyte counts,inclu-ding agglutinated cells in the presence of cold agglutinins(Oyamatsu et al.,19;Riley et al.,2001), autofluorescence of RBC(porphyria,drugs)and diag-nostic intravenousfluorescent dyes(Riley et al., 2001),high amount of paraproteins,and haemolysis (Riley et al.,2001).

Although depending on the software gate correc-tions,in most of the above described situations the HA show abnormalflags,prompting the technologist to perform a manual count.However,exceptions to

Ó2007The Authorsflagging have been reported,at least in cold agglutinin disease(Oyamatsu et al.,19)and in beta-thalas-saemia major(Ghevaert et al.,1997;see Table2).

CONCLUDING REMARKS

The widespread use of HA has led to major improve-ment of cellular haematology,because of quick and accurate results found in most instances,and now preanalytical and analytical variables should be con-sideredfirst within the laboratory when spurious results from the HA are found(see also concluding remarks from part I).In most situations,HA generate flags or peculiar scattergrams in response to clinical or artifactual parameters,independent of the technology used.Inadequate blood sample may be responsible for various anomalies,including initiation of coagulation within the tube because of difficult venepuncture or to low bloodflow,excess of EDTA salt result of insuf-ficientfilling of the tube,which generate PLT and/or WBC aggregates,fibrin precipitates,or others.Over-filling of blood sample and time elapsed from samp-ling to analysis must also be considered at times.In several instances the disease itself generates changes that disturb measurements performed by HA(RBC agglutination,cryoprecipitates).Some anomalies, however,are intrinsic to the technology used for ana-lysis,and have led manufacturers to develop technical changes in analysing blood cells,one example corres-ponding to the use of hydrodynamic focusing,which dramatically improved measurement of MCV in impedance-type HA.Some protocols for measurement of blood cells have been discarded,such as PLT counts performed after RBC lysis,leaving intraglobular com-ponents free and enumerated together with PLT. Improvements in analysis of PLT and RBC parameters has led to an increase in the accuracy of results and has also led to the generation of severalflags in abnormal situations.The enumeration of PLT in thrombocytopenias,the identification of NRBC leading to spurious PLT and WBC counts,and other situa-tions,have led manufacturers to optimise computer-ized analysis for the detection and analysis of every blood cell,and moreover to develop further methods for their specific identification and their specific enu-meration on the same sample and on the same HA. The improvement of WBC analysis and the careful study of WBC differential scattergrams have led to another major improvement in blood cell analysis, allowing WBC differential to be performed automatic-ally but also to exhibit and to give explanation for several anomalous CBC results.Several peculiar WBC scattergrams generated were progressively identified, and related to spurious counts.As discussed all along both reports,inaccurate identification,analysis,or enumeration of one or several components from the CBC leads in many instances to an abnormal WBC differential scattergram.By the rule the WBC differen-tial isflagged or invalidated,so a blood smear and an optical count are often needed.Automated abnormal WBC differentials related to spurious counts must be included together with the various but insufficiently reported situations related to the inability of the HA to perform WBC differential or to identify specifically one or several cells from the WBC differential.All these situations should certainly need a specific report.

So,if some spurious counts were found to be numerous enough to generate technical improve-ments to identify them clearly and create other meth-ods for measurement,several other spurious counts either do not generate specificflags to identify the anomaly or even do not generate anyflag at all. Moreover,if the most recent and powerful HA are able to generate severalflags related to at least a part of spurious counts,it is stressed that simpler HA, namely those without any WBC differential scatter-gram,will not.So,if acquiring a new HA is a personal choice,each haematologist must know how his machine will react when(at least)the situations reported here occur.

Table2.Situations leading to possible interference

with automated methods for reticulocyte analysis Inaccurate gating of RBCs:giant PLTs,PLT clumps, abnormal WBCs,abnormal number of WBCs,WBC fragments,nucleated red blood cells

Intraerythrocytic particles:Howell-Jolly bodies, Pappenheimer bodies,basophilic stippling,Heinz bodies, sickle cells,spherocytes,Haemoglobin H inclusions, plasmodium,babesia

Others:cold agglutinin disease,autofluorescence of RBCs (drugs,porphyria),paraproteins,haemolysis,diagnostic intravenousfluorescent dyes

Ó2007The AuthorsACKNOWLEDGEMENTS

We are indebted to Mathilde LINARD for kindly reviewing English manuscript.CONFLICT OF INTEREST No conflict of interest.

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