Coulter CLS Clinical Case Studies

Coulter VCSTechnologyClinical Case StudiesBulletin # 3008TM1IndexIntroduction 31.Sample Stability Studies with Normal Blood 42. Left Shift 63.Left Shift 84. Hypereosinophilia Syndrome 105.Infectious Mononucleosis 126.Agranulocytosis 147.Acute Myeloid Leukemia FAB M0 168.Acute Myeloid Leukemia FAB M4 189.Acute Myeloid Leukemia FAB M5a 2010. C-ALL, FAB L2 2211. Refractory Anemia (RAEB-t) 2412. CML in Blast Crisis 2613. Non-Hodgkins Lymphoma 2814. Prolymphocytic Leukemia (PLL) 3015. Pernicious Anemia 3216. Hereditary Spherocytosis 3417. Anisocytosis 3618. Essential Thrombocytemia 38IntroductionThe quantitative and qualitative analysis ofblood cells, one of the oldest medical labo-ratory procedures, remains one of the mostimportant basic tests of medical diagnosis. Theterm "blood picture" vividly reflects the fact thatpathophysiologically correct and clinically rele-vant interpretation requires comparative andcontrastive evaluation of the number andappearance of various blood cells. Changes inthe blood picture, or blood count, may be thenormal reactions of the blood cell system todemands placed on it by the environment or bydisease, or they may be the expression of a pri-mary or secondary disease of the hematopoieticsystem itself. Quantitative and qualitativechanges may be related to disorders of cell for-mation, cell disintegration or cell distribution.The methodological basis for interpreting theblood count is to determine as accurately andrapidly as possible, with a minimum of interfer-ing factors, the concentrations of various bloodcells, which differ in their origin, function andmorphological properties. Classical chambercounting methods combined with microscopicdifferentiation of fixed, stained blood cells onlypartially fulfilled these requirements, and wereextremely time-consuming. The standardization of hemoglobin determina-tions by Heilmeyer and Wintrobe's hematocritmethod, as well as use of the impedancemethod of performing blood cell counts,described as the "Coulter principle," made itpossible for the first time to determine withadequate precision, the concentration of redblood cells, nucleated cells (white blood cells)and platelets, in the course of routine medicaldiagnosis. From these counts, it was also possi-ble to determine fairly precisely the size andhemoglobin content of individual red bloodcells, two important parameters in the diagno-sis of anemia.However, there was one essential challenge ofblood cell diagnosis, which these methods wereunable to solve, the biological heterogeneity ofnucleated cells, which result from different ter-minal differentiation pathways and have differ-ent functions. The improved precision obtainedby automating the white cell count was lostagain due to the "error of the small sample size"which occurred during conventional differentia-tion of white cells by stained blood smear. Thenecessary consequence of improving countingaccuracy was therefore the development ofautomated methods of identifying cells withinthe nucleated blood cell group. Two completelydifferent approaches were taken to solve thisproblem. One attempt, largely unsuccessful,was based on conventional microscopy; itinvolved using a computer-controlled micro-scope to find and identify, based on structureand coloration, nucleated cells which had beenfixed and stained in a blood smear. This methodthus replaced the hand, which moves themicroscope stage with a computerized mecha-nism and the memory and decision-makingcapability of the human brain with a computer.Within the technological restraints of a practicaldiagnostic setting, it was not possible toincrease precision by substantially increasingspeed, nor to overcome the problems of algo-rithmic structural identification, i.e., the abilityof an experienced morphologist to recognizeand classify a structure was not then achieved,and has yet to be achieved by any instrument.A solution that was practical in the context ofmedical diagnosis was first achieved with theuse of flow cytometry, which is also based onparticle counting according to the Coulter prin-ciple. Today, blood counts are evaluated aroundthe world using instruments, which suspendnucleated blood cells in a suitable fluid andquantify them with a high level of precision.Different manufacturers detect different bio-physical, structural and biochemical parame-ters. All include cell volume. The solutionselected by Coulter in its VCS system for bloodcell differentiation, also includes conductivityand light scattering properties.A unique benefit of VCS Technology is that themeasurements selected, impedance, conductiv-ity and laser light scatter, analyze as closely aspossible, similar cellular characteristics thatenable morphologists to visually classify bloodcells. To achieve this, the system uses electro-optical analysis of individual cells in their near-native state to provide complete and detailedanalysis in a single channel.These Case Studies have been compiled in col-laboration with hematologists and physiciansspecializing in laboratory medicine, who havetheir own experience both in laboratory diagno-sis and in clinical hematological diagnosis. Theguide is intended to show, using concreteexamples, some of the possibilities of translat-ing conventional morphological and flowcytometry concepts, and to demonstrate thediagnostic conclusions which can be drawnfrom analytical results obtained with VCS tech-nology. It will help users recognize the benefitsthat can result if laboratory results are interpret-ed correctly and in a manner suited to the tech-nology. The examples cited will enable hema-tology laboratory personnel, hospital and pri-vate-practice physicians to take advantage ofthe results of this highly developed technologyand to examine them critically and intelligentlyfor the benefit of their patients. We hope thatthis short case study book, produced collabora-tively by Beckman Coulter and the hematologylaboratories of three university hospitals, doesjustice to these intentions.31.Sample Stability Studies with Normal BloodScatterplot and HistogramsBlood from a patient with normal values wastested 2, 12, 24, and 48 hours after drawingwith K3EDTA. The values were stable overthe entire period. The specimen was stored atroom temperature. Of note are the changesin the Scatterplot. After 2 hours, thedistribution is normal. After 12 hours,changes are due to changes in the cells, e.g.vacuolization, nuclear distortions, etc. After24 hours, suspect flags appear for "Blasts," "Atypical lymphocytes," and "ImmatureGranulocytes 2." Neutrophilic granulocytesshow distinct dispersion upward into thelarge-volume region of the Scatterplot andmoderate dispersion into the region belowthe population, the region of aging cells.After 48 hours, very many cells disperse into this region, and the suspect flags areidentical to those in the analysis after 24 hours.Clinical DataThe sample was collected from a healthy 46-year-old woman. The blood sample wascollected in K3EDTA and stored at roomtemperature (approx. 20C).4Suspect Flags Definitive FlagsWBC BlastsImmature Granulocytes 2Atypical lymphocytesRBCPLT2hr 12hr48hr 24hrCoulter VCSTMTechnologyClinical Case StudiesRBC morphologyDIAGNOSISCommentUpper: Lymphocyte and Segmented Neutrophil at 2 hrsLower: Monocyte and Granulocyte at 24hrs.Old bloodNo abnormalitiesAsthisexampleshowsexcellentanalyticalresultsarepossibleforupto1-2days, 3 days at most. To achieve this some samples may well require a period ofcorrect equilibration and this is especially true for poorly collected or pediatrictubes.Forsamplesthathavebeenlefttostandwithoutmixing,whatiscalled In-Vitrostasis,thereisalsoaperiodofequilibrationwhenthesampleisfirstmixed.Withcontinuousmixingthiscanbeshortenedto10-20minsinmostcases.For the results to be optimal, the differential should always be performed within24hrs of the blood being drawn.Thequantitativeresultsofthisnormalpatienttestwouldstillbereproducibleafter 48 hours.Microscopic Differential% x109/LSegmented 45 3.7Lymphocytes 41 3.4Monocytes 4 0.4Eosinophils 4 0.4Basophils 1 0.152. Left ShiftScatterplot and HistogramsThe status display on theprintout indicates abnormalWBC. A neutrophilpopulation shifted slightlyupward can be seen in theScatterplot. This is the causeof the Imm Gran 1 flag. At79% neutrophils, the valueindicates a slight elevation,likewise for the marginalWBC. The smear reveals a leftshift with 10% band cells asthe cause.The Plt and RBC histogramsare normal. Aside from a mildnormochromic anemia, theremaining parameters arenormal. The normal MCV ismarked with a flag as definedby the lab.Clinical Data35 year-old woman who had been coughingfor a week. She also complained of pain in thelimbs and fever as high as 39C.6Suspect Flags Definitive FlagsWBC Immature Granulocytes 1RBCPLTCoulter VCSTMTechnologyClinical Case StudiesAdditional TestsDIAGNOSISCommentReactive left shift with pneumonia. In addition to 2 segmented, neutrophil granulocytes, twoband neutrophils and a small lymphocyte arepictured in the detail.Pneumonia (pathogen not identified)None requiredReactive left shift with marginal leukocytosis. The course of the current diseaseistooshorttoexplainthemildnormochromicanemia. ThesuspectflagImmatureGran.1indicatesanincreaseinbandneutrophils.Insuchacase,an additional differential count should be done with a blood smear.Microscopic Differential% x109/LBands 10 1.8Segmented 62 5.5Lymphocytes 15 1.5Monocytes 9 0.9Eosinophils 1 0.1Plasma cells 3 0.3RBC morphologyAnisocytosis+73.Left ShiftScatterplot and HistogramsThe Scatterplot is remarkablefor a population in theneutrophil region extendinginto the larger-volumechannels, indicating the needto look for immatureprecursors on smear. Inaddition, the instrumentshows suspect flags "Blasts"and "Immature Granulocytes2." The total WBC count iselevated, as is the proportionof neutrophilic granulocytes.RBC and Plt histograms shownormal distribution. Amongthe values, the low Hgb andthe borderlinethrombocytopenia arenoteworthy. Clinical Data52-year-old male with testicular tumor, Status post chemotherapy and administrationof G-CSF.8Suspect Flags Definitive FlagsWBC Blasts LeukocytosisImmature Granulocytes 2 Neutrophilia %Neutrophilia #RBCPLTCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentPromyelocyte and segmented neutrophilgranulocyte. In the smear, only very fewthrombocyte agglutinates (lower right hand side in photo) were present.1.Leukocytosis with left shift as far as blasts on administration of G-CSF after chemotherapy 2.Testicular tumorGrowth-stimulating factors (in this case, G-CSF) may lead to significant leukocy-tosis with marked left shift all the way through blasts, which bears a clear mor-phologic resemblance to CML in its chronic phase.Microscopic Differential(n=200) % x109/LBlasts 1 0.3Promyelocytes 2 0.4Myelocytes 6 1.2Metamyelocytes 5 1.1Bands 4 0.8Segmented 71 14.5Lymphocytes 7 1.4Monocytes 4 0.8RBC morphology:Anisocytosis +Poikilocytosis +Polychromasia +Toxic granulation +94. Hypereosinophilic SyndromeScatterplot and HistogramsAt first sight, the Scatterplot is remarkable for the closelyopposed populations in theneutrophil and eosinophilregion. The suspect flag"Immature Granulocytes 2" is displayed, and confirmedon smear. Lymphocytes,monocytes, and basophilicgranulocytes make up only a small portion of thenucleated cells.The total WBC count iselevated. The differentialparameters show markedeosinophilia.The RBC histogram is shiftedinto the microcytic region,i.e. to the left, and the MCVis low. In addition, the curveis widened and the RDWincreased, indicatinganisocytosis. Both findingsare confirmed on smear. The patient is anemic (Hgb).Plt count and histogram areunremarkable.Clinical Data49-year-old female referred because offever, weight loss, dyspnea, andunproductive cough.10Suspect Flags Definitive FlagsWBC Immature Granulocytes 2 LeukocytosisEosinophilia %Eosinophilia #RBCPLTCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentA segmented granulocyte (with toxic granulation)and an eosinophilic granulocyte. The eosinophilicgranulocyte is larger than usual, the nucleus hasfour lobes, and the eosinophilic granules do notcompletely fill the cytoplasm. (In the photo thegranules are rather pale and do not have thecharacteristic reddish color that is typical underthe microscope.)Hypereosinophilic syndrome (HES)The peripheral blood shows leukocytosis, granulocytosis with left shift as far aspromyelocytes, and pronounced eosinophilia (15 x 109/L). The closely opposedpopulations of neutrophils and eosinophils are probably due to the greater lightscattering of the toxic granulation of the neutrophils and the lesser light scatterofthepaleeosinophilicgranulesseenonthefilm. Theeosinophiliahasbeenrecognized for years, but no cause (such as allergic reactions, parasitic diseases,specificskindisorders,orautoimmunediseases.)hasbeenfound.Infiltrateswere shown in the lungs and heart.Microscopic Differential% x109/LPromyelocytes 1 0.45Myelocytes 1 0.45Metamyelocytes 3 1.4Bands 9 4.1Segmented 46 21.0Lymphocytes 3 1.4Monocytes 3 1.4Eosinophils 33 15.0Basophils 1 0.45RBC morphologyAnisocytosis ++Poikilocytosis +Polychromasia +++115.Infectious MononucleosisScatterplot and HistogramsImmediately obvious on theprintout is a highly abnormaldistribution of the cells in theScatterplot. In addition to asmall population in thelymphocyte field, theoverlapping population oflymphocytes and monocytesis also noteworthy. Themicroscopic differentialcount reveals that the latter isdue to the large virocytestypical of mononucleosis.Only a small percentage(28%) is normal lymphocytes.Some of the abnormal cellswere classified in themonocyte region. The suspectflags Blasts and Atyp.Lymph give an indication ofthis alteration, as do thelymphocytosis andmonocytosis flags.This patient also hasleukocytosis andthrombocytopenia. The Pltcurve is not extrapolatedsince the Plt volume is mostlysmall. The value is thereforeflagged R. The RBC valuesand histogram show noabnormalities.Clinical Data26-year-old male with fever of 39C persistingfor the past 10 days.The patient also complained of difficulty withswallowing, night sweats and coughing.12Suspect Flags Definitive FlagsWBC Blasts Lymphocytosis %Atyp. Lymphocytes Monocytosis %RBCPLTCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentA Monocyte and Lympho-Monocytoid Virocyte.Infectious mononucleosis positivenegativepositivemarginally diminished Transaminases elevated.23 g/LTheinstrumentassignedthemorphologicallyabnormalpopulationofthelym-pho-monocytoid virocytes partially to the lymphocytes and, to a lesser extent, tothemonocytes.Still,thetotalpopulationismarkedwiththeflagblasts. Thesimultaneously existing massive left shift of the neutrophils was not flagged. Thereason for the predominance of small platelets is not clear. The absence of giantplatelets in this cell distribution is counterindicative of the presence of immunethrombocytopenia or other increases in peripheral turnover platelets.Virocytes occur in infectious mononucleosis and seldom in other viral diseasessuch as cytomegalovirus infections or viral hepatitisMicroscopic Differential% x109/LMetamyelocytes 2 0.3Bands 6 0.9Segmented 10 1.5Lymphocytes 28 4.1Virocytes40 5.9Monocytes 12 1.8Eosinophils 1 0.1Basophils 1 0.1Special Observations:Virocytes: Lympho-monocytoid cells and largeblast-like cells with nucleoli.Additional TestsMononucleosis quick test: Other viral serology:Direct Coombs' test:Haptoglobin:Liver and spleen enlarged:Gamma globulins elevated:136.Agranulocytosis (Neutropenia)Scatterplot and HistogramsSignificant in the Scatterplotis the total absence of anyneutrophil population. Thepercentage count is less than1%. The total WBC count islikewise greatly reduced. Based on the position andpercentage and/or absolutevalues, normal populations oflymphocytes, monocytes,eosinophil and basophilicgranulocytes are present. RBC and Plt histograms areunremarkable. Among thevalues, the borderline anemiaand the very small Plt (MPV)are noteworthy.Clinical Data59-year-old woman with hyperthyroidism, treated with Favistan.After an interval for convalescence, she comes in for routineexamination, where agranulocytosis (presumably from Favistan)is established. Presently in reverse isolation, receiving G-CSF.14Suspect Flags Definitive FlagsWBC Lymphocytosis %Neutropenia %Neutropenia #RBCPLTCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentLymphocyte at low magnification.Drug-induced agranulocytosis Thenewdevelopmentofanalmosttotalabsenceofneutrophilicgranulocytesamong otherwise mostly normal blood values was a significant criterion in thediagnosis. Thepatientwastakenoff ThiamazolandtreatedwithB-CSF.Fourdays later the granulocyte count had once again exceeded 1000.Microscopic Differential% x109/LLymphocytes 91 1.63Monocytes 6 0.11Eosinophils 1 0.02Basophils 2 0.04RBC morphologyAnisocytosis +Anulocytes* +Special Observations:No segmented granulocytesfound even after prolongedsearch.* extremely hypochromic RBCwith only a little peripheral circle of Hgb.157.Acute Myeloid Leukemia FAB M0Scatterplot and HistogramsThe Scatterplot displays adistinct population located in the region of lymphocytesand monocytes. Theneutrophilic granulocytescomprise only a small, poorlydefined population. The totalWBC count is elevated. Thelymphocyte fraction isrelatively and absolutelyincreased. Suspect flags areshown for "Blasts" and"Atypical lymphocytes." Forthe RBC, there are markedchanges in the histogram(widening, absence of normalascent and descent of thecurve) and in the counts.Based on the increased RDW,anisocytosis is to beexpected, and anemia ispresent in addition (Hgb).The Plt curve is notextrapolated, sincepredominantly very small Plt (MPV) are counted, andmoderate thrombocytopeniais present. Clinical Data54-year-old male with increasing fatigue and fever. No swelling of lymph nodes orsplenomegaly. Referred by private physicianfor possible acute leukemia. 16Suspect Flags Definitive FlagsWBC Blasts LeukocytosisAtypical lymphocytes Lymphocytosis %RBCPLT ThrombocytopeniaCoulter VCSTMTechnologyClinical Case StudiesAdditional TestsCytochemistry:Flow Cytometry:Cytogenetics: DIAGNOSISCommentBlast and lymphocytesAML-M0 according to FAB classificationBlasts: Peroxidase negative; Esterase negativeNoreactionofblastswithlymphaticmarkerscyCD22,CD19,CD10,cyCD3,CD5,CD2,SignificantreactionswithmyelomonocyticmarkersCD13 and CD33 Not doneIn this case the position of the Blasts in the Scatterplot gives no indication as towhich line the blasts belong. In the typical AML FAB M0 the blasts are usuallymediumtolarge,undifferentiatedwithfewgranules.Inthiscasethe VCS technologyhasplacedthemwheretheywouldbeexpectedbasedontheirstructure. The final diagnosis is based on flow cytometric data. An AML FAB M0is characterised by an expression of myelo-monocytic markers and negativity forother cell lineage antigens.Microscopic Differential(n=200) % x109/LBlasts 59 11.9Segmented 5 1.0Lymphocytes 35 7.0Monocytes 1 0.2RBC morphology:Anisocytosis +Poikilocytosis +Special Observations:Morphologically medium-sized blasts without granulesor Auer Rods.178.Acute Myeloid Leukemia FAB M4Scatterplot and HistogramsIn the Scatterplot, apopulation is easily identifiedin the monocyte region that isshifted fairly far to the right inthe direction of theneutrophils. Eosinophilicgranulocytes are notidentifiable as a population,and lymphocytes barely so.The instrument flags Blastsand Immature Granulocytes2. WBC count is elevated.The RBC curve is shifted intothe microcytic region and theMCV decreased. On the right-hand side it is atypicallyshaped and widened(RDW). Hypochromia ispresent (MCH). The reasonfor the slightly atypical shapeof the left-hand side of thecurve in the histogram is thepresence of microcytic andfragmented cells. These arealso seen in the > 20-fLregion of the Plt curve.Nevertheless, the instrumentcan separate RBC from Plt, asshown in the extrapolation.The Plt count is markedlydecreased. Clinical Data70-year-old female with bleeding tendency,otherwise feeling well.Clinical findings: Gingival hyperplasia,multiple hematomas.18Suspect Flags Definitive FlagsWBC Blasts LeukocytosisImmature Granulocytes 2 Monocytosis %Monocytosis #RBCPLT1. Secondary acute myelomonocytic leukemia (AML M4 by FAB classification).2. Status post multiple combined immunosuppressive therapy.DIAGNOSISCoulter VCSTMTechnologyClinical Case StudiesCommentUpper Right:Monocytoid Blasts, marked anisocytosis and poikilocytosis of the erythrocytes.Additional TestsBone marrow cytology:Cytochemistry:Flow Cytometry:Theatypicalmonocytepopulationisappropriatelylocatedinthemonocytewindow. The left shift of the neutrophils up to the blasts was also properly iden-tified.Althoughnoflagforgiantplateletswasdisplayedontheinstrument,careful observation of the thrombocyte histogram reveals a change in the curveintheregion>20fL.Interferencewiththeerythrocytesisnegligibleinthiscase. In general, attention should be paid to the histogram as well as to the flag.Microscopic Differential% x109/LBlasts 2 0.8Promyelocytes 1 0.4Myelocytes 2 0.8Metamyelocytes 3 1.2Bands7 2.8Segmented 21 8.3Lymphocytes 12 4.7Monocytes 51 20.2Eosinophils 1 0.4Normoblasts 1 0.4RBC morphologyAnisocytosis +Poikilocytosis +Polychromasia +Elliptocytes +Schistocytes +Teardrops +Basophilic stippling +Spicule cells +Special Observations:Atypical monocytesToxic granulation Giant PltLower Left:Bone Marrow Cytology: A maturing apparent left-shift of the granulopoiesis with a high percentage ofmaturing myelomonocytic blasts interspersedwith islets of maturing erythropoiesis anddimorphic megakaryocytes.19Cellularity markedly increasedMegakaryocytes decreasedMicromegakaryocytesGranulopoiesis markedly shifted to left70% blastsEosinophiliaThe leukemic blasts are peroxidase positive, nonspecific-esterase positive, PASnegative.The circulating blasts are CD11a positive, CD11c positive, CD13 positive,CD14 positive, CD33 positive, CD65s positive, HLA-DR positive, TdT negative.DIAGNOSISEarly relapse of acute monoblastic leukemia (AML, M5a accordingto FAB classification) with Trisomy 8.9.Acute Myeloid Leukemia FAB M5aScatterplot and HistogramsIn the Scatterplot, there is a noticeable upward shift in the monocyte population.The neutrophil populationalso shows strong dispersioninto the upper region of thedisplay. The instrumentindicates Blasts andImmature Gran. 2, whichare confirmed by smear. Afew small cells are found inthe debris region below theleukocyte threshold.Pronounced leukocytosis,neutrophil generation, andmonocytosis are identifiedwith their respective flags.The patient has a slightmicrocytic anemia. The RBCreadings indicatemicrocytosis (MCV) andanisocytosis (RDW). In thesmall volume thresholdregion ( 100 x103/L raises R)The thrombocyte count is atthe lower threshold of thereference region. Thedistribution curve indicatesthe presence of rather smallplatelets. The thrombocytevalue is flagged with an Ralert as a consequence ofleukocytosis.Clinical DataTwo years ago this 56-year-old male lost 15 kgand suffered from fatigue and weakness. InFebruary 1994, a diagnosis of CLL (Rai II) wasmade. One year later, splenomegaly and risingWBC were noted in addition.30Suspect Flags Definitive FlagsWBC Blasts Monocytosis %Immature granulocytes 2Atypical lymphocytesRBCPLTCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentProlymphocytesProlymphocytictransformation of CLLAdditional TestsCorrections of values forextremely high WBC countWBC (diluted) RBC (WBC subtracted) Hct (centrifuged) Hgb*MCV (recalculated) MCH (recalculated) MCHC (recalculated) Leukocytosis>100x103/Ldisturbsthehemoglobinmeasurement(turbidity).Thecloudingdependsontheleukocytecountandonthecelltype.Withavalue of 13.2 g/dL, a true HGB value > 10 g/dL can be assumed, although theexactvaluecanonlybedeterminedthroughphotometricmeasurementaftercentrifugation of the Hgb preparation. As an alternative, the hematocrit can bedetermined with the reference method (centrifuge) and the severity of the ane-mia thereby evaluated. Prolymphocytes are easiest to recognize in the thin partof the smear. The thrombocyte count was flagged with an R alert; if the throm-bocyte distribution is unremarkable, the value can be accepted and used.Microscopic Differential(n=200) % x109/LSegmented Neutrophils 2 5.0Lymphocytes 98 251RBC morphology:Anisocytosis +Poikilocytosis +Special Observations:The majority of lymphocytesare prolymphocytes 31256 x 109/L3.83x1012/L0.34 11.4 g/dL88.8 fL29.8 pg./cell33.5 g/dL*Determination by reference method (after centrifugation of specimen)15.Pernicious AnemiaScatterplot and HistogramsThe Scatterplot shows distinctpopulations in the lympho-cyte, monocyte, and neu-trophil regions. Only rarecells are distinguished in theeosinophil region. At 2.0 x 103/L, the leukocytecount is low, caused by a significant decrease in thenumber of neutrophil granu-locytes. The neutrophils dis-perse in the large-volumeregion (suspect flag:Immature Granulocyte 2). The lymphocyte count is atthe lower threshold of the ref-erence range.Compared with the micro-scopic differential, all valuescorrelate very well. The totalWBC count is decreased.Remarkable in this case arethe bimodal RBC curve andthe extremely high RDW(normal up to about 15%).The instrument signals"Dimorphic RBC population."All other RBC values are like-wise abnormal: Hgb marked-ly decreased, Hct decreased,MCV extremely high, i.e.macrocytic or megaloblasticRBC, and MCH extremelyhigh, i.e. hyperchromia ofRBC. Only the MCHC valueis normal. The Plt value andhistogram are abnormal. The curve does not approachthe x-axis in the > 20-fLregion and the value isflagged "R." most likely as aresult of the microcytic redcell. Chamber count confirmsthe low Plt count.Clinical DataThis 55-year-old woman consulted her physician because ofincreasing fatigue. He diagnosed pancytopenia and referredthe patient to the hospital. Admission data: WBC 2.1 x 109/L;Hgb 6.7 g/dL; MCV 129 fL; Plt 38 x 109/L.32Suspect Flags Definitive FlagsWBC Immature Granulocytes 2 Neutropenia %Neutropenia #RBC Dimorphic RBC populationPLT ThrombocytopeniaCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentMacrocytesMegaloblasticanemia.Additionaldiagnostictestsdemonstratepernicious anemia. Additional TestsPlt (chamber count): Bone marrow cytology:Vitamin B12:Folic acid:35 x 109/LHypercellularmarrow;erythropoiesismarkedlyincreased;megaloblasticchanges; abundant hyperlobular megakaryocytes; dysplastic hemopoiesis.DecreasedNormalMicroscopic Differential(n=200) % x109/LBands 3 0.06Segmented 37 0.72Lymphocytes 49 1.0Monocytes 5 0.1Eosinophils 4 0.08Basophils 2 0.04RBC morphology:Anisocytosis ++Poikilocytosis +Macro and megalocytes ++Uponadmissiontothehospital,thebloodcountshowedonlyanerythrocytepopulationwithamediumvolumeofapproximately130fL(measurementonCoulterSTKRinemergencylab;notpicturedhere). Thepatientreceivedtwoerythrocyteconcentrates,thenormalerythrocytesofwhichnowcanbeidenti-fiedintheregionofapproximately85fLintheerythrocytedistributioncurve.The two erythrocyte populations are also recognizable in the smear: clear aniso-cytosis with macrocytes and/or megalocytes.The low thrombocyte value was confirmed in the chamber count.3316.Hereditary SpherocytosisScatterplot and HistogramsIn the Scatterplot, thelymphocyte, monocyte, andneutrophilic granulocytepopulations are situated closetogether. This phenomenonmay occur when a specimenis left standing and unmixedfor several hours andanalyzed without prior re-equilibration as in this case.There are no suspect flags.There is good agreement withthe microscopic differential. Hemoglobin concentrationand the number oferythrocytes, as well as thesize distribution oferythrocytes, areunremarkable. The MCV iswithin the reference range.The mean corpuscularhemoglobin (MCH), however,is elevated in relation to themean corpuscular volume(MCV), which, comparedwith normal persons, resultsin an elevation of the meancorpuscular hemoglobinconcentration (MCHC).The Plt curve is normal, thecount elevated.Clinical Data26-year-old male for follow-up of knownspherocytosis. Recently performedsplenectomy; outpatient follow-up. 34Suspect Flags Definitive FlagsWBCRBCPLTCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentSpherocytesSpherocytic anemia (shortly after splenectomy)Additional TestsHct (centrifuged) MCV MCH MCHCReticulocytes 0.42 (repeatedly)79.6 fL31.9 pg/cell40.0 g/dL58%306 x 109/LSmearsfromnormalpersonsshow"spherocytes"roundtheperiphery.Whenspherocytosis is suspected, the thick part of the smear must be examined.Note: When spherocytosis is suspected, attention should be paid to the MCHCvalue on the instrument, which is borderline elevated!Becauseoftheirsphericalshape,impedancemeasurementcharacteristicallyyields MCV values too high for these cells therefore the MCHC determination isfalsely elevated. If spherocytosis is suspected, the hematocrit should always bemeasured in parallel with a reference method (centrifugation). Whereas in nor-malpersonsthemanualHctisalways1-2%higherthantheautomatedvalue("trapped plasma"), in spherocytosis it is lower. Microscopic Differential(n=200) % x109/LSegmented 45 3.9Lymphocytes 39 3.3Monocytes 11 0.9Eosinophils 3 0.3Basophils 2 0.2RBC morphology:Spherocytes ++Howell-Jolly bodies +3517.AnisocytosisScatterplot and HistogramsNoteworthy in the Scatterplotis the population in the lowerleft of the lymphocyte fieldand the population ofneutrophilic granulocytes.The instrument indicatesnucleated RBC and Pltaggregates. Both flags areconfirmed by the differentialcount.The *R alert for WBC, i.e., the measurement of particlesin the region of the 35 fLthreshold can be attributed tothe presence of lysis-resistanttarget cells and the plateletaggregates. In the chamber count, theWBC read was 5.1 x 109/L.The slight upward shift in theneutrophil populationindicates the presence of immature granulocytes, in this case only 5% bandsand 1% myelocytes.The RBC distribution curveshows an atypical wideningwith an increased RDW of 21.4%. This corresponds to the anisocytosis in themanual differential bloodcount.The Plt curve is normal, the Plt count diminished.Clinical Data58-year-old female in a state of incipientdelirium tremens following alcoholwithdrawal after many years of alcohol abuse.36Suspect Flags Definitive FlagsWBC Monocytosis %RBC Nucleated RBCPLT Platelet aggregatesCoulter VCSTMTechnologyClinical Case StudiesDIAGNOSISCommentAnisocytosis of the erythrocytes with macrocytes, target cells, basophilic stippling and pappenheimerbodies.Decompensated alcoholic cirrhosis of the liverDelirium tremensPortal vein thrombosisAdditional TestsWBC (chamber count):Epigastric sonogram:Bilirubin:Staining of blood smear for iron:5.1 x 109/LRecent portal vein thrombosis, cirrhosis of the liver, ascites, normal-sized spleen.Elevated: total 90 mol/L, direct 63 mol/LSiderocytes.Thenoteworthysmall-volumecellpopulationintheScatterplotiscomposedofnormoblasts, giant platelets, and thrombocyte aggregates as well as possible lyse-resistant macrocytes and target cells. Because of their volume and light scatteringproperties,thesecellswerecountedaslymphocytesandraisedtheleukocytecount by 2.3 G/L. The R alert and the suspect flags should be understood as arequest for controlling the leukocyte count and differentiation.TheHowell-Jollybodiesweretheresultofalossofspleenfunctionwithfreshportalveinthrombosis.Pappenheimerbodies(irondeposits)intheerythrocytes,for example, are an indication of sideroachrestic disorders in cirrhosis of the liver.Microscopic Differential% x109/LMyelocytes 1 0.1Bands 5 0.4Segmented 61 4.5Lymphocytes 8 0.6Monocytes 21 1.6Plasma cells 3 0.2Normoblasts 1 0.1RBC morphology:Anisocytosis+Poikilocytosis+Schistocytes+Target cells +Howell-Jollybodies +Basophilic stippling +Pappenheimerbodies +Siderocytes +Special Observations:Giant platelets and plateletaggregates3718.Essential ThrombocytemiaScatterplot and HistogramsThe Scatterplot shows thelymphocyte and neutrophilicgranulocyte populationspractically merging into oneanother. There are no suspectflags for the WBC.Microscopic values correlatewell with the automateddifferential. The Plt count isgiven as ++++, meaning thatthe linearity limit of 1000 x109/L has been exceeded. Thehigh Plt count may havecaused interference. For thisreason, the WBC count,MCH, MCHC, and Hgb havebeen flagged R. Afterdilution of the blood, a countof 1988 x 109/L is obtained.The RBC curve is widened,with an elevated RDW.Particles are identified in the< 50-fL region. This mayindicate aggregated or giantPlt. (With Plt counts of 2000 x109/L, there are always smallaggregates.) Anisocytosis isconfirmed by smear. MCV isborderline low. Clinical Data68-year-old female, suffering for several yearsfrom headache and dizziness.Clinical examination reveals slightsplenomegaly.38Suspect Flags Definitive FlagsWBC LeukocytosisRBCPLTDIAGNOSIS Chronic myeloproliferative syndrome (MPS with essential thrombo-cythemia)Coulter VCSTMTechnologyClinical Case StudiesCommentVery distinct thrombocytosis and aniscocytosis ofthe thrombocytes together with macrothrombocytes.Very little anisocytosis of the erythrocytes.Additional TestsPlt count (after dilution):Bone marrow cytology:Neutrophil alkaline phosphatase index:Serum erythropoietin:Serum ferritin:1988 x 109/L Bone trabeculae normal. Cellularity increased by 80%. Megakaryocytes signifi-cantly increased, some large forms, some nests. Erythropoietic and granulocyticmaturation, E:G = 0.2:1. Increased eosinophils.Normal (61)Significantly decreased Normal (58 g/ml)Amassiveincreaseinthrombocyteswasdetectedafterthebloodsamplewasdiluted. Micro thrombocytes and small thrombocyte aggregation was visible onthe blood smear. These aggregates are responsible for deforming the erythrocytehistogramintheregionbelow50fl. Theanisocytosisconfirmedthesefindings.Thesechangesarenotvisibleontheplateletdistributioncurve. Theleukocytecount and hemoglobin concentrations were increased possibly as a result of thethrombocytosisandaggregation.Nocomparisonwithreferencemethodswereperformed in the absence of any suggested enumeration differences.Microscopic Differential% x109/LBands5 1.1Segmented 84 18.1Lymphocytes 4 0.9Monocytes 3 0.7Eosinophils 1 0.2Basophils 3 0.7RBC morphology:Anisocytosis ++Poikilocytosis+Polychromasia +Target cells+Special Observations:Giant Plt +39Africa/Middle East/Eastern Europe: Switzerland, Nyon (41) 22 994 0707. Australia, Gladesville (61) 2 9844 6000. Canada, Mississauga (1) 905 819 1234.China, Beijing (86) 10 6515 6028. Hong Kong (852) 2814 7431, 2814 0481. France, Villepinte (33) 1 49 90 90 00. Germany, Krefeld (49) 2151 33 35. Italy, Milan (39) 02 953921.Japan, Tokyo (81) 3 5404 8424. Latin America (1) (305) 380 4709. Mexico, Mexico City (525) 575 6805. Netherlands, Mijdrecht (31) 297 230630. Singapore (65) 339 3633.South Africa, Johannesburg (27) 11 805 2014. Sweden, Bromma (46) 8 98 53 20. Switzerland, Nyon 0800 850 810. Taiwan, Taipei (886) 2 2378 3456.UK, High Wycombe (44) 1494 441181. USA, Brea, CA (1) 800 352 3433, (1) 714 993 5321.www.beckmancoulter.com Copyright 1999 Beckman Coulter, Inc. PRINTED IN EUROPETM