anemii anemii pdf 008

ABSTRACT

This review attempts to clarify the criteria used to diagnoseand classify anemias. Because Abbott Cell-Dyn masters most ofthe technologies involved in anemia diagnosis and classification,data generated on various Cell-Dyn hematology analyzers wereused to demonstrate the effect of technology differences on report-ed erythrocyte parameters. In this study, a pulse-editing system(Cell-Dyn 3700) and 2 hydrodynamic focused analyzers (Cell-Dyn 4000 and 3200) are included. The Cell-Dyn 4000 systemreports red cell parameters by impedance and the Cell-Dyn 3200system by light scatter analysis. Anemia is diagnosed by hemoglo-bin and hematocrit (H&H) using the World Health Organizationlimits for hemoglobin (male, 13 g/dL; female, 12 g/dL) and hema-tocrit (male, 0.39; female, 0.36). The various automated red cellparameters involved in anemia work-up are defined and referenceranges are provided. The value of the rule of 3 is discussed inmonitoring the performance of the H&H along with its relation-ship to the mean cell hemoglobin concentration (MCHC), or cellchromicity. Anemia classification based on the mean cell volume(MCV) and MCHC is presented, including various factors thataffect the MCV across the different technologies, such as physio-logic changes (oxygenation and blood aging) and various redblood cell pathologies, that result in inaccurate results. TypicalMCVs and red cell parameters for different anemias are given.Red cell deformation and viscosity effects on the MCV andMCHC measurements across the different technologies are dis-cussed in detail and show that the MCHC from hydrodynamical-ly focused systems is a clinically useful parameter, whereas theMCHC from pulse-editing systems should be used only as a qual-ity control parameter. The relevance of a clinically useful MCHCand accurate MCV in the classification of anemias is documented

with this evaluation’s Cell-Dyn data and compared with pub-lished information. In summary, hydrodynamically focusedhematology analyzers provide accurate MCV and MCHC resultsin the classification of anemias. Lab Hematol. 2000;6:93-108.

KEY WORDS: Anemia · Anemia classification ·Anemia diagnosis · Cell-Dyn ·H&H · HCT · Hb · IRF ·MCH · MCHC · MCV ·Microhematocrit · RDW · Retic

INTRODUCTION

Because many users expressed the need to better understand theCell-Dyn red cell indices and their usefulness in the diagnosis andclassification of anemias, we have attempted to provide a clinicallyoriented, up-to-date review of the red cell parameters, focusing onCell-Dyn technology.

ANEMIA DIAGNOSIS

Anemia is the most common hematologic disorder. Twenty per-cent of all hospital admissions among the elderly are due to anemia[1]. Anemia is best defined in relation to H&H (hemoglobin [Hb]and hematocrit [HCT]) levels below the normal reference range,because a patient’s symptoms and physiologic consequences are theresult of decreased oxygen-carrying capacity of the blood. Accord-ing to World Health Organization (WHO) criteria [2], anemia isdiagnosed in males when Hb is <130 g/L (13 g/dL) and HCT is<0.39 (39%); in females, when Hb is <120 g/L (12 g/dL) andHCT is <0.36 (36%).

Reference intervals for red cell measurements vary with testmethodology, environmental factors, sex, and age. Reference inter-vals are based on statistical studies of a representative sample of ahealthy population [3,4]. Table 1 shows recently published normal-range values for adult and pediatric specimens [5]. Table 2 summa-rizes the central 95% reference intervals obtained on Cell-Dyn ana-lyzers. Some minor differences, similar to published differences, are

Anemia Diagnosis, Classification, and Monitoring UsingCell-Dyn Technology Reviewed for the NewMillennium

L. VAN HOVE,1 T. SCHISANO,1 L. BRACE2

1Abbott Laboratories, Diagnostics Division, Hematology, Santa Clara, California; 2University of Illinois at Chicago, Department ofPathology, Division of Hospital Laboratories, Chicago, Illinois

Laboratory Hematology 6:93-108© 2000 Carden Jennings Publishing Co., Ltd.

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Correspondence and reprint requests: Luc Van Hove, Medical Director,Hematology, Abbott Laboratories, 5440 Patrick Henry Dr., Santa Clara,CA 95054Received 30 July 1999; accepted 30 July 1999

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Official Publication

94 L.Van Hove et al

observed between the 2 data sets [4,5]. We collected a confirmatorysample of 93 normal specimens to test the reference intervals on theCell-Dyn systems against the published data. In this data set, nor-mal is defined as healthy adult volunteers from Abbott Laboratoriesin Santa Clara, California, and includes specimens from 45 femalesand 48 males. The specimens were analyzed as fresh samples across 3Cell-Dyn systems and the spun microhematocrit. The Cell-Dyn4000 system was calibrated with Hemcal and used as the primaryinstrument. The other systems were whole blood cross-calibrated tothe Cell-Dyn 4000. The mean cell volumes (MCVs) were calibratedby whole blood cross-calibration to the uncorrected spun microhe-matocrit value from normal fresh samples. The red blood cell (RBC)count from the primary Cell-Dyn 4000 and the measured manualhematocrit were used to calculate the manual MCV.

Table 2 shows typical hemoglobin and hematocrit referenceintervals for healthy adult males and females derived from the dif-ferent Cell-Dyn systems. These Hb and HCT ranges agree with thepublished values and the WHO anemia definition. Moreover, thedata confirm the need to use separate Hb and HCT reference inter-vals for males and females. The slightly lower values at the upper

end of the normal limits could be the consequence of the smallpopulation size, blood collection in the morning after breakfast, ahigh frequency of runners (Californian fitness madness), and thelow frequency of smokers. Minor differences, which are not clini-cally significant, can be seen between the different automatedmethods. Spun HCTs and automated HCTs agree well within ±3%HCT units. Further investigation of a larger sample of a healthypopulation is required to better understand differences in red cellparameters for age, sex, ethnicity, and geography.

Conditions associated with anemia can be subdivided into 2groups on the basis of whether the change in red cell mass (RCM)is absolute or relative. In absolute anemia there is a true decrease inRCM. In relative anemia, there is a fluid shift from the extravascu-lar to the intravascular compartment, expanding the plasma volumeand diluting the RCM. This is usually seen in association withpregnancy and hyperproteinemia. Relative erythrocytosis is theresult of decreased plasma volume and is most commonly seen indehydration. These relative changes must be differentiated fromabsolute anemias [6]. Table 3 summarizes variables that affect thehematocrit.

TABLE 1. Representative Normal Reference Intervals by Patient Age and Sex, Central 95% Ranges*

Parameters Adult Male Adult Female Birth (Cord Blood) 1–3 Days (Capillary Blood) 0.5–2 Years 2–6 Years

RBC, �1012/L† 4.5–5.9 4.0–5.2 3.9–5.5 4.0–6.6 3.7–5.3 3.9–5.3Hb, g/dL†‡ 13.5–17.5 12.0–16.0 13.5–19.5 14.5–22.5 10.5–13.5 11.5–13.5HCT† 0.41–0.53 0.36–0.46 0.42–0.60 0.45–0.67 0.33–0.39 0.34–0.40MCV, fL 80–100 80–100 98–118 95–121 70–86 75–87MCH, pg 26–34 26–34 31–37 31–37 23–31 24–30MCHC, g/dL 31–37 31–37 30–36 29–37 30–36 31–37

*Reference intervals based on mean values ± 2SD [5]. RBC indicates red blood cell; Hb, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, meancell hemoglobin; MCHC, mean cell hemoglobin concentration.†RBC, Hb, and HCT are slightly lower in 3 significant situations: after age 50, in recumbency (a blood sample taken when the patient is lying down), and aftermeals (as much as 10% lower).Values may be significantly lower in runners than in nonrunners, but seldom reflect true anemia.‡Hb may be elevated in heavy smokers because of the increase in carboxyhemoglobin, which is not capable of carrying oxygen to tissues.

TABLE 2. Representative Normal Reference Intervals by Analyzer, Central 95% Ranges*

Cell-Dyn 4000 Cell-Dyn 3200 Cell-Dyn 3700

Male Female Male Female Male Female

RBC, �1012/L 4.3–5.5 4.0–5.3 4.3–5.5 4.0–5.0 4.3–5.6 4.0–5.0Hb, g/dL 13.2–17.0 12.2–14.8 13.2–15.9 12.3–15.1 13.1–15.9 12.2–15.0HCT* 0.39–0.47 0.36–0.45 0.40–0.48 0.37–0.44 0.39–0.47 0.36–0.44MCV, fL† 82–94 82–97 81–95 82.5–97 81–94 83.5–95MCH, pg 26.8–32.4 27.4–32.5 26.9–32.2 26.7–32.9 27.1–32.1 27.4–32.5MCHC, g/dL† 31.9–35.1 32.3–35.9 31.8–36.0 31.3–35 32.8–34.7 32.6–34.4RDW, %CV 10.9–14.2 10.8–13.6 11.4–15.4 11.5–15.4 12.1–16.1 12.1–16.3Retic, 109/L 36.6–108 33.9–112 — — — —Retic, % 0.5–2.0 0.8–2.6 — — — —IRF, ratio 0.16–0.35 0.14–0.31 — — — —

*Reference intervals based on the 2.5 and 97.5 percentiles of a healthy adult population in Silicon Valley consisting of 48 males and 45 females. RBC indicatesred blood cell; Hb, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; RDW,red cell distribution width; %CV, coefficient of variation; Retic, reticulocytes; IRF, immature reticulocyte fraction.†Manual reference intervals derived from the spun microhematocrit and the Cell-Dyn 4000 RBC count: HCT 0.395–0.505 (male), 0.36–0.46 (female); MCV82–96 (male), 83–98 (female); MCHC 31.3–35.5 (male), 31.5–35.3 (female).

Anemia Diagnosis, Classification, and Monitoring 95

DEFINITIONS AND TECHNOLOGIES

Hb: Hemoglobin concentration; an estimate of the oxygen-carryingcapacity of the blood. The International Committee for Standardiza-tion in Hematology (ICSH) [7] and the National Committee forClinical Laboratory Standards (NCCLS; document H15-A 2) [8]recommend the cyanmethemoglobin method for Hb determination.On the Cell-Dyn systems tested, 2 cyanide-free Hb methods areused. The Cell-Dyn 4000 system measures hemoglobin with amethemoglobin method using imidazole as the heme-ligand. Themethod is fast (10 seconds) and sensitive and has less interference

TABLE 3. Variables That Affect the Hematocrit

Increased Hematocrit Decreased Hematocrit

Dehydration Volume overloadCapillary specimens Supine positionProlonged tourniquet stasis Capillary tube leakageExposure to cold Excess anticoagulantIncreased muscular activity Automated techniquesUpright position PregnancyCentrifugation techniques Hyperproteinemia

A B

FIGURE 1. Automated red blood cell (RBC) analysis by different technologies. A. Electrical resistance or impedance measurementusing the Coulter principle has several inherent limitations, including coincident passage loss, nonaxial passage, and cell recircula-tion. B. Impedance analysis with the Von Behrens transducer minimizes the recirculation of cells. C. Impedance measurement ofhydrodynamically focused and sphered RBCs passing through the aperture of a Cell-Dyn 4000 transducer generates superior RBCparameter results. D. Optical light scatter analysis of hydrodynamically focused and sphered RBCs passing through the laser lighton Cell-Dyn 3200 or Cell-Dyn 4000 systems. The Cell-Dyn 3200 system measures all parameters except hemoglobin by opticallight scatter analysis. The Cell-Dyn 4000 uses a combination of light scatter, impedance, and fluorescence analysis. The reportableRBC count and RBC indices are impedance-derived. An optical RBC count is provided for autovalidation purposes.

DC

96 L.Van Hove et al

from a high white blood cell (WBC) count than the cyanmethe-moglobin method. The reagent (NaOH and lauramine oxide) dis-solves all cellular particles and lipids and destroys bilirubin. Themethod, therefore, has no Hb turbidity interference with WBC<250�109/L, lipids (cholesterol and triglycerides) <700 mg/dL,bilirubin <33 mg/dL, or paraproteins found in Waldenstrom’smacroglobulinemia and multiple myeloma. Consequently, thismethod provides more reportable results than any other method.The Cell-Dyn 3000 systems use a methemoglobin method withhydroxylamine as a direct oxidant and a quaternary ammoniumsalt for stable chromogen formation. Both cyanide-free methodscorrelate well (r = 099) [9,10] with the cyanmethemoglobin refer-ence method.HCT: hematocrit value or ratio; also called packed cell volume(PCV). A measure of the relative volume occupied by RBCs in cap-illary or venous whole blood samples [11]. The HCT reflects, there-fore, the body’s RCM divided by the total blood volume. The HCTis used to detect the presence or absence of anemia and poly-cythemia. The HCT can be determined by 2 methods: mechanical-ly, by spinning the cells in a microcentrifuge, and electronically,using an automated cell counter. The spun microhematocrit suffersfrom an inherent problem of plasma trapping and is often slightlyhigher (0.01–0.03 or 1–3%) than the automated hematocrit [12].This phenomenon causes an erroneously high manual HCT in sam-ples with deformed RBCs such as sickle cells. In normal samples,the microhematocrit and the electronic HCT should agree within±3% (HCT units). Although the spun microhematocrit has limita-tions, it is the reference method for HCT measurements. The hema-tocrit on Cell-Dyn hematology analyzers is a calculated parameterexpressed as percent or L/L (SI units) derived from the followingequation: HCT (L/L) = RBC (1012/L) � MCV (fL)/1000.RBC: Red blood cell count; a measure of the number of red cellsper liter of whole blood. RBCs carry hemoglobin into close contactwith the tissues for a successful oxygen exchange. Cell-Dyn systemsgenerate an electronic RBC count by measuring changes in imped-ance through a small aperture or light scatter of cells passingthrough a laser light. The electrical resistance or impedance count-ing principle is an accepted standard in cell counting. This technolo-gy, however, does have inherent problems of nonaxial passage, coin-cident passage, and recirculation of cells during the measurementprocess [13] (Figure 1A). These erratic flow patterns cause erroneouspulses and, therefore, inaccurate results. To minimize these limita-tions, cell counters employ pulse-editing circuitry to remove thelarge and aberrant pulses. The best technique, however, is toimprove the flow pattern through the transducer. The Cell-Dyn3500 and 3700 systems use pulse-editing circuitry in combinationwith the Von Behrens transducer (Figure 1B), which reduces therecirculation of cells. The counted cell stream continues through asecond opening, and the cells are trapped behind a plate preventingrecirculation. Hydrodynamic focusing, an alternative method, wasdeveloped to control both the number and orientation of cells pass-ing through the sensing zone [13]. A sheath stream forces the cells toenter the sensing zone in single file and provides a high number ofsingle cells for analysis without the erroneous pulses found in tradi-tional impedance counting. This focusing technology is used forsome electronic impedance methods and for optical flow cytometry(Table 4). Sphering the red cells further optimizes the pulse meas-urement, which is a necessity for optical RBC analysis. Manufactur-ers of cell counters are using different technologies to count RBCs

and measure red cell volume (RCV). Technologists familiar with aparticular technology may not be aware that another model uses adifferent technology with different limitations. Table 4 illustrates thetechnology differences in RBC analysis.MCV: Mean cell volume; a measure of cell volume (in fL) that ismore reproducible than the RBC size information obtained from theblood film. Clinicians arbitrarily use a range of 80–100 fL as normal.MCV is very useful in determining the anemia type, because only afew pathologies cause abnormal MCV results. In conditions of rapidblood loss or chronic disease, the MCV will be normal with a lowHCT. Iron deficiency and thalassemia show a decreased MCV. Folateor vitamin B12 deficiency, alcoholism, and myelodysplastic syndromeresult in an increased MCV. Because only the mean cell volume ismeasured, a small but significant degree of microcytosis or macrocy-tosis and the presence of anisocytosis can be missed.

On the tested Cell-Dyn hematology analyzers, MCV analysis isslightly different, depending on the technology used. Minor differ-ences are observed between male and female specimens (Table 2),and the 95% reference intervals of the systems are tighter than thearbitrary MCV limits. The upper limit of the Cell-Dyn 3500 and3700 systems (pulse-editing) is lower compared with the systemsthat use hydrodynamic focusing and the reference method.

The Cell-Dyn 4000 system measures RBC volume by imped-ance on hydrodynamically focused and sphered RBCs (Table 4).The RBC, MCV, and red cell distribution width (RDW) are meas-ured on the platelet (PLT)-RBC histogram using 2 dynamic thresh-olds to separate RBCs from PLT and WBC events.

The Cell-Dyn 3200 system measures RBC volume by 3-dimen-sional optical light scatter analysis on hydrodynamically focusedand sphered RBCs (Table 4). Individual RBCs are identified bytheir light scatter characteristics at the 0-, 90-, and 10-degreeangles. Multiparametric analysis of the RBC is used to define theMCV. WBC and coincident events are automatically excludedfrom the MCV analysis.

The Cell-Dyn 3500 and 3700 systems measure RBC volume byimpedance on RBCs maintained in their native state. Erroneouspulses of nonaxial passage, coincident passage, and recirculation areeliminated by pulse-editing circuitry. The MCV is defined as themean of the RBC histogram that is derived from the RBC popula-tion between the lower PLT-RBC threshold and channel 254.RDW: Red cell distribution width; a measure of red cell hetero-geneity or anisocytosis. On Cell-Dyn systems, it is expressed as the

TABLE 4. Technology Differences in Red Blood Cell Analysis*

Instrument Technology Editing Focusing Sphering

Abbott Cell-Dyn 4000 Impedance • •Abbott Cell-Dyn 3200 Optical • •Abbott Cell-Dyn 3500 Impedance •Abbott Cell-Dyn 3700 Impedance •Bayer Advia 120 Optical • •Sysmex SE-9000 Impedance •Beckman-Coulter Gen•S Impedance •

*Cell-Dyn is a registered trademark of Abbott Laboratories;Advia is a reg-istered trademark of Bayer Corporation; SE-9000 is a trademark of ToaCorporation; Gen•S is a trademark of Beckman-Coulter.


coefficient of variation (%CV) of the MCV after excluding the tailof the size distribution curve. RDW helps to distinguish betweeniron deficiency (high RDW) and thalassemia minor (normalRDW), provided a normal reference interval is established. Table 2shows typical normal range values for the different Cell-Dyn sys-tems tested. Note that hydrodynamically focused systems havelower normal range values than electronic pulse-editing systemsbecause fewer erroneous pulses are generated. MCH: Mean cell hemoglobin; a measure of the average amount ofHb in each individual RBC, using the equation MCH (in pg) =Hb (g/L)/RBC (1012/L).MCHC: Mean cell hemoglobin concentration; a measure of thecell chromicity or Hb concentration in each RBC. MCHC is alsoused as a quality control parameter to monitor stable instrumentperformance similarly to the rule of 3. Table 2 shows minor differ-ences between male and female specimens and a tighter normalrange for pulse-editing analyzers as for hydrodynamically focusedsystems. MCHC is calculated using the following equation:MCHC (g/L) = Hb (g/L) � 1000/RBC (1012/L) � MCV (fL).Reticulocytes: Transitional red cells between nucleated red cells andso-called mature erythrocytes [14]. A reticulocyte is an erythrocytethat, when stained with a supravital or fluorescent dye, containsstainable nucleic acids (cellular RNA). The reticulocyte countmeasures erythropoietic activity and therefore the bone marrowresponse to anemia. The reticulocyte count helps to distinguishbetween peripheral bleeding or hemolysis and decreased marrowproduction. The immature reticulocyte fraction (IRF) measureschanges in the rate of the erythropoietic response. Cell-Dyn analyz-ers enumerate reticulocytes by 2 different methods.

The Cell-Dyn 4000 system uses a proprietary CD4K530 nucle-ic acid fluorochrome [15,16] that binds efficiently to RNA andDNA, allowing reticulocytes to stain in 20 seconds and eliminatinginterference from DNA-containing cells. This system allows fullautomation of the reticulocyte assay. RBCs and reticulocytes areidentified on the reticulocyte cytogram in the RBC region by7-degree light scatter and RNA green fluorescence. A valley-findalgorithm separates the fluorescent reticulocytes from the nonfluo-rescent RBCs on the RNA fluorescence histogram. Highly fluores-cent immature reticulocytes (IRF) are separated from more maturereticulocytes by a discriminator line set 30 channels above theRBC-reticulocyte threshold. This results, on the average, in 65 �106/L reticulocytes ± 42 (2SD) or 1.4% reticulocytes ± 0.93 (2SD)and a mean IRF of 0.24 ± 0.11 (2SD) for normal specimens.Because reticulocytes are rare event counts that show a non-Gauss-ian distribution (data not shown), the central 95% reference inter-val is best expressed by the 2.5 and 97.5 percentiles as shown inTable 2. Moreover, adult males and females have slightly differentreticulocyte values as shown.

Cell-Dyn 3500 and 3700 systems enumerate reticulocytes by asupravital new methylene blue procedure [17]. Reticulocytes arestained offline for a minimum of 15 minutes. They are then run onthe analyzer, and the degree of reticulum is measured using lightscatter analysis. RBCs and reticulocytes are separated from non-erythroid cells using 3-dimensional radial analysis of the light scat-tered across the 0-, 10-, and 90-degree angles. Light scattering retic-ulocytes are separated further from RBC by radial analysis of the 0-and 10-degree scatter. Immature reticulocytes (IRF) are separatedfrom more mature reticulocytes by histogram analysis using the 0-and 10-degree light scatter to define the discriminator line. On a

limited data set of 32 normal samples, an average reticulocytecount of 87 � 106/L ± 31 (2SD), a percent reticulocyte of 1.8 ±0.6 (2SD) and a mean IRF of 0.29 ± 0.6 (2SD) was obtained.These preliminary reference intervals, in general, are in agreementwith the published values for the new methylene blue method[4,14] but are higher than those for the fluorescence methods.Supravital stains are known to lack the sensitivity of the fluorescentRNA stains in identification of low numbers of reticulocytes and indiscrimination of weakly stained reticulocytes from RBCs [14],resulting in higher reference intervals.

THE RULE OF 3

Over the years, clinicians and technologists have used a quickmathematical check to ensure that the patient’s Hb and HCT valuesare consistent. The HCT should be roughly 3 times the Hb [18].This simple formula only applies to normocytic normochromicspecimens, which can be monitored by the HCT/Hb ratio, typically3 ± 0.08 (1SD) on Cell-Dyn systems. On the tested Cell-Dyn 4000,3700, 3500, and 3200 systems, this ratio was within the range of2.8 to 3.2. The calculated HCT, derived from multiplying the Hbtimes 3, and the reported HCT values (H&H) from Cell-Dyn sys-tems are typically within the narrow range of ±3% (HCT units). Ifthe calculated HCT does not agree with the reported HCT, the sys-tem could be miscalibrated or malfunctioning or the sample haspathology requiring further investigation.

ANEMIA CLASSIFICATION

Anemia diagnosis, based on Hb and HCT values below the nor-mal reference ranges, is followed by anemia classification based on1) MCV, 2) mechanism, and 3) patient’s symptom list.

Examination of the peripheral blood film is not better thanRBC indices from the automated hematology analyzer in helpingdiagnose anemia types [1,6,19]. RBC morphology providesunique diagnostic information in only 4 to 6% of anemia casesand assists diagnosis in 25% [1]. Also, automated RBC indices areassociated with too many false-positive and false-negative results tobe of great practical use on their own. The diagnosis of a specificanemia will, therefore, depend on the test characteristics and theprevalence of the anemia. The most common type of anemia isiron-deficiency anemia, followed by thalassemia and anemia ofchronic disease [1]. An effective diagnostic workup therefore startswith the question: Does this patient have iron-deficiency anemia?Seventy five percent of all hospital anemias are caused by iron defi-ciency or by a chronic disease [1].

The following automated RBC parameters are important in thediagnosis of anemia types: Hb, HCT, MCV, RDW, MCH,MCHC, reticulocytes, and IRF.

As early as 1934, Wintrobe [20] presented a scheme for classify-ing anemias morphologically, based on calculated RBC indices; thescheme became the basis for classifying anemia into 3 categories: 1)normocytic (MCV 80 to 100 fL); 2) microcytic (MCV <80 fL),and 3) macrocytic (MCV >100 fL). By convention, a broad normalrange is used that helps identify the etiology, because anemias withabnormal MCVs are caused by only a few conditions. Table 5shows the morphologic classification of anemias in a format widelyused for many years. Today, RBC indices are measured directly orcomputed automatically by sophisticated electronic cell counters.

98 L.Van Hove et al

Although results are now more reliable, the reported indices are stillonly as good as the methods used to measure and calculate them.Electronic determination of RBC indices has increased the clinicalusefulness of the MCV and decreased that of the MCH andMCHC. The MCV is derived directly from RBC size distributiondata. The HCT, MCH, and MCHC are calculated. The MCHvaries in a linear relationship with the MCV and provides no addi-tional diagnostic information. On analyzers using the pulse-editingprinciple, the MCHC is clamped around the mean [22] and losesits sensitivity as an indicator of iron deficiency. Although the MCHand MCHC have lost their clinical value on such analyzers, all

RBC indices are useful quality control tools and aid in the detec-tion of instrument malfunctions.

MEAN CELL VOLUME

Of the measured red cell parameters, the MCV analysis variesthe most between technologies. To generate accurate MCV results,several requirements have to be met: the correct amount of bloodhas to be collected in a K3EDTA- or K2EDTA-anticoagulated con-tainer to prevent anticoagulant under- or overfilling effects [23,24];the right reagent mix has to be used; and the instrument gain set-ting has to be optimal.

The last is achieved by running latex particles of a particularsize, typically 5 µm. The generated impedance or optical scattersignal has to be set in a particular channel to allow algorithms tocorrectly measure the RBC size information. The instrument set-ting is then refined by the calibration procedure. Typically, stabi-lized blood with assigned values is used for calibration. Theassigned values are derived from whole blood cross-calibration of amanufacturer’s reference instrument to the reference methods. ForMCV, the reference method is the spun microhematocrit. A biasin assigned values on a calibrator will be propagated into thepatient results. This explains the minor differences seen in calibra-tor-calibrated analyzers vs. whole blood cross-calibrated systemsagainst a primary analyzer. In a laboratory with multiple hematol-ogy analyzers, typically the most advanced instrument will beselected as the primary analyzer and be calibrated with the manu-facturer’s recommended calibrator. The other analyzers are thenwhole blood cross-calibrated against the primary instrument. Thisapproach allows MCV reporting across technology platforms with-in a 2-fL difference for normal specimens (Table 6). It also helpsreduce variation in the calculated HCT, which is within 0.02 (2%)for normal samples. Under these conditions, the rule of 3 (dis-cussed above) runs tightly around the mean HCT value ±3%.Paired difference analysis of the RBC parameters across hematol-ogy analyzers allows monitoring of tight cross-calibration of theinstruments. The clinical advantage of maintaining accurate cali-brated platforms is the identification of acute bleeding demon-strated by a drop of 3% or more in the HCT value.

TABLE 6. Typical Data From Normal Specimens Run on Cross-Calibrated Analyzers*

Instrument RBC, �1012/L Hb, g/dL HCT MCV, fL MCH, pg MCHC, g/dL CV of RDW, %

Cell-Dyn 4000-1† 4.67 13.8 0.417 89.4 29.6 33.1 12.1Cell-Dyn 4000-2 4.65 13.9 0.412 89.5 29.9 33.4 12.1Cell-Dyn 3200-1 4.66 13.8 0.417 89.8 29.7 33.1 13.0Cell-Dyn 3200-2 4.69 13.9 0.422 90.2 30.0 32.8 13.1Cell-Dyn 3500-1 4.66 13.8 0.415 89.2 29.6 33.2 14.2Cell-Dyn 3500-2 4.71 13.8 0.420 89.3 29.4 32.9 14.5Spun hematocrit 0.420 90.1‡ 32.8‡

*Data derived from 40 healthy in-house donor specimens run in duplicate on all systems. RBC indicates red blood cell; Hb, hemoglobin; HCT, hematocrit;MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; RDW, red cell distribution width; CV, coefficient of vari-ation.†Reference instrument calibrated with Hemcal lot #118 and whole blood calibrated for MCV against the spun microhematocrit. The other systems arewhole blood cross-calibrated against the reference instrument.‡Manual MCV and MCHC derived from microhematocrit packed cell volume and the Cell-Dyn 4000-1 RBC and Hb.

TABLE 5. Morphological Classification of Anemias*

Microcytic (MCV <80 fL)Commonly microcytic Occasionally microcytic

Iron deficiency Anemia of chronic diseaseThalassemia HemoglobinopathiesHereditary sideroblastic anemia

Macrocytic (MCV >100 fL)Commonly macrocytic Occasionally macrocytic

Folic acid deficiency Hypoproliferative anemiaVitamin B12 deficiency Refractory anemiaLiver diseaseHemolytic anemiaBlood loss anemia

Normocytic (MCV 80–100 fL)Commonly normocytic Occasionally normocytic

Hypoproliferative anemia Early iron deficiencySecondary anemia in malignanciesRefractory anemia in myelodysplasiaHemolytic anemiaHemoglobinopathiesBlood loss anemiaAnemia of chronic diseaseAcquired sideroblastic anemia

*From Knapp [21]. MCV indicates mean cell volume.


PHYSIOLOGIC CHANGES IN MCV VALUES

Several conditions affect the MCV analysis. The most frequentare discussed below.

OxygenationA literature search revealed only 1 study describing the oxygena-

tion/deoxygenation effects on impedance analyzers [25]. Therefore,further investigation was performed using impedance and opticalCell-Dyn analyzers.

Freshly drawn, closed specimens are only partially oxygenated.This state represents the typical sample analyzed on the hematologyanalyzer. When fresh normal samples are run immediately or after10 minutes of intense oxygenation on different Cell-Dyn systems, adownward MCV shift of 0.9 fL can be seen on the Cell-Dyn 4000system between partially and fully oxygenated blood. The Cell-Dyn3500 system (without hydrodynamic focusing) reports a drop ofonly 0.6 fL and lacks the sensitivity to discriminate between the 2results. The Cell-Dyn 3200 system (with hydrodynamic focusing)reports a change of 1.3 fL, whereas the true MCV change as deter-mined by the microhematocrit is 1.9 fL. Therefore, the Cell-Dyn3200 result best reflects truth. Similar observations were made in arepetitive sampling experiment on these analyzers. Samples wererun in open mode after airing and inversion-mixing (Figure 2).After 2 hours, the MCV dropped on both the Cell-Dyn 4000 and3200 systems by 1.6 fL, by 0.5 fL on the Cell-Dyn 3700, and by2.5 fL on the manual MCV method. After 4 hours, the MCV onthe Cell-Dyn 4000 and 3200 systems dropped by 2.3 fL, by 0.7 fLon the Cell-Dyn 3700, and by 5.9 fL on the manual MCVmethod. The MCV decrease leveled on the Cell-Dyn 4000 and3200 systems after 4 hours, whereas on the Cell-Dyn 3700 andmanual MCV method, the decrease continued to 1.6 fL and8.2 fL, respectively, after 4.5 hours. A similar pattern to that of thestressed oxygenation experiment is seen: hydrodynamically focusedsystems are more sensitive to real oxygenation changes in red cellvolume. Moreover, in this experiment on the automated systems,

the MCV changes after 4 hours of aeration are not clinically signifi-cant and are limited to half the change of the reference method. Upto 4 hours, HCT and MCHC values on the automated systems didnot change by more than 0.5 and 1.4 g/dL, respectively. After4 hours, blood analyzed in closed tubes shows no significantly dif-ferent MCV results (<3 fL). The magnitude of the oxygenationeffect depends on the analyzer, specimen tube type, intensity, andtime of aeration. Up to 4 fL MCV change was observed on hydro-dynamically focused systems.

Similar oxygenation/deoxygenation effects on the MCV, HCT,and MCHC have been described for Sysmex impedance analyzers[25]. Our data indicate that for short-term imprecision studies ofMCV and HCT, either the sampling from a single tube should berestricted to 5 measurements and a second tube should be used forthe next 5 measurements, or only completely oxygenated bloodshould be used. Blood should be oxygenated for at least 4 hourswhen mixed in an opened container or stress-oxygenated for>1 hour. Because the MCV is stable on a freshly collected andcapped specimen, the first option is the simplest and represents bestthe typical workflow in the routine hematology laboratory. It is,therefore, also recommended that the MCV on hematology analyz-ers be calibrated with freshly collected and capped whole bloodspecimens from normal donors. Analyzers should be calibratedagainst the microhematocrit, through either assigned calibrator val-ues or whole blood cross-calibration. The use of completely oxy-genated blood for calibration, as suggested in the literature [25],would introduce an oxygenation bias (up to 4 fL) in the clinicalspecimens, because they are only partially oxygenated. Our dataalso show that the oxygenation effect is best measured by hydrody-

FIGURE 2. Oxygenation effect. K3EDTA-anticoagulated nor-mal blood was pooled in a Falcon 50-mL plastic tube anddirectly sampled, after aeration and inversion-mixing, at dif-ferent time intervals on the different analyzers. MCV indi-cates mean cell volume.

FIGURE 3. Sample aging effect on mean cell volume (MCV)analysis. After storage at room temperature, 5 normal sam-ples were analyzed in duplicate over a period of 48 hours onthe different systems. The average results are shown. At 48hours, the MCV duplicate delta did not exceed 0.7 fL formanual analysis, 0.3 fL on Cell-Dyn 4000 system, 0.4 fL onCell-Dyn 3200 system, and 0.3 fL on Cell-Dyn 3700 sys-tem. For comparison, the MCV changes on the Cell-Dyn4000 system after refrigerated storage are shown for thesame time interval. The other methods showed similarchanges on refrigerated samples.

100 L.Van Hove et al

namically focused systems. The MCV changes that are induced byoxygenation/deoxygenation are primarily due to configurationchanges in the hemoglobin molecule [26]. The hemoglobintetramer has a tighter configuration and a smaller central cavity inthe oxygenated state. Because hemoglobin is the major intracellularprotein in RBCs, this is a logical explanation. A second mechanismconsisting of a shifted water balance might also be involved [25],because the remaining content of the RBC is largely water.

Blood AgingA literature search revealed only a few articles describing the

effects of storage on MCV analysis [23,24,27–30]. These studiesdemonstrate that MCV values increase at different degrees withincreasing storage times at room temperature, independent of thetechnology used. MCVs on samples stored at 4°C are stable for upto 72 hours, which explains the recommendation for storing bloodin the refrigerator.

K3EDTA-anticoagulated blood collected from healthy donorsand stored at room temperature or refrigerated was analyzed over aperiod of 48 hours on impedance and optical Cell-Dyn systems.The microhematocrit method was used as reference. Five sampleswere run in duplicate; the average results are shown in Figure 3.Duplicate deltas were largest at 48 hours of storage and did notexceed the values described in Figure 3. The study demonstrates thathydrodynamically focused systems, using either impedance (Cell-Dyn 4000) or optical (Cell-Dyn 3200) technology, are measuringMCV changes accurately over time compared with the referencemethod. The Cell-Dyn 3700, with pulse-editing, was underestimat-ing the MCV changes. The calculated HCT and MCHC valueswere affected to a lesser extent. Refrigerated blood showed MCVchanges of <3 fL over 48 hours, which is clinically insignificant.Focused flow systems are preferred when clinicians expect to receiveaccurate MCV values. Therefore, when MCV values have to bereported from aged samples stored at room temperature for24 hours, an MCV increase of 5 fL over the fresh sample result hasto be expected. The MCV increases by 8 to 9 fL on the Cell-Dyn4000 and 3200 systems and the reference method after 48 hours.Stable MCV results are obtained by storing the samples in the refrig-erator or at 4°C or analyzing them within 24 hours on less sensitivesystems using pulse-editing. After 24 hours of storage at room tem-perature, the MCV increase becomes clinically significant for theCell-Dyn 4000 and 3200 systems. After 48 hours of storage at roomtemperature, the MCV on the Cell-Dyn 3700 increased by 6 fL.

MCV Inaccuracies Resulting From PathologyIn the literature, several clinical conditions are identified that

affect the MCV analysis to different degrees on all hematology ana-lyzers [31–33]. Briefly, falsely high MCVs can be caused by highWBC counts or the presence of agglutinins. These interferences arecorrected by removal of the buffy coat and replacement with iso-tonic salt solution and repeat estimation. Changes in plasma osmo-larity also affect the MCV analysis. RBCs from patients with severehypernatremia or severe hyperglycemia are hyperosmolar. Whensuch blood is run on an automated hematology analyzer, RBCsswell rapidly because water is moving in faster than electrolytes,glucose, or urea can move out, resulting in an artificially increasedMCV and HCT, with a corresponding decrease in MCHC. Suchphenomena have been observed in hypernatremic dehydration,severe uremia in renal failure, and hyperglycemia due to uncon-

trolled diabetes or intravenous feeding with concentrated carbohy-drates. The opposite effect is seen in hypo-osmolar states such ashyponatremia in chronic alcoholics and patients with inappropriatesecretion of antidiuretic hormone. Dilution of the blood sample inan isotonic solution before analysis allows red cells to equilibrate tonormal external conditions and results in more accurate MCVs.Falsely low MCVs can also be generated when significant numbersof large platelets are present.

MCV in AnemiasTypical RBC parameter results for different anemias are pub-

lished in hematology textbooks [31]. Wintrobe’s ranges for differ-ent anemias are summarized in Table 7 for hematology analyzersbased on the pulse-editing principle. Table 8 shows typical RBCparameter results obtained for different anemia types using a varietyof Cell-Dyn analyzers. All systems, as described above, are wholeblood cross-calibrated to the primary Cell-Dyn 4000 system. TheMCVs on all analyzers are whole blood calibrated to the spunmicrohematocrit.

The data demonstrate that for microcytic and normocytic ane-mias, MCV differences up to 6 fL can be expected between systemsusing different measurement technologies. In macrocytic anemias,the MCV differences can exceed 10 fL. This variation is the conse-quence of the different technologies used. The microcytic and nor-mocytic MCV deltas do not exceed the clinical limit of ±3% HCTand, therefore, are clinically insignificant. If clinicians expect closerMCV results across different hematology analyzers, the only solu-tion is to duplicate the preferred instrumentation. MCV deltas onmacrocytic anemias are driven by the limitations of the differenttechnologies. Pulse-editing methods eliminate many large pulsesand underestimate the MCV on some samples with results in therange of 100–125 fL compared with the manual reference value.Hydrodynamic focusing and sphering methods provide a betterestimate in that range. The Cell-Dyn 4000 system agrees best withthe reference value. The Cell-Dyn 3200 system corrects the MCV

TABLE 7. Typical Ranges for Specific Anemia Pathologies With Pulse-Editing Impedance Technology*

Condition Hb, g/dL MCV, fL MCHC, g/dL

Normal males 16 (14–18) 89 (82–100) 34 (32–36)Normal females 14 (12–16) 89 (82–100) 34 (32–36)Iron deficiency anemia 8 (4–12) 74 (53–93) 28 (22–31)Anemia of chronic disorders 10 (8–13) 86 (70–95) 32 (26–36)Thalassemia minor 12 (9–14) 68 (56–75) 31 (29–33)Thalassemia major (2–7) (48–72) (23–32)Thalassemia hemoglobin H 9 (7–11) 70 (53–88) 25 (24–28)Abnormal Hb, E trait (EA) 14 (12–17) 73 (71–78) 33 (28–36)Abnormal Hb, E disease (EE) 12 (11–15) 64 (58–76) 33 (32–34)Abnormal Hb, C disease (CC) 10 (7–14) 74 (55–93) 32 (23—38)Hereditary sideroblastic 6 (4–10) 77 (49–104) 25 (14 – 30)

anemiaIdiopathic refractory 10 (7–12) 104 (83–118) 32 (26–36)

sideroblastic anemia

*Ranges according to Wintrobe [31]. Hb indicates hemoglobin; MCV, meancell volume; MCHC, mean cell hemoglobin concentration.


value for the hemoglobin viscosity effect (discussed in more detailbelow), resulting in the largest deltas found between the Cell-Dyn3200 and 3700 systems. Figures 4 and 5 provide an explanation forthis discrepancy. On the Cell-Dyn 3200 system, macrocytic, nor-mochromic RBCs (up to 140 fL) agree well with the manual refer-ence value. As shown in Figure 5B, for macrocytic, hypochromicRBCs, the uncorrected MCV is overestimated, driving the value inthe opposite direction for an MCV result affected by the hemoglo-bin viscosity effect. On impedance analyzers, this difference is well

described [22]. On hypochromic RBCs, an underestimated MCVcan be generated by these systems. The effect is greatest in macro-cytic anemias. Figures 6 and 7 demonstrate these differences intechnology. Most of the macrocytic samples analyzed on the Cell-Dyn 3200 system agree with the manual reference values (Figure6A and C and Figure 7B). The Cell-Dyn 3200 3-dimensionalMCV algorithm corrects for this chromicity effect. Figure 7Cdemonstrates that for the Cell-Dyn 3700 system, several macrocyt-ic MCV values are located below the regression line, suggesting a

TABLE 8. Typical Red Blood Cell Results for Different Anemias Analyzed on a Variety of Cell-Dyn Hematology Analyzers*

RBC, Hb, HCT, MCV, MCH, MCHC, CV of Retic, IRF,Condition �1012/L g/dL % fL pg g/dL RDW, % �106/L % Retic ratio

Normal femaleManual — — 38.6 92.1 — 32.6 — — — —CD4000 4.19 12.6 37.8 90.3 30.1 33.3 13.1 73.1 1.8 0.25CD3200 4.15 12.9 37.4 90.1 31 34.4 13.5 — — —CD3700 4.14 12.7 37.2 89.9 30.7 34.1 16.1 — — —

Microcytic hypochromic anemiaManual — — 23.8 56.1 — 31.9 — — — —CD400 4.24 7.6 24.9 58.8 17.9 30.5 19 79.3 1.9 0.55CD3200 4.37 7.42 26.4 60.5 17 28.1 24.8 — — —CD3700 4.23 7.38 25 59.1 17.5 29.5 20.2 — — —

Iron-deficiency anemiaManual — — 35.1 70.3 — 31.3 — — — —CD4000 4.99 11 37 74.2 22.1 29.8 16.8 40.7 0.8 0.34CD3200 4.92 10.7 35.3 71.7 21.8 30.4 21.5 — — —CD3700 4.83 10.6 33.6 69.6 22 31.6 19.4 — — —

�-ThalessemiaManual — — 35.9 63.2 — 32.6 — — — —CD4000 5.68 11.7 36.3 63.9 20.6 32.2 14.5 151 2.7 0.42CD3200 5.67 11.6 36.9 65.1 20.5 31.5 18.9 — — —CD3700 5.48 11.3 35.5 64.7 20.7 31.9 16.9 — — —

Normocytic normochromic anemiaManual — — 32 100.9 — 32.2 — — — —CD4000 3.17 10.3 31.1 98 32.5 33.2 17.5 108 3.4 0.47CD3200 3.35 10.5 33.7 101 31.2 31 18.6 — — —CD3700 3.33 10.5 31.2 93.7 31.5 33.6 18.5 — — —

Sickle cell anemia, crisisManual — — 22.1 97.4 — 35.7 — — — —CD4000 2.27 7.9 21.8 96.3 34.8 36.2 22.3 384 16.9 0.44CD3200 2.21 7.5 21.2 93.5 33.9 36.3 26.8 — — —CD3000 2.29 8 22.7 98.8 34.7 35.1 21.5 — — —

Resistant red cell pathologyManual — — 41.3 91.2 — 34.6 — — — —CD4000 4.53 14.3 42.6 94 31.5 33.5 12.3 160 3.53 0.32CD3200 4.56 14.3 44.2 95.7 29.5 30.8 13.5 — — —CD3000 4.46 14 42.1 92.4 30.7 33.2 13.4 — — —

Macrocytic normochromic anemiaManual — — 38.5 125.4 — 35.1 — — — —CD4000 3.07 13.5 39.4 128 43.8 34.2 13.2 19.8 0.64 0.35CD3200 3.15 13.4 39.6 126 42.6 33.9 13 — — —CD3700 3.15 13.5 37.9 121 42.8 35.5 13.7 — — —

*RBC indicates red blood cell; Hb, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin con-centration; RDW, red cell distribution width; CV, coefficient of variation; Retic, reticulocyte; IRF, immature reticulocyte fraction.


hypochromic effect. On the Cell-Dyn 4000 system, the same pop-ulation clusters around the regression line because RBC deforma-tion interferes less when hydrodynamic focusing with red cellsphering is used.

RED CELL DEFORMATION AND MCHC MEASUREMENT

The other red cell parameter in anemia classification is meancell hemoglobin concentration, used to express chromicity of theRBCs. Before the clinical utility of the MCHC is discussed, red celldeformation and viscosity have to be explained.

Erythrocytes are known to change shape when exposed to rapidacceleration in a suspending fluid [34]. The RBCs change from a

biconcave discoid shape to an elongated, somewhat cigar-likeshape. As depicted in Figure 4A, this phenomenon occurs when redcells pass through the aperture of an impedance transducer. Thisgenerates an electrical resistance pulse, which is essentially propor-tionate to the cell profile or cross sectional area, rather than its truevolume. For spheres, the electrical resistance is directly related tovolume. However, for particles of other shapes, a shape factor andparticle orientation have to be included in the equation [35]. Redcell deformation is not constant; it changes with the internal viscos-ity of the RBC, which is in turn determined by the hemoglobinconcentration. Thus the MCHC can influence the MCV measure-ment on hematology analyzers. As depicted in Figure 5A, erythro-cytes with a true high MCHC are more viscous and less deformableand offer a wider cross-sectional area during passage through the

FIGURE 5. Red blood cell viscosity affects mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC) measure-ments. A. Impedance. B. Optical scatter.

FIGURE 4. A. Red blood cell (RBC) deformation during electrical resistance (impedance) measurement. As the electrolyte diluentapproaches the impedance transducer, its flow rapidly increases with a consequent acceleration of the suspended erythrocytes. Theshear forces on the RBCs cause maximum cellular deformation at the very point of resistance measurement. B. RBC deformationduring hydrodynamic focused flow measurement of sphered red cells on an optical system. The cell suspension is moving at a con-stant velocity inside a sheath stream of rapidly moving fluid. The sphered cells are subject to a limited amount of deformation atthe measurement point.

A B

A B


aperture, resulting in overestimation of the MCV. Conversely, cellswith a true low MCHC are more deformable and present a smallercross-sectional profile. Therefore, a falsely low estimate of the MCVis generated for a RBC whose MCHC is low. Because the MCV isused to calculate the MCHC, there is a consequent overestimationof the automated MCHC. The net result of this impact of the trueMCHC on the measured MCV is that measured MCHC rarelychanges. This explains why impedance analyzers generate differentdegrees of clinically unresponsive or “clamped” MCHCs, as is welldocumented [22,36].

Initial studies focused on methods to test red cell deformation.One of the better approaches is to compare automated MCHC val-ues with the reference value [22], since that determines the clinicalusefulness of the reported MCHC. Reference MCHCs are obtain-able in 2 ways. First, the MCHC is derived from the spun microhe-

matocrit [36], using the hemoglobin result of a hematology analyz-er. The true MCHC is estimated by dividing Hb by PCV (packedcell volume). This approach was followed in our MCHC evalua-tion. The second method is based on multiple MCHC determina-tions via hemoglobin measurements [37]. The effect of red celldeformation can also be studied through the correlation of theMCH to the MCV [36]. The closer the correlation between the 2parameters, the greater the influence of cell deformation on theMCV measurement. From these studies, it became clear thathydrodynamically focused systems generate automated MCHCsthat agree best with the reference value.

Light scatter carries information about the size, shape, orienta-tion, internal structure, and refractive index of a cell [38,39]. Tomeasure erythrocytes on an optical system, they have to be spheredand presented at the measurement point by a hydrodynamically

FIGURE 6. Anemia classification by mean cell hemoglobin concentration (MCHC) versus mean cell volume (MCV). A. Spunmicrohematocrit reference method. B. Cell-Dyn 4000. C. Cell-Dyn 3200. D. Cell-Dyn 3700. Typical population of 241 samplesplotted, including 100 healthy donor specimens from Abbott volunteers and 141 pathology samples. MCV and MCHC thresh-olds defined by arbitrary classification definition: normal MCV, 80–100 fL; Normal MCHC, 31–36 g/dL. Region 1: normocytic,normochromic RBCs; region 2: microcytic, hypochromic RBCs; region 3: normocytic, hypochromic RBCs; region 4: macrocytic,normochromic RBCs; region 5: macrocytic, hypochromic RBCs; region 6: normocytic, hyperchromic RBCs; region 7: macrocyt-ic, hyperchromic RBCs. The following typical pathology results are highlighted: iron-deficiency anemia (green), thalassemia (pur-ple), sickle cell anemia (red), lyse-resistant RBCs (blue), normocytic normochromic anemia (yellow), and macrocytic nor-mochromic anemia (teal). The Cell-Dyn 3700 MCHC analysis (electronic editing) is clamped.


focused flow with constant low velocity to minimize orientation anddeformation effects [40,41]. Figures 4B and 5B depict the opticalanalysis of erythrocytes and illustrate the effect of the internal viscos-ity on the MCV and MCHC measurements. When the cell passesthrough the laser light, light is scattered in all directions. No angle ofmeasured light scatter contains all the volume information; there-fore, optical volume analysis requires a minimum of dual scatteranalysis. Forward scatter (0- to 3-degree) is essentially proportionateto the cell profile or cross-sectional area, rather than its true volume.Intermediate-angle (5- to 15-degree) and right-angle (90-degree)scatter hold useful information in measuring the effect of cell viscos-ity on volume and corrections for it. The transformation map usedin Bayer systems [42] to transform scattered light intensity to cellvolume and cell hemoglobin illustrates this. Figure 5B illustratesthat for optical systems, erythrocytes with a true high MCHC (vis-cosity) are seen as a smaller cross-sectional area, contrary to the elec-trical resistance measurement. Therefore, the uncorrected MCV willbe underestimated, and consequently the calculated MCHC will beoverestimated. With increased red cell viscosity, the cell deforms lessand the changed refractive index results in less light scattered in theforward direction and more in the intermediate- and right-angledirection. Combining both pieces of information at the cell-by-celllevel is an attempt to correct for this effect. The accuracy of thereported MCV depends on the quality of this correction. The oppo-site effect happens with true low MCHCs. The measured MCV willbe overestimated, and consequently the calculated MCHC will be

underestimated. At very low hemoglobin concentrations, the meas-ured MCV will be underestimated, and consequently the calculatedMCHC will be overestimated.

MCHC, A CLINICALLY USEFUL PARAMETER IN ANEMIA CLASSIFICATION

Anemia is classified based on MCV (size) and MCHC(chromicity) data, as discussed above. To study the clinical utility ofthe MCHC parameter in anemia classification on the Cell-Dyn4000 and 3200 systems, a data set of 241 samples was collected,100 specimens from healthy donors in our Santa Clara facility, and141 pathology specimens. Some of the pathology specimens—inparticular, the sickle cell and resistant red cell samples—are derivedfrom the Cell-Dyn 4000, 3200, and 3000 systems of the Hematol-ogy Laboratory, Department of Pathology, University of Illinois atChicago, after confirmation that these systems performed equally aswell as ours on normal samples. Where possible the samples wererun in duplicate for paired precision analysis.

Figure 6 shows the different bivariate plots of the MCHCexpressed against the MCV. The MCHC and MCV data derivedfrom spun microhematocrit are used as reference (plot A). The fol-lowing arbitrary limits were used: normal MCV, 80–100 fL, andMCHC, 31–36 g/dL. There was no discussion on the MCV thresh-olds, because clinicians are told by Wintrobe that these are the val-ues for anemia classification. The thresholds for the MCHC were set

FIGURE 7. Linear regression analysis of automated mean cell volumes (MCVs) compared and plotted against the manual referenceMCV. A. Cell-Dyn 4000. B. Cell-Dyn 3200. C. Cell-Dyn 3700. Specimen population described in Figure 6. — indicates regressionline. Y Int indicates Y intercept.


based on our data obtained with the reference method (Table 2) andare in agreement with the published data shown in Table 1. Thelower limit of 31 g/dL in our opinion does reflect truth better thanthe 32 g/dL published by Wintrobe (Table 7) using pulse-editingsystems. The upper limit of 36 g/dL agrees with Wintrobe. Usingthese limits, 9 quadrants are defined, the most important ones beingNormocytic, Normochromic, (containing the normal samples andthe normocytic anemias), Microcytic, Hypochromic (including theiron-deficiency anemias and thalassemias), and Macrocytic, Nor-mochromic (where most macrocytic anemias are found).

Figure 6A demonstrates that the manual method has great sensi-tivity to identify microcytic hypochromic anemias, which are themost prevalent types. The manual MCHC clearly has clinical utilityin the early identification of iron-deficiency anemia. This plot showsthat hyperchromic cells were found on only rare occasions.

Figure 6B demonstrates anemia classification based on the auto-mated MCVs and MCHCs generated by the Cell-Dyn 4000 system,which uses hydrodynamically focused impedance. Excellent agreementis observed with the reference method. Early stages of hypochromicityare identified, such as found in iron-deficiency anemia.

Figure 6C shows anemia classification based on the Cell-Dyn3200 system’s MCVs and MCHCs. This optical method useshydrodynamic focusing and sphered RBCs. Again, agreement withthe reference method is found with excellent sensitivity to identifyearly stages of iron-deficiency anemia. The majority of the macro-cytic samples agreed well with the spun microhematocrit. In the

normocytic and microcytic regions, no significant differences werefound. A hereditary spherocytosis specimen that was run only onthe Cell-Dyn 3200 system (therefore not included in the data set)reported a MCV of 73.8 fL and MCHC of 42.7 g/dL, demonstrat-ing the clinical utility of these parameters.

Figure 6D shows the anemia classification based on the MCVsand MCHCs from the Cell-Dyn 3700 system, which uses imped-ance with pulse-editing. As mentioned before, the MCHC is unre-sponsive (or clamped) with pathology specimens run on pulse-edit-ing instruments. The sensitivity to identify hypochromic anemiasearly on is lost. Most pathologies, including the real hypo- andhyperchromic anemias, report normal MCHC results. Therefore,such MCHC results have no clinical value and can only be used asquality control parameters.

LINEAR REGRESSION ANALYSIS OF THE AUTOMATED MCHC VERSUS THE MANUALREFERENCE

Bull et al [22] stated that the primary value of the MCHC is fordiagnostic use. They perceived the unresponsiveness or clampedMCHC of pulse-editing systems as an undesirable state of affairsand requested that manufacturers address the viscosity impact onMCV analysis.

Bull et al. [22] showed that the MCHC regression analysis cor-related between various hematology analyzers against the manual

FIGURE 8. Graphs of the linear regression analysis of automated mean cell hemoglobin concentrations (MCHCs) versus reference man-ual MCHC. A. Cell-Dyn 4000. B. Cell-Dyn 3200. C. Cell-Dyn 3700. Specimen population described in Figure 6. A clamped MCHCis noted for the electronic pulse-editing method versus the reference. — indicates regression line. Y Int indicates Y intercept.


reference MCHC. He explained that the square of the correlationcoefficient (r 2) for each of the tested methods approximatelyreflects the fractional information about true MCHC available inthe estimated MCHC of a particular analyzer. The study showedan r 2 of 0.73 for impedance systems (Sysmex NE-8000) usinghydrodynamic focusing, an r2 of 0.56 for optical systems (Techni-con H1) using hydrodynamic focusing with sphering, and an r2 of<0.30 for pulse-editing systems (Coulter S Plus IV and Cell-Dyn3000). Paterakis et al. [36], in a similar study, showed an r2 of 0.53and 0.60 for Coulter STKS and Abbott Cell-Dyn 3000 MCHCmeasurements, respectively. He observed an r2 of 0.78 for SysmexNE-8000 MCHCs and an r2 of 0.66 for Bayer H1.

To test the improvements introduced in Cell-Dyn hematologysystems, linear regression analysis and correlation studies were per-formed on the data set presented above. Figure 8 shows the resultsof the MCHC regression studies. For the Cell-Dyn 4000 system,which employs impedance, hydrodynamic focusing and RBCsphering, an r2 of 0.75 is obtained. This is equal to the best pub-lished data. For the Cell-Dyn 3200 system, which employs opticalanalysis of sphered RBCs in a hydrodynamic focused flow, an r2 of0.64 was calculated. The Cell-Dyn 3700 MCHC, which employs apulse-editing system, resulted in an r2 of 0.48, which is in agree-ment with the Paterakis et al. study [36], as are our slope and inter-cept data.

Paterakis et al. [36] also proposed the MCH correlation withMCV as a method to study red cell shape effect on volume meas-urement and showed the highest correlations for pulse-editing sys-tems and the lowest for optical systems. We confirmed that obser-vation with the following regression data for the MCH regressed

against the MCV: Cell-Dyn 4000, r2 = 0.96, r = 0.98, slope 0.40,Y-intercept –6.4; Cell-Dyn 3200, r2 = 0.77, r = 0.88, slope 0.34, Y-intercept –1.0; Cell-Dyn 3700, r2 = 0.98, r = 0.99, slope 0.39, Y-intercept –4.9. Our data show that the smallest shape effectoccurred on the optical MCV analysis.

The 3 studies demonstrate superior performance for impedancemethods using hydrodynamic focusing, with clinical value of suchautomated MCHCs in the classification of anemias. The 3 studiessurprisingly show consistently lower correlations for the opticalmethods. This finding might be the consequence of difficultieswith the calibration of these methods or inconsistencies in the algo-rithm equations. The Cell-Dyn 3200 MCHC is corrected for theviscosity effect in macrocytic anemias and therefore has clinical use-fulness. The Cell-Dyn 3700 MCHC is unresponsive or clamped tothe viscosity effect and therefore has no clinical usefulness to classi-fy pathologies. However, the reduced physiologic range makes thisMCHC more sensitive to identify drift in analyzer calibration.

MCHC USED AS A QUALITY CONTROL TOOL

One of the advantages of using Cell-Dyn technology is the useof the Quality Control files, which allow monitoring and access ofdata to spot potential instrument drift that may ultimately requiretroubleshooting of the system. The MCHC from specimens can bemonitored in this manner and across all Cell-Dyn technologieswith some slight variation in the types of specimens used.

Pulse-editing technologies such as the Cell-Dyn 3700 cause aclamping effect on the MCHC value, decreasing its ability to beused clinically, but still allow it to be used as a quality control tool.

FIGURE 9. Examples of Levey-Jennings graphs generated from a quality control file using normal specimens to show the movement ofdata along with the mean cell hemoglobin concentration (MCHC) mean, the 2 SD limits, and the Westgard rules violated. A. Cell-Dyn 4000. B. Cell-Dyn 3200. C. Cell-Dyn 3700.

A B

C


Moreover, this clamping effect of the MCHC allows the use of nor-mal and abnormal samples in the X-B moving average files [43] tomonitor the system. On the other hand, focused flow systems suchas the Cell-Dyn 4000 and 3200 show wider variation around theMCHC mean when pathologic specimens are included in the X-Bmoving averages. This still allows identification of instrument drift;however, their MCHC performance can best be monitored by theuse of normal samples only. Figure 9 demonstrates this alternativequality control approach, using normal retention samples.

With specimens run in a quality control file, multirule Westgardrules [44] can be activated, which are a better method to alert theuser to violations and the need for action. As shown in Figure 9,Levey-Jennings graphs can be generated and printed from the quali-ty control file to show the movement of data along with the MCHCmean, the 2SD limits, and the Westgard rules violated. This alterna-tive method shows the advantage of using the quality control filesystem with its easy access to the data and monitoring of the system.

Normal specimens run with pulse editing systems yield MCHCvalues within 32 to 35 g/dL, whereas the same specimens run on afocused flow system yield MCHC values within 31 to 36 g/dL(Table 2).

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