PERIPHERAL BLOOD FILM REVIEW

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1 INTERPRETATION OF THE PERIPHERAL BLOOD FILM /02 $ PERIPHERAL BLOOD FILM REVIEW The Demise ofthe Eyecount Leukocyte Differential Robert V. Pierre, MD Peripheral blood film review refers to checking or verifying flagged or abnormal results obtained by an automated hematology analyzer. The usual mechanism for this review is to have an expert human inspect a stained blood film with identification and enumeration of100 or more consecutive leukocytes. This function also includes inspection of red cell and platelet morphology with comments and interpretation ofthe findings, when indicated. Before the introduction of automated leukocyte differential instruments, it was common practice to prepare, stain, and examine a blood film and perform a 100- or 200-cell eyecount leukocyte differential count (ECLDC) on all samples submitted for a complete blood count (CBC), whether done by old-fashioned manual techniques or modern automated hematology analyzers. This practice was labor intensive requiring highly trained technologists, and added greatly to the cost ofproviding laboratory results. This practice took place because it was a widely held beliefthat it was necessary, to prevent significant errors caused by false-negative results by the instruments. The complete blood cell count and leukocyte differential count traditionally have been the highest volume tests in the clinical laboratory. Today, the common laboratory practice is to review only those specimens from the automated instruments that are flagged for review, because ofdistributional, morphologic, or instrument failure flags. Numerous studies have documented that this approach can markedly reduce the costs of the CBC and leukocyte differential, and provide greater sensitivity for detection ofabnormalities ofthe CBC and leukocyte differential. 1, 5 The following issues are covered in this article: 1. The development of automated leukocyte differential instruments and the evolution oftheir use in the clinical laboratory. From the Department ofpathology, Los Angeles County/University ofsouthern California Medical Center, Los Angeles, California CLINICS IN LABORATORY MEDICINE VOLUME 22 NUMBER 1 MARCH

2 280 PIERRE 2. The rationale and justification ofcriteria established for slide review. 3. Elimination of proportional (percentage) differential results. 4. The rationale for discontinuation ofthe 100-cell ECDLC as part ofthe review process. 5. The role ofrestrictions oftest requests to reduce the laboratory workload. BACKGROUND The Coulter Counter Model S (Beckman Coulter, Brea, CA) introduced automation ofthe CBC (total white blood cell and red cell parameters) using anticoagulated whole blood in Other manufacturers soon introduced instruments with similar capabilities (Ortho Elt-8, Ortho Diagnostics, Rahway, NJ; Sysmex Corporation, Long Grove, IL). The routine inclusion ofthe platelet count as part ofthe CBC first occurred in 1977, with the introduction ofthe Coulter Counter Model S Plus. The diagnosis ofa hematologic disorder is dependent on the patient history and physical examination finding, laboratory testing, and the intelligent interpretation ofthese results by a clinician. The clinician is aided in this role by the laboratorian, who can provide expert interpretation ofthe laboratory results. A mutual, cooperative approach by the clinician and laboratorian serves the best interest ofthe patient. The automated CBC with platelet count combined with an ECDLC and scanning ofthe blood film for red cell, white cell, and platelet abnormalities became the primary laboratory cornerstone ofthe diagnosis ofhematologic disease. The CBC using the automated instruments was inexpensive, fast, precise, and accurate; however, the ECLDC remained an expensive, slow, imprecise, inaccurate, and labor-intensive procedure. The ECLDC is expensive because it requires highly trained personnel and is time consuming. The time required to perform and record a 100-cell ECLDC has been reported as short as 1.9 minutes to as great as 6 minutes. 2, 6 The first attempts at automation ofthe ECLDC occurred in the early 1970s. The instruments attempted to reduce the labor-intensive nature ofthe procedure, and to increase precision and accuracy. Image processing differential (IPD) instruments used an automated stage microscope with an analog gray-scale scanning device to capture the leukocyte images. Scene segmentation techniques were used to separate the leukocyte images from the red cells. These instruments digitized the captured leukocyte image, did feature extraction, and matched the image features with a library of features of the normal leukocyte types to determine the leukocyte identity. Some ofthe instruments used analog video cameras (Corning Larc, Corning Glass Company, Corning, NY; Abbott ADC 500, Abbott Diagnostics Division, Dallas, TX; Coulter Diff 3, Beckman Coulter, Brea, CA; Hitachi-806, Hitachi, Tokyo, Japan; and the Omron Micron). 7 The Hematrak by Geometric Data (Wayne, PA) used a flying spot scanner to acquire the image. The IMI Micro 21 (Beckman Coulter, Brea, CA), LAFIA system (Sysmex Corporation, Kobe, Japan), 10 and Diffmaster (Cella Vision, Lund, Sweden) 4 used charged couple devices to capture the leukocyte images and a neural network system to do image analysis. The objective ofthe IPD devices was to complete a 100- or 200-cell differential count in approximately 1 minute, and to classify the differential as normal or abnormal with an accuracy comparable with that ofthe ECLDC. Extensive evaluation studies 6, 16 proved that these devices were as accurate and precise as the ECLDC. Unfortunately, they did not increase efficiency to as great a degree as hoped. They were first introduced into the clinical laboratory in the early 1970s, and widely used until the mid-1980s.

3 PERIPHERAL BLOOD FILM REVIEW 281 The Technicon Hemalog D (Technicon Instruments, Tarrytown, NY) was the first instrument to perform automated leukocyte differential counts. This instrument used flow-through cytochemical differential staining with white light scatter and absorption to distinguish between stained and unstained cells. Three cytochemical-staining modules were used. The first used myeloperoxidase staining to identify neutrophils and eosinophils. Lymphocytes were identified in this channel by their lack ofstaining and small light scatter (size). Large peroxidasenegative cells were called large unidentified cells. The large unidentified cells were composed ofmainly variant lymphocytes or blast cells. Eosinophils were identified by their intense peroxidase staining and large size. Monocytes were identified in a second module by use ofa specific -naphthol butyrate esterase stain. The basophils were identified in a third channel using alcian blue to stain the basophils selectively. Evaluations ofthis instrument showed that much greater precision of the differential results could be achieved because the differential was based on 10,000 cells identified in each ofthe three channels. Figure 1 shows the increased precision of the 10,000-cell versus the 200-cell differential. 11 Studies also showed that the false-normal (negative) flagging rate was comparable with the ECLDC; however, the false-positive (abnormal) flagging rate was higher than the ECLDC. 11 The Hemalog D90 was used at the Mayo Clinic from 1975 to Only samples that were flagged by the instrument were subjected to a microscopic slide review. The Hemalog D substantially reduced the workload ofthe leukocyte differential function of the laboratory, because nearly 70% of all samples were normal, within distributional checking limits, or unflagged and did not require preparation ofa stained slide and microscopic review. Technicon introduced an automated slide maker stainer (Autoslide) that was connected to the Hemalog D, which used an aliquot ofthe aspirated sample to prepare stain, label, and coverslip a blood film on each sample. The device produced a wedge blood film of excellent quality, but it suffered from a fatal flaw. The blood film was only one halfthe width ofthe glass slide. This made the slides impossible to use on the Hematrak image processing instrument that was being used to review slides. The Hematrak scan pattern expected a full slide width blood film. The instrument scan would go offthe edge ofthe blood film and get lost, requiring intervention by a human operator. The Autoslide was an excellent idea that failed because of a design error, which the manufacturer would not correct. Automated slide maker stainers have recently been introduced by all major hematology instrument manufacturers for use in conjunction with their analyzers. The initial evaluation studies ofthe Hemalog D developed the concept of categorizing differential counts as being normal, having distributional abnormalities, or morphologic abnormalities; this approach was later incorporated into the National Committee for Clinical Laboratory Standards (NCCLS) reference (H20A) 10 method for leukocyte differentials. In 1983, the Coulter Counter Model Plus IV 5 was introduced. This instrument produced a three-part leukocyte differential count. The three classes of leukocytes were (1) lymphocytes, (2) mononuclear cells, and (3) granulocytes. The leukocytes were exposed to a reagent, which stripped the cytoplasm from the cells leaving the bare nucleus. The instrument determined nuclear size and a nuclear volume histogram was displayed. Cells between 35 and 90 fl were identified as lymphocytes. Cells between 90 and 160 fl were termed mononuclear cells, and cells greater than 160 fl were called granulocytes. This system did not specifically measure or report the eosinophils, basophils, or monocytes. Evaluation studies ofthe Coulter Counter Plus IV, however, gave classification of

4 282 PIERRE Second 200-cell count First 200-cell count 100 Second HDL-cell count A First HLD-cell count Figure 1. See legend on opposite page

5 PERIPHERAL BLOOD FILM REVIEW Second 200-cell count First 200-cell count 50 Second HDL-cell count B First HLD-cell count Figure 1 (Continued). Comparison of duplicate 200-cell eyecount differentials performed on the same slide by two observers versus duplicate 10,000 cell differentials performed on the same blood sample by the Hemalog D90. Results are shown for neutrophils (A) and lymphocytes (B).

6 284 PIERRE samples as having normal results, distributional abnormalities, or morphologic abnormalities comparable with that observed with the Hemalog D90 five-part differential instrument. The lower cost and ease of operation of the three-part differential systems with comparable flagging efficiency for samples that needed review led to replacement ofthe Hemalog D90 at the Mayo Clinic by Coulter Counter Model S-Plus IV instruments. During the period ofuse ofthe Hemalog D90 and the evolution to the Coulter three-part differential system, the Mayo Clinic hematology laboratory used Hematrak (IPD) instruments as devices to review samples that were flagged for review. The Hematrak (model 590) had automated sample loading. Each slide s identity, cassette location, and the slide cordinates ofeach flagged cell were stored in the instrument s memory. The instrument could go to the appropriate cassette, slide, and cell location to present a cell image to the human observer for final identification and modification of the differential result if necessary. Using the Hematrak instruments in this fashion made review of the flagged specimens more efficient and reduced technologist effort. A serious blow was dealt to the development ofthe IPD instruments, however, by the introduction of flow-based three-part differential systems, such as the S Plus IV, which was reinforced with the introduction of the early five-part differential flow instruments, such as the Coulter STKS, the Sysmex 9000 (Sysmex Corporation, Long Grove, IL), and the Cell Dyne 3500 (Abbott Diagnostics, Abbott Park, IL) instruments. The Achilles heel of the image processing differential systems was not the ability ofthe instruments to identify cells correctly, but that only a 100- or 200- cell based differential could be performed in an acceptable time frame of 1 or 2 minutes. These instruments also did not provide an adequate substitute for the microscopic evaluation ofred cell and platelet morphology. The manufacture and maintenance ofthe existing image processing instruments ceased, except for the IMI Micro 21 in the United States, and the Hitachi-806 and the Omron Micron in Japan, because they could not compete with the throughput, precision, and accuracy of the dedicated flow differential instruments. A meeting was arranged by the College ofamerican Pathologists (CAP) in Aspen, Colorado, in 1977 in which evaluation data and information pertaining to the use of automated leukocyte differential instruments were presented. The information was published in a book. 8 At this conference, the participants with representatives of NCCLS committed to preparing a reference method for leukocyte counting. Ifa reference method exists, a new test instrument can be classified under product class II ofthe Food and Drug Administration regulations and requires only a 5 to 10K submission by the manufacturer. After preparation of the Reference NCCLS H20P in 1981, a petition hearing requested for reclassification of the automated differential instrument from a proposed class III to the product class II status was granted. Since that time there has been continued development and innovation in the five-part differential instruments. A working group ofthe International Society oflaboratory Hematology has collaborated with NCCLS to develop introduction of an expanded differential on these instruments with methods to detect and report nucleated red blood cells, variant lymphocytes, blasts, and immature granulocytes. CAUSES OF ERRORS IN EYECOUNT LEUKOCYTE DIFFERENTIAL COUNTS There are four sources of error in the performance of ECLDC: 1. Observer errors 2. Slide distribution errors 15

7 PERIPHERAL BLOOD FILM REVIEW Statistical sampling error Recording errors The observer misclassification ofleukocyte cell types accounts for a small proportion ofcounting errors when well-trained technologists perform the ECLDC. Subsequent studies reported by the CAP Hematology Resource Committee have convincingly demonstrated that bands cannot be distinguished reliably from segmented neutrophils on blood films by human observers. 3 The CAP published a recommendation that band counts not be counted and reported on leukocyte differential results (see the article by Cornbleet elsewhere in this issue). Slide distribution errors are caused by the nonrandom distribution ofleukocyte types on blood film prepared by the wedge-type technique. 15 This technique results in a greater concentration ofleukocytes on the edges ofthe blood film and in the feather end. In addition, large cell types, such as monocytes, eosinophils, and neutrophils, tend to be concentrated along the edges and end ofthe film, whereas the small lymphocytes tend to be in the central area ofthe film. The battlement scan technique was developed to compensate for this maldistribution ofcell types. This maldistribution ofleukocyte cell types is not observed on blood films made with slide centrifuges. Statistical sampling error is the greatest source oferror in the ECLDC. The leukocytes on a peripheral blood film are considered to be a random sample of the circulating blood leukocytes. The leukocytes counted in the differential are considered to be a random sample ofthose in the blood film and that all cells are correctly identified. Rümke 14 discussed the statistically expected variability in differential leukocyte counting and pointed out the 95% confidence limits for various percentages ofblood cells ofa given type when 100, 200, 500, or 1000 cells are differentiated. For example, if a patient has a true neutrophil concentration of50%, the 95% confidence limits for observed values ofneutrophils on a 100-cell differential are 39% to 61% (Table 1). When cells of low frequency are counted, the errors can be much larger. Ifone considers that the 200-cell ECLDC normal limit for basophils is 0% to 1.5%, the normal limit for eosinophils is 0.5% to 8% and monocytes are 3% to 11%, and if one considers the effect of statistical sampling error, a true basophil concentration of1% could be reported correctly as 0% to 4%, a true eosinophil concentration of4% could be reported as 1% to 8%, and a true monocyte concentration of7% as 3% to 12%. It is even more informative to look at duplicate 200-cell ECLDC of basophils, eosinophils, and monocytes (Figs. 2 4); the results nearly fill the normal range areas for each cell type. The variability in a comparison of 100-cell differential is even greater. This suggests that the values obtained for these minor cell types in a normal patient are a random number within each cell type s normal range. Attempts to use the white blood cell count and the proportional differential leukocyte count to generate absolute leukocyte subtype counts suffer by multiplication ofthe cell counting errors by the error associated with the leukocyte count itself. This was especially true in the past when chamber eye counts were used to obtain leukocyte counts because ofthe imprecision ofthe chamber method. REFERENCE METHOD DIFFERENTIAL COUNT (PROPORTIONAL) AND EVALUATION OF INSTRUMENT METHODS The reference method defines the qualification of blood film examiners, description ofthe morphology and the nomenclature ofthe normal leukocyte

8 286 PIERRE Table 1. NINETY-FIVE PERCENT CONFIDENCE LIMITS FOR VARIOUS PERCENTAGES OF BLOOD CELLS OF A GIVEN TYPE AS DETERMINED BY DIFFERENTIAL COUNTS N 100 N 200 N 500 N N number ofcells counted; observed percentage ofcells ofa given type. Adapted from Rümke CL: The statistically expected variability in differential counting. In Koepke JA (ed): Differential Leukocyte Counting. CAP Conference/Aspen, Skokie, IL, College of American Pathologists, 1978, pp 39 45; with permission. Figure 2. Monocytes percent. Comparison ofduplicate 200-cell eyecounts performed on the same slide by two observers (from the specimens reported in Fig. 1). A line of identity is shown as a solid line. The dashed lines describe the upper normal limits for monocytes. Diamond Series 1.

9 PERIPHERAL BLOOD FILM REVIEW 287 Figure 3. Eosinophils. Comparison ofduplicate 200-cell eyecounts performed on the same slide by two observers (from the same specimens reported in Fig. 1). A line of identity is shown as a solid line. The dashed lines define the upper normal limit for eosinophils. Figure 4. Basophils. Comparison ofduplicate 200-cell eyecounts performed on the same slide by two observers (from the same specimens reported in Fig. 1). A line of identity is shown as a solid line. The dashed lines define the upper normal limits for basophils.

10 288 PIERRE cell types, preparation and staining ofa blood film, and the requirements for an acceptable blood film. The battlement-scanning pattern is discussed. An outline ofthe experimental protocol for determination ofclinical sensitivity ofa test instrument is shown in Figure 5. The clinical sensitivity study should be performed on a minimum of 200 specimens; ofthese, 100 or more should be from normal subjects and 100 or more from a variety of clinical conditions described in the protocol. The 200 samples are studied by both the reference method and the test method study. The reference method consists of a 400-cell ECLDC performed as a 200-cell count by each oftwo qualified examiners on one oftwo slides prepared from the sample. In the earlier versions ofthe standard, an 800-cell ECLDC was used composed of four 200-cell differentials performed by four qualified examiners. Two individual test determinations are made by the test method on at least 100 samples. Bands are added to the total neutrophils when the instrument does not report bands as a separate parameter. Clinical sensitivity studies include three separate activities: 1. Determination of reference (normal) values for the test and reference methods; central 95th percentile results are used. 2. Determination ofsensitivity to finding abnormal samples with distributional and morphologic abnormalities. 3. Arbitration ofresults by an arbitrator when test and reference methods disagree. Figure 5. A flowchart ofthe clinical sensitivity testing protocol from NCCLS H20A.

11 PERIPHERAL BLOOD FILM REVIEW 289 EVALUATION OF THE 100-CELL EYECOUNT DIFFERENTIAL BY THE NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS REFERENCE METHOD In the initial discussions ofevaluation ofthe automated differential systems, it was stated that the instrument only had to be as good as the eyecount differential; it did not have to be better. This raised the question of how good is a 100-cell eyecount differential? Koepke et al 9 performed a study of the 100-cell leukocyte differential compared with the NCCLS reference 800-cell differential (Table 2). They compared cell ECLDCs with the 800-cell reference method. The results were quite startling. The large error rates for distributional abnormalities reflect the imprecision ofthe 100-cell eyecount caused primarily by sampling error. The morphologic false-abnormal rate was 0% reflecting that well-trained technologists do not identify normal cells as abnormal cells. The morphologic false-normal (negative rate) of 18.6%, however, does not suggest that technologists cannot identify abnormal cells, but that it also is caused by sampling error. These data, combined with the observations that the distributional false-normal (negative) and distributional false-abnormal (positive) rates for the automated instruments were lower than that of the 100 or 200 ECLDC, suggested that there was little rational reason to review abnormal distributional values from the instruments by means of an ECLDC in an attempt to confirm the distributional abnormality. This, however, is not an absolute rule; some distributional abnormalities ofnormal cell types are an indication to review a blood film. For example, in lymphocytosis it is important to determine the morphologic features of the lymphocytes and provide a description and comment on the significance ofthe finding; a 100- or 200-cell eyecount leukocyte differential contributes nothing. Table 3 from a study by Cox et al 5 also revealed that the morphologic falseabnormal (positive) rate for the three-part differential instruments was higher than for the eyecount method; this might suggest a severe deficiency of the automated methods compared with the eyecount method. A false-positive result for a morphologic flag, however, is not a serious instrument error. The finding ofa morphologic flag triggers a slide review and causes the technologist to Table 2. COMPARISON OF 100-CELL EYECOUNT DIFFERENTIAL WITH NCCLS H CELL REFERENCE DIFFERENTIAL 100-Cell Eyecount Leukocyte Differential Count (No. of Cases) 800-Cell Distributional Morphologic Reference Method Normal Abnormality Abnormality Total Normal Distributional abnormality Morphologic abnormality Total Distributional false-abnormal (positive) 8.8% Distributional false-normal (negative) 29.9% Morphologic false-abnormal (positive) 0% Morphologic false-normal (negative) 18.6 From Koepke JA, Dotson MA, Shifman MA: False positive/false negative rates for the eyecount leukocyte differential count. Blood Cells 11:173, 1985; with permission.

12 290 PIERRE Table 3. CLINICAL SENSITIVITY TESTING: COMPARISON OF MORPHOLOGIC AND DISTRIBUTIONAL ABNORMALITIES FOR 100-CELL EYECOUNT AND THREE-PART AUTOMATED DIFFERENTIAL MEASURED AGAINST H20P REFERENCE METHOD 100-Cell Eyecount Toa E-500 Coulter S-Plus IV % %* % Distributional false-abnormal Distributional false-normal Morphologic false-abnormal Morphologic false-normal *Toa E-5000, Sysmex, Long Grove, IL. Coulter S-Plus IV, Coulter Electronics, Hialeah, FL. 100-Cell Eyecount data from Koepke JA, Dotson MA, Shifman MA: False positive/false negative rates for the eyecount leukocyte differential count. Blood Cells 11:173, search the slide for the presence of the indicated abnormality. The lower morphologic false-normal (negative) results of the instruments indicate that the instruments flag abnormal samples more dependably than an eyecount differential. One can argue that if an eyecount differential is performed, the technologist scans the slide and likely finds the morphologic abnormality. This argument is negated by the Duke study 9 ; the laboratory technicians performing the 100-cell ECLDC were following their standard laboratory procedure, which included a slide scan and comment on red cell and platelet morphology, yet their morphologic false-normal (negative) rate was higher than that of the instrument. Clinical sensitivity study results ofthe five-part automated systems, the Coulter STKS, Sysmex NE-8000, and the Technicon H1, are shown in Table 4 and are compared with the 100-cell eyecount data from the Koepke et al 9 study. These data were the result ofside-by-side comparison data on a common sample set carried out using the NCCLS H20A protocol. THE DEVELOPMENT OF FLAGGING CRITERIA FOR SLIDE REVIEW OF AUTOMATED LEUKOCYTE DIFFERENTIAL RESULTS During development ofthe Hemalog D instrument, discussions on how to compare the instrument results with eyecount differential results were held. It Table 4. CLINICAL SENSITIVITY TEST: COMPARISON OF FIVE-PART AUTOMATED DIFFERENTIALS AND 100-CELL EYECOUNT WITH NCCLS H20A REFERENCE METHOD 100-Cell STKS NE8000 H2 % % % % Distributional false-abnormal Distributional false-normal Morphologic false-abnormal Morphologic false-normal STKS Coulter Counter Model STKS; NE-8000 Sysmex Model NE-8000; H2 Technicon Model H Cell Eyecount data from Koepke JA, Dotson MA, Shifman MA: False positive/false negative rates from the eyecount leukocyte differential count. Blood Cells 11:173, 1985.

13 PERIPHERAL BLOOD FILM REVIEW 291 was proposed that central 95th percentile reference limits for each measured parameter of normal cells for both the instrument and reference method be developed. Values outside these limits are considered distributional abnormalities. If the test method classification differed from the reference method classification, it was considered a distributional disagreement. 12 The difference between a 10,000-cell and a 200-cell eyecount, however, also had to be recognized. Table 5 shows the acceptable agreement limits. The presence ofany immature granulocyte (metamyelocytes, myelocytes, promyelocytes, and myeloblasts) beyond the established normal limits for these cells on the eyecount differential is called a morphologic disagreement. Note that two metamyelocytes and one myelocyte on a 200-cell eyecount differential were accepted as being within normal limits, whereas any promyelocyte or myeloblast was a morphologic abnormality. The presence ofnucleated red cells, any type ofblast cell, qualitatively abnormal cells, such as variant lymphocytes, or Pelger-Huët neutrophil megakaryocytes, increased bands, or cells containing microorganisms were considered morphologic abnormalities on the eyecount differential. Instrument flags on any of the automated instruments that indicated the presence ofimmature granulocytes, blasts, nucleated red cells, or inability to give results because offailure ofthe cell populations to meet the instrument s algorithms were considered cause for review. In addition, many laboratories established slide review criteria for red cell parameters and platelet parameters. The review criteria for the Coulter Counter Model STKS in use at the Los Angeles County (LAC)/University of Southern California (USC) Medical Center are shown in Table 6. Many laboratories also developed criteria for review based on the instrument scatterplots or histograms ofcell populations, because some patterns suggested or identified abnormalities (e.g., a high take-offofthe histogram population on the Coulter three-part differential histogram suggested platelet clumping or failure to lyse red cells). Abnormalities in the plots ofred cells or platelets have also been used as indications for blood film review. The specific review criteria for each type of analyzer in use in the laboratory must be established, because different analyzers may provide parameters not found on other instruments. Some instruments produce the same parameters, but the methods ofmeasurement and reference values can differ substantially. Table 5. CRITICAL PERCENTAGE DIFFERENCE BETWEEN EYECOUNT DIFFERENTIAL AND 10,000-CELL AUTOMATED DIFFERENTIAL Number of Cells Counted Instrument (% of Cells)

14 292 PIERRE Table 6. CHECKING LIMITS FOR COULTER COUNTER MODEL STKS Paramater Low High WBC 10 6 /L RBC 10 9 /L 2 8 Hbg/dL 5 18 Hct % MCH pg MCHC % MCV fl RDW 18.5 Platelets 10 6 /L Neutrophils 10 6 /L Lymphocytes 10 6 /L 5.5 Monocytes 10 6 /L 1.5 Eosinophils 10 6 /L 1 Basophils 10 6 /L 0.5 Morphologic flags vote out; there is failure ofagreement ofat least two apertures. incomplete computation 999 values beyond the linearity limits ofthe instrument Blast flag Immature granulocyte flag Band flag Atypical lymphocyte flag Nucleated red cell flag Visual inspection ofthe scatterplot reveals an unusual pattern The criteria for review include the following: 1. Failure ofthe instrument to provide a result for a parameter. 2. Instrument flags that indicate a possible instrument or sample analytic error, such as a clot or plug ofan aperture. 3. Instrument flags that suggest the presence ofimmature or abnormal cells (e.g., a blast cell flag). 4. A set ofreview criteria created by a laboratory, such as values above or below a specified range for a particular cell type. 5. Indications for review of instrument results may also be based on clinical paradigms. Individual clinicians or clinician groups may suggest rules for review of instrument results, such as review of blood films in all neonates or infants with fever. Hematologists and infectious disease specialists may want blood films reviewed on all patients. Certain protocols written for drug therapy or drug toxicity evaluation require leukocyte differential counts at specified intervals, often with little regard for their actual necessity or value. Such clinical paradigms or requirements from outside groups, such as cooperative study groups, may pose significant problems for the laboratory in attempts to reduce the numbers ofreviews performed. The validity ofthese requests must be evaluated in a cooperative and consensus manner with the clinicians or other groups to reach an accord based on scientific evidence and efficacy studies, not on habit or traditional practices. The nature ofthe illnesses ofpatients being treated or studied in a particular hospital or clinic in part also determines the necessary review rate. Institutions with a high percentage ofacutely or seriously ill patients or hematologic and infectious disease patients may have higher review rates. The percentage of

15 PERIPHERAL BLOOD FILM REVIEW 293 patients with normal differentials or results within review limits were found to be similar at the Mayo Clinic and LAC/USC Medical Center (approximately 70% in each). As a result ofthese many factors, the percentage ofautomated leukocyte differential results that are reviewed by preparation of a stained blood film and reviewed by an expert human varies from institution to institution. TEST RESTRICTIONS Another technique for reduction of the review rates of automated hematology instrument results involves restriction ofthe ordering ofleukocyte differential counts. A study was performed at the Mayo Clinic in 1991 in which all patients who had a CBC with automated differential ordered by a clinician were identified over a consecutive 5-day period. A total of4840 patients were identified. The purpose ofthe study was to identify clinical services that seemed to order CBC with automated differential inappropriately. The study was conducted by reviewing the medical records of4312 patients to determine whether the repeat differential counts were medically indicated. The remaining records were not readily available for review for various reasons. The total number of CBC and differential results were determined for each patient over the 5-day period. The period consisted ofthe inpatient or outpatient care period ofthe patient included in the 5-day period in which the CBC and data were collected. A total of 83.9% of patients had a single automated CBC-differential performed, 13.8% had two to five tests done, and 2.3% had 6 to 26 tests performed. The largest number oftests performed on a single patient was 26. In nearly all of the repeat studies done, the differential count provided no clinically important data. The repeat counts were done because the patients were bleeding or postoperative and the primary indication was to follow the hemoglobin levels. In other patients the primary indication for the CBC was to follow the total white count because of known or suspected infection or to monitor the effect of chemotherapy. In other cases the platelet count was the primary parameter ofinterest in patients being treated for thrombocytopenia or to monitor the effects of chemotherapy. When clinicians were asked why they ordered differential counts with the CBC results with differential counts routinely, they said they had always done it that way or their mentor had told them that it should be done. As a result ofthis study, the question was asked, at what interval and under what circumstances is a repeat differential indicated in a patient under continuous care, provided the clinician can obtain a measure ofthe erythroid values, the total white count, and platelet count whenever it is desired on a stat or routine basis. A proposal was made to the staffofthe LAC/USC Medical Center that the reporting and review of leukocyte differential counts from automated hematology analyzer results be restricted to once every 7 days. A physician group made up ofmembers ofall the major clinical services was formed and the issue discussed. A consensus was reached that the policy of not reporting differential counts at intervals of less than 1 week was reasonable. It was decided that there were few, if any situations, where the actual differential count was required for clinical management, if the erythroid values, white count, and platelet count were available whenever needed. The initial objection to this policy was that differentials had traditionally been provided as part of the automated instrument results. Another common statement was that differential results were required at daily or frequent intervals by drug testing or chemotherapy protocols. The test restrictions were implemented by the laboratory information system, which flagged all CBC orders ofless than 7 days interval. The

16 294 PIERRE technologists did not perform slide reviews on these cases even though the results indicated a slide review. An exception to this rule was provided ifthe clinician informed the laboratory of a specific indication for the slide review. A follow-up of this policy at the institution revealed no reports of adverse effects on patient management over a 3-year period. The request for daily CBC and differential counts on patients undergoing drug testing or chemotherapy protocols should be reviewed by institutions to determine the medical necessity of such testing and require the individuals or agencies that request such information to provide justification for the practice. In addition, whether reporting ofabsolute leukocyte values rather than percentage leukocyte values had an effect on the number of patient results that would generate review ofsmears in the Mayo Clinic study of4840 patients needed to be determined. Of these patients the leukocyte differential results were scored in each patient (Table 7) to determine the effect of using absolute leukocyte values rather than percentage leukocyte values as an indication for slide review. The use ofabsolute values led to a reduction in reviews of7%. The reduction reflected a reduction in patients classified as distributional abnormalities and shift ofmost ofthese patients to the normal classification from the distributional abnormality outside checking limits (score 3). This also suggests that there is no reason to continue to report automated differential results as proportional (percentage) results, which is a tradition established when only a proportional count could be measured on a stained blood film. This also raises the issue of elimination of the percentage differential results from automated instrument reports, because it reduces the incidence offlagging ofresults for slide review and erroneous classification ofpatients by clinicians relying on the percentage differential. The ongoing development ofthe automated instruments with the imminent implementation of methods for an extended differential, such as detection and counting ofnucleated red cells, detection and counting ofblast cells, and flagging ofabnormal lymphoid cells, provides even more accurate flagging. Provision ofreticulocyte counts and immature reticulocyte fraction and improvements in platelet counting only enhance accurate screening ofblood samples greater than can be provided by an actual 100- or 200-cell eyecount differential. The development ofmethods to decrease the number ofblood film reviews performed by the laboratory to decrease personnel needs and cost must not be approached solely by the laboratory, but must be made in concert and collaboration with the clinicians. The clinicians must understand and concur with the Table 7. USE OF PERCENTAGE VERSUS ABSOLUTE DIFFERENTIAL LEUKOCYTE VALUES AS AN INDICATION FOR SLIDE REVIEW Percentage Absolute Scores Number Percentage Scores Number Percentage Score of1 all results within reference limits Score of2 at least one result outside reference limits, but within checking limits Score of3 at least one value outside ofchecking limits Score of4 a morphologic flag was present

17 PERIPHERAL BLOOD FILM REVIEW 295 laboratory actions, because they are the users ofthis information. Hematologic diagnoses are not made in the laboratory; the laboratory provides only one of the elements needed for the diagnosis and management of a patient. An interesting insight is that at the time ofthe study at the Mayo Clinic ofthe 4840 patients, the average incidence ofmorphologic abnormalities was 14% among the approximately 1000 CBC-differential tests run each day. At that time, clinicians were requesting differentials with 28% of the CBC requests. Of the 140 morphologic abnormalities seen each day, 130 were included in the cases in which the clinicians had requested differentials. This suggests that clinicians are good predictors of which patients have an abnormal differential. Unfortunately, the author did not attempt to determine whether the other 10 cases represented cases in which the clinician already knew about the abnormality from the clinical information or previous tests or whether the abnormalities were clinically unsuspected. SUMMARY The automated hematology analyzer with CBC and differential results has replaced the traditional manual or individual assay methods for hematologic parameters and the eyecount leukocyte differential as the initial screening and detection system for hematologic abnormalities in modern hospitals and clinics. The traditional review ofall automated hematology instrument results by preparation, staining, and microscopic examination ofa blood film has disappeared in most institutions. The reasons are the more accurate detection ofspecimens with distributional or morphologic abnormalities by the instruments than by the traditional eyecount method. The opportunity for a clinician to request a microscopic examination ofa blood film, whether or not it is flagged, must be preserved, because the clinician s knowledge ofthe patient s history, physical findings, and current or prior therapy may indicate review to discover an abnormality that may not have been apparent from the instrument results alone. There has also been a dramatic reduction ofthe numbers ofmedical technologists and technicians in medical laboratories. Automation ofthe CBC and differential counts has reduced the number of technologists needed for performance of these tests. But other factors have had a negative effect, such as the necessity to reduce costs. Consolidation ofhematology and chemistry laboratories in core laboratories may produce savings in labor costs, but may also create problems ofcreating and maintaining areas ofexpertise, such as hematologic morphology, because ofthe cross-training required and the necessity of personnel to do all things. This article suggests and documents a number ofmeasures that can be stituted by the laboratory and by clinicians to reduce the number ofeyecount differentials and blood film reviews that need to be performed. The first effort is to convince clinicians that valid data exist that confirm that a policy of allowing the laboratory to initiate blood film review based on findings ofthe CBC and automated differential is a more sensitive and accurate method of detecting patients with blood film abnormalities than routine blood film review ofall specimens by technologists. Clinicians need to recognize that daily differential results or differentials at intervals ofless than a week are not medically necessary in most patients. The laboratory, however, must provide opportunities for the clinician to request differentials at any time for specific medical reasons. The laboratory must establish the validity ofscreening criteria for detection

18 296 PIERRE ofdistribution and morphologic abnormalities ofleukocytes by clinical correlation studies or adopt criteria established by laboratories with the same instrumentation and which have conducted clinical evaluations. A final observation on the eyecount differential is that it was the only way to identify cell types and their relative proportion for nearly 100 years. Cells were identified by their shape, intracellular structures, and staining characteristics. Many studies were able eventually to correlate some aspect ofeach cell type s function with their morphologic appearance. It has also been learned that the bone marrow is the source ofproduction ofmost circulating cells and a great deal ofthe controls ofcell production and release into the peripheral blood have been learned. But leukocytes have many functions, almost none of which are performed in the peripheral blood. The peripheral blood is mainly a conduit from the bone marrow to the tissues where the leukocytes perform their function in the case ofthe neutrophils and monocytes. It is mainly a recirculation and redistribution system for lymphocytes that usually receive their instructions from antigen processing cells in the tissues and allow these modified cells to home to sites where their functions occur. Cellular morphology and staining characteristics tell little about the maturation stage and functional capabilities of leukocytes. One cannot tell the difference between a band and a segmented neutrophil or whether a lymphocyte is a T or B cell on the conventional eyecount differential. One cannot tell the mature granulocyte of a patient with chronic myeloid leukemia from a normal mature neutrophil. Increasingly, techniques are being developed to identify better the maturation stages of cells and association with specific functional capabilities by flow cytometric techniques. The neoplastic nature ofsome normal-appearing leukocytes can be identified by techniques, such as fluorescent in situ hybridization. With the rapid advances in many approachs to understand the nature and functional capability of leukocytes, the eyecount differential with the traditional Romanowsky stain may be past the apogee ofits ascent and beginning its trip into history along with the hemocytometer counting chamber and the Sahli pipet. The development and implementation ofnew laboratory cornerstone techniques for diagnosis ofhematologic disease are eagerly awaited. On the other hand, the red cells and platelets exist to function in the peripheral blood. More emphasis is needed in the development ofautomated methods ofdetermining the nature and functional capabilities of these true blood cells as part ofthe CBC. References 1. Bentley SA, Johnson A, Bishop C: A parallel evaluation offour automated hematology analyzers. Am J Clin Pathol 100: , Bull BS, Korpman RA: The logistics of the leukocyte differential count (implications for automation): In Koepke JA (ed): Differential Leukocyte Counting. CAP Conference/ Aspen Skokie, IL, College ofamerican Pathologists, 1978, pp CAP hematology and clinical microscopy committee report. CAP TODAY May, Cella Vision: Leaflet of Diffmaster Octiva. Lund, Sweden, Cella Vision AB, Cox CJ, Habermann TM, Payne BA, et al: Evaluation ofthe Coulter Counter Model S- Plus IV. Am J Clin Pathol 84: , Dutcher TF, Jakubowski D, Orser B: A comparative evaluation ofautomated blood cell differential analyzers: Hematrak, Larc and Hemalog D. In Koepke JA (ed): Differential Leukocyte Counting. CAP Conference/Aspen Skokie, IL, College of American Pathologists, 1978, pp Kobayashi T: Cell analyzer, Microx. Medical Electronics and Biotechnology 17:64, 1979

19 PERIPHERAL BLOOD FILM REVIEW Koepke JA (ed): Differential leukocyte counting CAP Conference/Aspen, Skokie, IL, College ofamerican Pathologists, Koepke JA, Dotson MA, Shifman MA: False positive/false negative rates for the eyecount leukocyte differential count. Blood Cells Mol Dis 11: , Mitsuhashi T, Kwai Y, Arai T, et al: Telehematology trials using the Sysmex blood cell imaging filing system. Sysmex J International 5:77 84, NCCLS Document H20-A. Reference Leukocyte Differential Count (Proportional) and Evaluation ofinstrument Methods. Villanova, PA, National Committee for Clinical Laboratory Standards, Pierre RV: The Technicon Hemalog D Automated Leukocyte Differential System. In Koepke JA (ed): Differential Leukocyte Counting. CAP Conference/Aspen Skokie, IL, College ofamerican Pathologists, 1978, pp Pierre RV, O Sullivan MB: Evaluation of the Hemalog D automated differential leukocyte counter. Mayo Clin Proc 49: , Rümke CL: The statistically expected variability in differential counting. In Koepke JA (ed): Differential Leukocyte Counting. CAP Conference/Aspen, Skokie, IL, College ofamerican Pathologists, 1978, pp Stiene-Martin EA: Causes for poor leukocyte distribution in manual spreader-slide blood films. Am J Med Tech 46: , Trobaugh FE Jr, Bacus JW: Design and performance of the LARC Automated Leukocyte Classifier. In Koepke JA (ed): Differential Leukocyte Counting. CAP Conference/Aspen Skokie, IL, College ofamerican Pathologists, 1978, pp Address reprint requests to Robert V. Pierre, MD 1519 Majestic Way Glendale, CA rpierre@earthlink.net