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EXERCISE #8: Automation MLAB 1415 Hematology

HEMATOLOGY AUTOMATION

LAB OBJECTIVE

1. Cite the electrical impedance principle of cell counting.2. Identify instruments that utilize electrical impedance.3. State the principles of light scatter used in cell counting.4. Identify instruments that utilize light scatter for analysis.5. Categorize cell parameters as directly measured and whether they are derived from

a histogram, scattergram or calculation.6. Identify and interpret CBC parameters and correlate patient results with various

disease states. HISTORY Automated cell counts began in the mid 1950’s. Blood cell counters replaced manual hematocytometer blood cell counts, spun hematocrits, spectrophotometrically determined hemoglobins and peripheral blood smear preparations. The major advantage of these instruments was they provided data with increased reliability, precision and accuracy. The two main principles of blood cell counting currently used by hematology instruments are impedance and optical light scatter.

Electronic Impedance:Principle:Based on the increased resistance that occurs when a blood cell with poor conductivity passes through an electrical field. The number of pulses indicate the blood cell count and the amplitude (height) of each pulse is proportional to the volume of each cell. Originally developed by Coulter Electronics, it is often referred to as the Coulter principle.

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Procedure:1. EDTA sample is divided into four aliquots. The first aliquot is delivered to the RBC/platelet

dilution chamber where blood cells are diluted in an electrically conductive diluent. Within the dilution chamber is an external electrode and three apertures, each of which has an internal electrode. As the individual cells pass through the aperature, there is an increase in the resistance between the two electrodes proportional to the volume of the cell. In the chamber that counts RBCs and platelets, particles that are between 2-20 fL are counted as platelets and those greater than 36 fL are counted as RBCs. A steady stream of diluent flows behind each aperature to prevent cells from re-entering the aperature- this is called the sweep flow.

2. Since there are three aperatures within the chamber, three erythrocyte counts are obtained. These counts are compared and evaluated. If there is agreement, the reported RBC count represents the average of the counts from these three aperatures.

3. At this point, an erythrocyte histogram is created. The erythrocyte histogram (size distribution curve) is based on cell volume and relative cell number. Each pulse of the X axis represents size in fL, the Y-axis is the relative number of cells.

The histogram allows the visualization of changes in size within the RBC population. In a homogeneous cell population, the curve assumes a symmetrical bell-shaped or Gaussian distribution. A wide or more flattened curve is seen when the standard deviation from the mean is increased. Histograms provide information about erythrocyte, leukocyte and platelet frequency and distribution as well as depict the presence of subpopulations. Shifts in one direction or another can be of diagnostic importance. Larger than normal cells will cause the histogram to shift to the right and smaller than normal cells will cause the histogram to shift to the left. A bimodal peak illustrating a dimorphic RBC population (camel humps) can be seen in such situations as cold agglutinin disease, after transfusion of red blood cells into a person with abnormally sized RBCs, treated iron deficiency anemia, as well as other conditions.The RBC histogram displays cells as small as 24fL, but only those between 36-360 fL are counted as RBCs.

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This histogram represents a normal red cell distribution. The small tail to the right of the curve represents coincidence, multiple cells passing through the aperture at the same time.

4. The platelet count is also obtained from the RBC/platelet dilution chamber. In the electrical impedance system, the analyzer’s computer classifies particles that are greater than 2 fL or less than 20 fL as platelets. The raw platelet histogram is electronically smoothed and extrapolated over 0 to 70 fL. The final platelet count is derived from this extrapolated histogram. The MPV (mean platelet volume) and the PDW (platelet distribution width) are obtained from the platelet histogram as well.

Platelet Histogram

5. The second aliquot is delivered to the WBC/hemoglobin dilution chambers. The WBC count is directly measured by electronic impedance from the leukocyte dilution after lytic agent is added. The lytic agent lyses the RBCs and converts hemoglobin to cyanmethemoglobin and shrinks the leukocyte cell membrane and cytoplasm. Therefore, the WBC count represents a measure of cell volume not native cell size. Particles greater than 35 fL are counted as WBCs. The WBC count is obtained from three aperatures and averaged.

6.The WBC histogram represents the size distribution curve for the leukocyte data and allows for

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visualization of subpopulations based on their relative sizes. The leukocyte histogram is the basis of the three-part differential identifying cells on the following:

lymphocytes between 35 and 90 fL mononuclear cells as cells between 90 and 160fL granulocytes as cells between 160 and 450 fL

7. The instrument examines the WBC histogram for the presence of interferences and uses a system of flags to alert the technologist. A valley or depression should be seen between each population. Abnormal sized cells or abnormal particles can cause abnormal patterns and the instrument will print an alert specific for the region where the abnormal pattern exists.

8. The hemoglobin concentration is determined by measuring the absorbance of cyanmethemoglobin in the WBC/hemoglobin chamber at 525 nm.

9. The third aliquot is delivered to the orbital mixing chamber. Here blood is lysed to remove the erythrocytes and leave the leukocytes in their native state. This dilution is sent to the VCS (volume,conductivity, scatter) flow cell for determination of the five-part differential.

Volume Analysis –Determined by impedance as they pass through the aperture. Conductivity - Cell walls act as conductors to high-frequency current. As the current

passes through the walls and through each cell interior, it detects differences in the insulating properties of cell components. It characterizes the nuclear and granular constituents and the chemical composition of the cell interior.

Scatter - Leukocytes are hydrodynamically focused and passed in a steady stream through a sensing zone on which a laser light is focused. As each cell passes through the sensing zone of the flow cell, it scatters and reflects the focused light which is detected by a photodetector. The patterns of scatter are measure at various angles (forward scatter at

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180 and right angle scatter at 90). Scattered light provides information about cell structure, shape and reflectivity. The characteristics are used to differentiate the various types of WBCs and to produce scatterplots with a five-part differential.

10. The fourth aliquot is used to measure reticulocytes.

Examples of instruments using electronic impedance:a. Beckman-Coulter (Gen-S, LH-750)b. Sysmex XE-2100c. Abbott CELL_DYN Sapphire

Optical Light Scatter:Principle:Based on light scattering measurements obtained as a single blood cell passes through a beam or light (optical/laser). Blood cells create forward scatter and side scatter that photodetectors detect. These light signals are converted into electrical signals. The number of signals indicates the number of cells. The degree of forward scatter is a measurement of cell size. The degree of side scatter is a measurement of cell complexity and granularity.

Procedure:1. Instrument has five measurement channels:

Erythrocyte/platelet channel: determined by light scattering measurements as cells pass through a helium-neon laser beam Hemoglobin channel: Cyanmethemoglobin determined at 546 nm Peroxidase channel: Id of neutrophils, monocytes and eosinophils by the degree of peroxidase activity and degree of forward light scatter. Lymphs and unstained larger cells not stained by peroxidase Basophil/lobularity channel: Basophils retain cytoplasm after diluent lyses other cells Reticulocyte channel

Examples of Instruments using Light Scatter:a. Siemens ADVIA- 120b. Siemens ADVIA 2120

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Parameter = a statistical term that refers to any numerical value that describes an entire population.

Parameters and their derivation for the Beckman-Coulter System

Parameter Unit of reporting

Derivation Calculation

WBC n x 103 cells/μL

# of leukocytes measured directly, multiplied by the calibration factor

NA

RBC n x 106 cells/μL

# of red cells measured directly, multiplied by the calibration factor

NA

Hemoglobin g/dL A beam of white light shines through the lysed WBC solution and then through an optical filter. The transmittance of light (525 nm) as compared to a reagent blank is converted to absorbance, then converted to g/dL using a calibration factor

NA

MCV fL Derived from the RBC histogram by multiplying the # of RBCs by the size of RBCs and multiplied by a calibration constant.

NA

Hematocrit % Calculated RBC x MCV 10

MCH pg Calculated Hgb X 10RBC

MCHC g/dL Calculated Hgb X 100Hct

RDW % Derived from RBC histogram NA

Platelet n x 103 cells/μL

# of platelets derived from the Plt histogram and multiplied by a calibration constant

NA

MPV fL Derived from the platelet histogram NA

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Instrument manufacturers and their websites are listed below:

Manufacturer Instruments available Web address

Abbot Cell-Dyn 1200, 3200, 4000 abbot.com

Siemens Diagnostics (Bayer) Advia 60 and 120 bayerdiag.com

Beckman-Coulter STKS, Gen-S beckmancoulter.com

Sysmex SE-Series Sysmex.com

SPECIMENEDTA-anticoagulated blood

QUALITY CONTROL1. Commercial low, normal and high controls

Monitor the CBC and differential parameters. Latron controls monitor the performance of the volume, conductivity and light scatter for the automated differential. Control values are stored in the instrument computer and can be monitored with the generation of a Levey-Jennings graph for each parameter.

2. XB analysisPatient results are monitored with continuous XB analysis (weighted moving averages), which uses the patient’s own data to monitor population values and instrument performance. Batches of 20 samples are used to track MCV, MCH, and MCHC values. This method can be used to detect changes in sample handling, reagents, or instrument performance. Batches of 20 are automatically printed out. The analyzer is considered to be in control when the MCV, MCH and MCHC determined on a batch of 20 patients by use of the XB algorithm are within 3% of the expected mean indices of the population.

3. Delta ChecksAnother method of quality control is the use of delta checks (delta means “difference”) which compares a patient’s own values with their most previous results. If the difference between the two is greater than laboratory-set limits, the current result is immediately flagged for review. These can only be used if the instrument is interfaced with a host Laboratory Information System (LIS).

4. Mode to ModeA selected specimen is run in both primary and secondary mode and results must fall within a specified limit.

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REFERENCE RANGES

REPORTING RESULTS

Parameter Normal Range

WBC 3.0-9.0 x 103/μL

RBC Male 4.5-5.5 x 106/μLFemale 4.0-5.0 x 106/μL

Hemoglobin Male 14-17.4 g/dlFemale 12-16 g/dl

Hematocrit Male 42-52%Female 36-46%

MCV 80-100 fl

MCH 28-34 pg

MCHC 32-36 g/dl or %

RDW 12.0-14.6%

Platelets 150,000 - 450,000/μL

MPV 6.8-10.2 fl

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LINEARITY LIMITSThe instrument will be accurate as long as the results fall within a certain range known as the linearity range. Linearity ranges very by instrument.

Linearity Range Table

Parameter Coulter STKS Bayer Advia

WBC 0-99.9 x 103/μL 0.02-400 x 103/μL

RBC 0-7.0 x 106/μL 0.0-7.0 x 106/μL

Hgb 0-18 g/dL 0-22.5 g/dL

MCV 50-200 fL NA

Plt 0-999 x 103μL 5.0-3500 x 103μL

MPV 5-20 fL NA

INSTRUMENT CODES

Code Cause Action indicated

+++++ Result exceeds printable range

Dilute 1:2 and rerun. Continue further dilutions if necessary until the result falls within the linearity range (See “Handling Abnormal Results)

H Result is higher than the laboratory-set patient high action limit

Review result

L Result is lower than the laboratory-set patient low action limit

Review result

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INTERFERENCES THAT MAY CAUSE ERRONEOUS RESULTS

Parameter Interfering agent

WBC Unusual RBC abnormalities that resist lysisNucleated RBCsFragmented WBCsUnlysed particles greater than 35 fLVery large or aggregated pltsSpecimens containing fibrin, cell fragments or other debris (esp pediatric/oncology specimens

RBC Very high WBC (greater than 99.9)High concentration of very large plateletsAgglutinated RBCs, rouleaux will break up when Istoton is addedRBCs smaller than 36 fLSpecimens containing fibrin, cell fragments or other debris (esp pediatric/oncology specimens

Hgb Very high WBC countSevere lipemiaHeparinCertain unusual RBC abnormalities that resist lysingAnything that increases the turbidity of the sample such as elevated levels of triglyceridesHigh bilirubin

MCV Very high WBC countHigh concentration of very large plateletsAgglutinated RBCsRBC fragments that fall below the 36 fL thresholdRigid RBCs

RDW Very high WBCHigh concentration of very large or clumped plateletsRBCs below the 36 fL thresholdTwo distinct populations of RBCsRBC agglutinatesRigid RBCs

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Parameter Interfering agent

Plt Very small red cells near the upper thresholdCell fragmentsClumped plateletsCellular debris near the lower platelet threshold

MPV Known factors that interfere with the platelet count and shape of the histogramKnown effects of EDTA

Hct Known factors that interfere with the parameters used for computation, RBC and MCV

MCH Known factors that interfere with the parameters used for computation, Hgb and RBC

MCHC Known factors that interfere with the parameters used for computation, Hgb, RBC and MCV

Diff parameters

Known factors that affect the WBC count as listed above, high triglycerides that affect lysing

HANDLING ABNORMAL RESULTS

Plts < 40,000 Check the integrity of the specimen (look for clots, short draw, etc.)Confirm count with smear review for clumps, RBC fragments, giant platelets, very small RBCs

WBC ++++ Dilute 1/2 with Isoton or further until count is within linearity (for final result, multipy diluted result by dilution factor); subtract final WBC from RBC; perform spun hct, calculate MCV from correct RBC & Hct (MCV = Hct/RBC x 10), do not report HGB, MCH, MCHC. Plt counts are not affected by high WBC. Add comment, “Unable to report Hgb, MCH, MCHC due to high WBC.”

Plt ++++ Check smear for RBC fragments or microcytes.1. If present, perform plt estimate. If they do not agree, perform manual plt count. 2. If not present, dilute specimen 1:2 with Isoton or further until count is within linearity, multiply diluted result by dilution factor.

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RBC > 7.0 Dilute 1:2 with Isoton or further until count is within linearity, multiply dilution result by dilution factor; perform spun hct, review Hgb, recalculate MCH, MCHC

MCHC > 36.5

Perform manual hct.1. If it stays the same, check plasma for lipemia, icteremia or other color interference. If present, perform Isoton replacement. Pour 3 ml blood into 10x75 tube. Mark level of top of blood. Centrifuge at high speed for 10 minutes. Pipette off plasma being careful not to disturb red cells. Replace plasma with Isoton to mark. Mix well and rerun to obtain correct Hgb and MCH, MCHC. RBC should be within 0.2 of previous result. Add comment, “results corrected for lipemia”.

2. If it is significantly higher, check the specimen for cold agglutinin by looking for RBC clumping. If present, warm the specimen at 37C for 5 minutes, mix well and repeat. If results are acceptable, report. If cold agglutinin persists, report the spun hct and mark through the RBC, MCV, MCH, MCHC results. Add comment, “specimen warmed before running”.

3. If the above conditions are not found, check the smear for spherocytes or lyse-resistant red cells. If present, ensure correct instrument operation by running controls and report result.

MCHC < 36.5

Perform spun hct. Verify proper instrument operation by running a previous patient. Decreased MCHC may be caused by swollen hyperglycemic red cells. Perform isoton replacement or correct values using spun hct.

Low plts, “Giant plts” or EDTA clumpers

Confirm with smear review.1. If clumps are present, check with phlebotomist to see if the phlebotomy was difficult. If not, recollect in blue top tube (Na citrate anticoagulant). If platelet clumps disappear and platelet count is acceptable, multiply plt result by 1.1 to account for the dilution factor.

2. If there are no clumps, but giant platelets are present, perform plt estimate from smear. Perform manual platelet count if smear estimate and instrument count do not agree.

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PROCEDURE NOTES1. The approximate relationship of the hemoglobin level to hematocrit is 1:3, a ratio that

may vary with the cause of the anemia and the effect of that cause on the RBC indices, particularly the MCV. This is referred to as the rule of 3.

Hct (2) = 3 x HgbAlso, the RBC is usually about a the hemoglobin. If the ratios are not appropriate, there is either something wrong with the instrument or there is something unusual with the specimen which must be resolved before reporting results. Some typical instrument problems that cause the H&H not to match include inadequate delivery of lysing reagent, inadequate delivery of diluent, clogs in tubing or blood sampling valve, inadequate draining of baths, etc. To differentiate between an instrument problem and a specimen problem, run a control or repeat a specimen from earlier in the day.

REFERENCESHarmening., Denise, Clinical Hematology and Fundamentals of Hemostasis, 3rd edition, pp. 593-

599.Turgeon, Mary Louise, Clinical Hematology - Theories and Procedures, 3rd edition, pp373, 376-

382.Rodak, Bernadette, Diagnostic Hematology, 1st edition, p.605-606.Coulter STKS Operating ManualMcKenzie, Shirlyn, Clinical Laboratory Hematology, 2nd edition,pp 813-829.

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STUDY QUESTIONS

Name ________________________________

Date_________________________________

1. State the principle of electronic impedance. (2 pts)

2. Label the cell populations on the following histograms: (6 pts)

3. Sketch the appearance of histograms associated with each of the following: (6 pts)macrocytosis MCV 120 fl

Microcytosis MCV 65 fl

Dimorphic red cell population

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4. List the parameters provided by automated analysis and the derivation (direct measurement or calculated). (10 pts)

5. What is coincidence correction? (2 pts)

6. How is the RDW derived? List three causes of an increased RDW. (5 pts)

For each of the following conditions, state whether the test would be falsely ↑, or not affected at all:

7. WBC count (6 pts)NRBCs

giant plts

clumped plts

hemolysis

lipemia

↑ RBC

8. RBC count (6 pts)

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cold agglutinin

rouleaux

lipemia

greatly ↑ WBC

clotted specimen

↑ background count

9. Hemoglobin (4 pts)

lipemia

icteremia

greatly ↑ WBC

↑ background count

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EXERCISE #8: Automated CBC MLAB 1415 Hematology

10. Calculate indices from the following results and state the expected RBC morphology (macro, micro, normocytic and hypo or normochromic). (24 pts)

RBCX 106/L

Hgbg/dL

Hct%

MCVfL

MCHpg

MCHC%

Expected RBC morphology

A 4.66 17.7 52.3

B 2.95 9.1 29.9

C 3.64 10.8 32.0

D 4.90 15.1 43.8

E 3.50 8.0 26.2

F 5.04 10.0 30.1

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