Rodak's Hematology Clinical Principles and Applications

PART III

Laboratory Evaluation of Blood Cells

CHAPTER 14

Manual, semiautomated, and point-of-care testing in hematology

Karen S. Clark , Teresa G. Hippel

OUTLINE

Manual Cell Counts

Equipment

Calculations

White Blood Cell Count

Platelet Count

Red Blood Cell Count

Disposable Blood Cell Count Dilution Systems

Body Fluid Cell Counts

Hemoglobin Determination

Principle

Microhematocrit

Rule of Three

Red Blood Cell Indices

Mean Cell Volume

Mean Cell Hemoglobin

Mean Cell Hemoglobin Concentration

Reticulocyte Count

Principle

Absolute Reticulocyte Count

Corrected Reticulocyte Count

Reticulocyte Production Index

Reticulocyte Control

Automated Reticulocyte Count

Erythrocyte Sedimentation Rate

Principle

Modified Westergren Erythrocyte Sedimentation Rate

Wintrobe Erythrocyte Sedimentation Rate

Disposable Kits

Automated Erythrocyte Sedimentation Rate

Point-of-Care Testing

Point-of-Care Tests

Objectives

After completion of this chapter, the reader will be able to:

1. State the dimensions of the counting area of a Neubauer ruled hemacytometer.
2. Describe the performance of manual cell counts for white blood cells, red blood cells, and platelets, including types of diluting fluids, typical dilutions, and typical areas counted in the hemacytometer.
3. Calculate dilutions for cell counts when given appropriate data.
4. Calculate hemacytometer cell counts when given numbers of cells, area counted, and dilution.
5. Correct white blood cell counts for the presence of nucleated red blood cells.
6. Describe the principle of the cyanmethemoglobin assay for determination of hemoglobin.
7. Calculate the values for a standard curve for cyanmethemoglobin determination when given the appropriate data, describe how the standard curve is constructed, and use the standard curve to determine hemoglobin values.
8. Describe the procedure for performing a microhematocrit.
9. Identify sources of error in routine manual procedures discussed in this chapter and recognize written scenarios describing such errors.
10. Compare red blood cell count, hemoglobin, and hematocrit values using the rule of three.
11. Calculate red blood cell indices (mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration) when given appropriate data, and interpret the results relative to the volume and hemoglobin content and concentration in the red blood cells.
12. Describe the principle and procedure for performing a manual reticulocyte count and the clinical value of the test.
13. Given the appropriate data, calculate the relative, absolute, and corrected reticulocyte counts and the reticulocyte production index; interpret results to determine the adequacy of the bone marrow erythropoietic response in an anemia.
14. Describe the procedure for performing the Westergren erythrocyte sedimentation rate and state its clinical utility.
15. Describe the aspects of establishing a point-of-care testing program, including quality management and selection of instrumentation.
16. Discuss the advantages and disadvantages of point-of-care testing as they apply to hematology tests.
17. Describe the principles of common instruments used for point-of-care testing for hemoglobin level, hematocrit, white blood cell counts, and platelet counts.

CASE STUDIES

After studying the material in this chapter, the reader should be able to respond to the following case studies:

Case 1

The following results are obtained for a patient with normocytic, normochromic red blood cells on a peripheral blood film:

RBC count = 4.63 × 10¹²/L

HGB = 15 g/dL

HCT = 40% (0.40 L/L)

1. Using the rule of three, given the hemoglobin concentration above, what is the expected value for the hematoctrit?
2. What could cause the hemoglobin to be falsely elevated or the hematocrit to be falsely low?
3. What would you do to correct for the interferences you listed in question 2?

Case 2

For another patient, the following results are obtained:

RBC count = 3.20 × 10¹²/L

HGB = 5.8 g/dL

HCT = 18.9% (0.19 L/L)

1. Calculate the red blood cell indices.
2. How would you describe the red blood cell volume and hemoglobin concentration based on these indices?
3. How should you verify this?

Case 3

The following results are obtained for a patient using a point-of-care device that employs the conductivity method to measure the hematocrit:

Sodium = 160 mmol/L (Reference interval: 135 to 145 mmol/L)

Potassium = 3.6 mmol/L (Reference interval: 3.5 to 5.5 mmol/L)

HCT = 17.0% (0.17 L/L)

HGB = 6.0 g/dL

1. Which electrolyte concentration could affect the hematocrit?
2. Would this electrolyte concentration falsely decrease or increase the hematocrit value?
3. What other factors can decrease the hematocrit value using this point-of-care device?

Clinical laboratory hematology has evolved from simple observation and description of blood and its components to a highly automated, extremely technical science, including examination at the molecular level. However, some of the more basic tests have not changed dramatically over the years. This chapter provides an overview of these basic tests and presents the manual and semiautomated methods that can be used in lieu of automated instrumentation. Included in this chapter is a discussion of point-of-care testing in hematology.

Manual cell counts

Although most routine cell-counting procedures in the hematology laboratory are automated, it may be necessary to use manual methods when counts exceed the linearity of an instrument, when an instrument is nonfunctional and there is no backup, in remote laboratories in Third World countries, or in a disaster situation when testing is done in the field. Although the discussion in this chapter concerns whole blood, body fluid cell counts are also often performed using manual methods. Chapter 18 discusses the specific diluents and dilutions used for body fluid cell counts. Chapter 15 discusses automated cell-counting instrumentation in detail.

Manual cell counts are performed using a hemacytometer, or counting chamber, and manual dilutions made with calibrated, automated pipettes and diluents (commercially available or laboratory prepared). The principle for the performance of cell counts is essentially the same for white blood cells (WBCs), red blood cells (RBCs), and platelets; only the dilution, diluting fluid, and area counted vary. Any particle (e.g., sperm) can be counted using this system.

Equipment

Hemacytometer

The manual cell count uses a hemacytometer, or counting chamber. The most common one is the Levy chamber with improved Neubauer ruling. It is composed of two raised surfaces, each with a 3 mm × 3 mm square counting area or grid (total area 9 mm²), separated by an H-shaped moat. As shown in Figure 14-1, this grid is made up of nine 1 mm × 1 mm squares. Each of the four corner (WBC) squares is subdivided further into 16 squares, and the center square subdivided into 25 smaller squares. Each of these smallest squares is 0.2 mm × 0.2 mm which is ¹/₂₅ of the center square or 0.04 mm². A coverslip is placed on top of the counting surfaces. The distance between each counting surface and the coverslip is 0.1 mm; thus the totalvolume of one entire grid or counting area on one side of the hemacytometer is 0.9 mm³. Hemacytometers and coverslips must meet the specifications of the National Bureau of Standards, as indicated by the initials “NBS” on the chamber. When the dimensions of the hemacytometer are thoroughly understood, the area counted can be changed to facilitate the counting of samples with extremely low or high counts.

FIGURE 14-1 Hemacytometer and a close-up view of the counting areas as seen under the microscope. The areas for the standard white blood cell count are labeled W, and the areas for the standard red blood cell count are labeled R. The entire center square, outlined in blue, is used for counting platelets. The side view of the hemacytometer shows a depth of 0.1 mm from the surface of the counting grid to the coverslip.

Calculations

The general formula for manual cell counts is as follows and can be used to calculate any type of cell count:

Or

*Reciprocal of depth.

The calculation yields the number of cells per mm³. One mm³ is equivalent to one microliter (μL). The count per μL is converted to the count per liter (L) by multiplying by a factor of 10⁶.

White blood cell count

The WBC or leukocyte count is the number of WBCs in 1 liter (L) or 1 microliter (μL) of blood. Whole blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) or blood from a skin puncture is diluted with 1% buffered ammonium oxalate or a weak acid solution (3% acetic acid or 1% hydrochloric acid). The diluting fluid lyses the nonnucleated red blood cells in the sample to prevent their interference in the count. The typical dilution of blood for the WBC count is 1:20. A hemacytometer is charged (filled) with the well-mixed dilution and placed under a microscope and the number of cells in the 4 large corner squares (4 mm²) is counted.

Procedure

1. Clean the hemacytometer and coverslip with alcohol and dry thoroughly with a lint-free tissue. Place the coverslip on the hemacytometer.
2. Make a 1:20 dilution by placing 25 μL of well-mixed blood into 475 μL of WBC diluting fluid in a small test tube.
3. Cover the tube and mix by inversion.
4. Allow the dilution to sit for 10 minutes to ensure that the red blood cells have lysed. The solution will be clear once lysis has occurred. WBC counts should be performed within 3 hours of dilution.
5. Mix again by inversion and fill a plain microhematocrit tube.
6. Charge both sides of the hemacytometer by holding the microhematocrit tube at a 45-degree angle and touching the tip to the coverslip edge where it meets the chamber floor.
7. After charging the hemacytometer, place it in a moist chamber (Box 14-1) for 10 minutes before counting the cells to give them time to settle. Care should be taken not to disturb the coverslip.
8. While keeping the hemacytometer in a horizontal position, place it on the microscope stage.
9. Lower the condenser on the microscope and focus by using the low-power (10×) objective lens (100× total magnification). The cells should be distributed evenly in all of the squares.
10. For a 1:20 dilution, count all of the cells in the four corner squares, starting with the square in the upper left-hand corner (Figure 14-1). Cells that touch the top and left lines should be counted; cells that touch the bottom and right lines should be ignored (Figure 14-2). See Figure 14-3for the appearance of WBCs in the hemacytometer using the low-power objective lens of a microscope.
11. Repeat the count on the other side of the counting chamber. The difference between the total cells counted on each side should be less than 10%. A greater variation could indicate an uneven distribution, which requires that the procedure be repeated.
12. Average the number of WBCs counted on the two sides. Using the average, calculate the WBC count using one of the equations given earlier.

FIGURE 14-2 One large corner square of a hemacytometer indicating which cells to count. Cells touching the left and top lines (solid circles) are counted. Cells touching bottom and right (open circles) are not counted.

FIGURE 14-3 White blood cells as seen in the hemacytometer under low power (10× objective) 100× total magnification.

BOX 14-1

How to Make a Moist Chamber

A moist chamber may be made by placing a piece of damp filter paper in the bottom of a Petri dish. An applicator stick broken in half can serve as a support for the hemacytometer.

Example using the first equation

When a 1:20 dilution is used, the four large squares on one side of the chamber yield counts of 23, 26, 22, and 21. The total count is 92. The four large squares on the other side of the chamber yield counts of 28, 24, 22, and 26. The total count is 100. The difference between sides is less than 10%.

The average number of cells of the two sides of the chamber is 96. Using the average in the formula:

Alternately, a 1:100 dilution may be used counting the number of cells in the entire counting area (9 large squares, 9 mm²) on both sides of the chamber (Table 14-1). As an example, if an average of 54 cells were counted in the entire counting area on both sides of the chamber:

TABLE 14-1

Manual Cell Counts with Most Common Dilutions, Counting Areas

General reference intervals for males and females in different age groups can be found on the inside front cover of this text. Reference intervals may vary slightly according to the population tested and should be established for each laboratory.

Sources of error and comments

1. The hemacytometer and coverslip should be cleaned properly before they are used. Dust and fingerprints may cause difficulty in distinguishing the cells.
2. The diluting fluid should be free of contaminants.
3. If the count is low, a greater area may be counted (e.g., 9 mm²) to improve accuracy.
4. The chamber must be charged properly to ensure an accurate count. Uneven flow of the diluted blood into the chamber results in an irregular distribution of cells. If the chamber is overfilled or underfilled, the chamber must be cleaned and recharged.
5. After the chamber is filled, allow the cells to settle for 10 minutes before counting.
6. Any nucleated red blood cells (NRBCs) present in the sample are not lysed by the diluting fluid. The NRBCs are counted as WBCs because they are indistinguishable when seen on the hemacytometer. If five or more NRBCs per 100 WBCs are observed on the differential count on a stained peripheral blood film, the WBC count must be corrected for these cells. This is accomplished by using the following formula:

Report the result as the “corrected” WBC count.

7. The accuracy of the manual WBC count can be assessed by performing a WBC estimate on a Wright-stained peripheral blood film made from the same specimen (Chapter 16).

Platelet count

A platelet count is the number of platelets in 1 liter (L) or 1 microliter (μL) of whole blood. Platelets adhere to foreign objects and to each other, which makes them difficult to count. They also are small and can be confused easily with dirt or debris. In this procedure, whole blood, with EDTA as the anticoagulant, is diluted 1:100 with 1% ammonium oxalate to lyse the nonnucleated red blood cells. The platelets are counted in the 25 small squares in the large center square (1 mm²) of the hemacytometer using a phase-contrast microscope in the reference method described by Brecher and Cronkite.¹ A light microscope can also be used, but visualizing the platelets may be more difficult.

Procedure

1. Make a 1:100 dilution by placing 20 μL of well-mixed blood into 1980 μL of 1% ammonium oxalate in a small test tube.
2. Mix the dilution thoroughly and charge the chamber. (Note: A special thin, flat-bottomed counting chamber is used for phase-microscopy platelet counts.)
3. Place the charged hemacytometer in a moist chamber (Box 14-1) for 15 minutes to allow the platelets to settle.
4. Platelets are counted using the 40× objective lens (400× total magnification). The platelets have a diameter of 2 to 4 μm and appear round or oval, displaying a light purple sheen when phase-contrast microscopy is used. The shape and color help distinguish the platelets from highly refractile dirt and debris. “Ghost” RBCs often are seen in the background.
5. Count the number of platelets in the 25 small squares in the center square of the grid (Figure 14-1). The area of this center square is 1 mm². Platelets should be counted on each side of the hemacytometer, and the difference between the totals should be less than 10%.
6. Calculate the platelet count by using one of the equations given earlier. Using the first equation as an example, if 200 platelets were counted in the entire center square,
7. The accuracy of the manual platelet count should be verified by performing a platelet estimate on a Wright-stained peripheral blood film made from the same specimen (Chapter 16).

General reference intervals for males and females according to age groups can be found on the inside front cover of this text.

Sources of error and comments

1. Inadequate mixing and poor collection of the specimen can cause the platelets to clump on the hemacytometer. If the problem persists after redilution, a new specimen is needed. A skin puncture specimen is less desirable because of the tendency of the platelets to aggregate or form clumps.
2. Dirt in the pipette, hemacytometer, or diluting fluid may cause the counts to be inaccurate.
3. If fewer than 50 platelets are counted on each side, the procedure should be repeated by diluting the blood to 1:20. If more than 500 platelets are counted on each side, a 1:200 dilution should be made. The appropriate dilution factor should be used in calculating the results.
4. If the patient has a normal platelet count, the 5 small, red blood cell squares (Figure 14-1) may be counted. Then, the area is 0.2 mm²on each side.
5. The phenomenon of “platelet satellitosis” may occur when EDTA anticoagulant is used. This refers to the adherence of platelets around neutrophils, producing a ring or satellite effect (Figure 16-1). Using sodium citrate as the anticoagulant should correct this problem. Because of the dilution in the citrate evacuated tubes, it is necessary to multiply the obtained platelet count by 1.1 for accuracy (Chapter 16).

Red blood cell count

Manual RBC counts are rarely performed because of the inaccuracy of the count and questionable necessity. Use of other, more accurate manual RBC procedures, such as the microhematocrit and hemoglobin concentration, is desirable when automation is not available.

Table 14-1 contains information on performing manual WBC, platelet, and RBC counts.

Disposable blood cell count dilution systems

Capillary pipette and diluent reservoir systems are commercially available for WBC and platelet counts. One such system is LeukoChek™ (Biomedical Polymers, Inc., Gardner, MA). It consists of a capillary pipette (calibrated to accept 20 μL of blood) that fits into a plastic reservoir containing 1.98 mL of 1% buffered ammonium oxalate (Figure 14-4). Blood from a well-mixed EDTA-anticoagulated specimen or from a skin puncture is allowed to enter the pipette by capillary action to the fill volume. The blood is added to the reservoir making a 1:100 dilution. After mixing the reservoir and allowing 10 minutes for lysis of the red blood cells, the reverse end of the capillary pipette is placed in the reservoir cap making a dropper. The first 3 or 4 drops of the diluted sample is discarded, and the capillary pipette is used to charge the hemacytometer.

FIGURE 14-4 LeukoChek™ blood diluting system for manual white blood cell and platelet counts. It consists of a 20 µL capillary pipette and plastic reservoir containing 1.98 mL of 1% buffered ammonium oxalate that makes a 1:100 dilution of whole blood. Source: (Courtesy Biomedical Polymers, Inc., Gardner, MA.)

Both WBC and platelet counts can be done from the same diluted sample. WBCs are counted in all 9 large squares (9 mm²) using low power (100× total magnification). Platelets are counted in the 25 small squares in the center square (1 mm²) using high power (400× total magnification). The standard formula is used to calculate the cell counts.

Body fluid cell counts

Body fluid cell counts are discussed in detail in Chapter 18.

Hemoglobin determination

The primary function of hemoglobin within the red blood cell is to carry oxygen to and carbon dioxide from the tissues. The cyanmethemoglobin (hemoglobincyanide) method for hemoglobin determination is the reference method approved by the Clinical and Laboratory Standards Institute.²

Principle

In the cyanmethemoglobin method, blood is diluted in an alkaline Drabkin solution of potassium ferricyanide, potassium cyanide, sodium bicarbonate, and a surfactant. The hemoglobin is oxidized to methemoglobin (Fe³⁺) by the potassium ferricyanide, K₃Fe(CN)₆. The potassium cyanide (KCN) then converts the methemoglobin to cyanmethemoglobin:

The absorbance of the cyanmethemoglobin at 540 nm is directly proportional to the hemoglobin concentration. Sulfhemoglobin is not converted to cyanmethemoglobin; it cannot be measured by this method. Sulfhemoglobin fractions of more than 0.05 g/dL are seldom encountered in clinical practice, however.³

Procedure

1. Create a standard curve, using a commercially available cyanmethemoglobin standard.
2. When a standard containing 80 mg/dL of hemoglobin is used, the following dilutions should be made:
3. Transfer the dilutions to cuvettes. Set the wavelength on the spectrophotometer to 540 nm and use the blank to set to 100% transmittance.
4. Using semilogarithmic paper, plot percent transmittance on the y-axis and the hemoglobin concentration on the x-axis. The hemoglobin concentrations of the control and patient samples can be read from this standard curve (Figure 14-5).
5. A standard curve should be set up with each new lot of reagents. It also should be checked when alterations are made to the spectrophotometer (e.g., bulb change).
6. Controls should be run with each batch of samples. Commercial controls are available.
7. Using the patient’s whole blood anticoagulated with EDTA or heparin or blood from a capillary puncture, make a 1:251 dilution by adding 0.02 mL (20 μL) of blood to 5 mL of cyanmethemoglobin reagent. The pipette should be rinsed thoroughly with the reagent to ensure that no blood remains. Follow the same procedure for the control samples.
8. Cover and mix well by inversion or use a vortex mixer. Let stand for 10 minutes at room temperature to allow full conversion of hemoglobin to cyanmethemoglobin.
9. Transfer all of the solutions to cuvettes. Set the spectrophotometer to 100% transmittance at the wavelength of 540 nm, using cyanmethemoglobin reagent as a blank.
10. Using a matched cuvette, continue reading the % transmittance of the patient samples and record the values.
11. Determine the hemoglobin concentration of the control samples and the patient samples from the standard curve.

FIGURE 14-5 Standard curve obtained when a cyanmethemoglobin standard of 80 mg/dL is used. A blank (100% transmittance) and four dilutions were made: 5 g/dL (72.9% transmittance), 10 g/dL (53.2% transmittance), 15 g/dL (39.1% transmittance), and 20 g/dL (28.7% transmittance).

General reference intervals can be found on the inside cover of this text.

Sources of error and comments

1. Cyanmethemoglobin reagent is sensitive to light. It should be stored in a brown bottle or in a dark place.
2. A high WBC count (greater than 20 × 10⁹/L) or a high platelet count (greater than 700 × 10⁹/L) can cause turbidity and a falsely high result. In this case, the reagent-sample solution can be centrifuged and the supernatant measured.
3. Lipemia also can cause turbidity and a falsely high result. It can be corrected by adding 0.01 mL of the patient’s plasma to 5 mL of the cyanmethemoglobin reagent and using this solution as the reagent blank.
4. Cells containing Hb S and Hb C may be resistant to hemolysis, causing turbidity; this can be corrected by making a 1:2 dilution with distilled water (1 part diluted sample plus 1 part water) and multiplying the results from the standard curve by 2.
5. Abnormal globulins, such as those found in patients with plasma cell myeloma or Waldenström macroglobulinemia, may precipitate in the reagent. If this occurs, add 0.1 g of potassium carbonate to the cyanmethemoglobin reagent. Commercially available cyanmethemoglobin reagent has been modified to contain KH₂PO₄salt, so this problem is not likely to occur.
6. Carboxyhemoglobin takes 1 hour to convert to cyanmethemoglobin and theoretically could cause erroneous results in samples from heavy smokers. The degree of error is probably not clinically significant, however.
7. Because the hemoglobin reagent contains cyanide, it is highly toxic and must be used cautiously. Consult the safety data sheet (Chapter 2) supplied by the manufacturer. Acidification of cyanide in the reagent releases highly toxic hydrogen cyanide gas. A licensed waste disposal service should be contracted to discard the reagent; reagent-sample solutions should not be discarded into sinks.
8. Commercial absorbance standards kits are available to calibrate spectrophotometers.
9. Handheld systems are commercially available to measure the hemoglobin concentration. An example is the HemoCue^{4, 5}(HemoCue, Inc., Brea, CA) (Figure 14-19) in which hemoglobin is converted to azidemethemoglobin and is read photometrically at two wavelengths (570 nm and 880 nm). This method avoids the necessity of sample dilution and interference from turbidity. It is discussed later in the section on point-of-care testing. Another method that has been used in some automated instruments involves the use of sodium lauryl sulfate (SLS) to convert hemoglobin to SLS-methemoglobin. This method does not generate toxic wastes.

FIGURE 14-19 The HemoCue ® Hb 201+ System for measuring hemoglobin. Source: (Courtesy HemoCue, Inc., Brea, CA.)

Microhematocrit

The hematocrit is the volume of packed red blood cells that occupies a given volume of whole blood. This is often referred to as the packed cell volume (PCV). It is reported either as a percentage (e.g., 36%) or in liters per liter (0.36 L/L).

Procedure

1. Fill two plain capillary tubes approximately three quarters full with blood anticoagulated with EDTA or heparin. Mylar-wrapped tubes are recommended by the National Institute for Occupational Safety and Health to reduce the risk of capillary tube injuries.¹⁰Alternatively, blood may be collected into heparinized capillary tubes by skin puncture. Wipe any excess blood from the outside of the tube.
2. Seal the end of the tube with the colored ring using nonabsorbent clay. Hold the filled tube horizontally and seal by placing the dry end into the tray with sealing compound at a 90-degree angle. Rotate the tube slightly and remove it from the tray. The plug should be at least 4 mm long.¹⁰
3. Balance the tubes in a microhematocrit centrifuge with the clay ends facing the outside away from the center, touching the rubber gasket.
4. Tighten the head cover on the centrifuge and close the top. Centrifuge the tubes at 10,000 gto 15,000 g for the time that has been determined to obtain maximum packing of red blood cells, as detailed in Box 14-2. Do not use the brake to stop the centrifuge.
5. Determine the hematocrit by using a microhematocrit reading device (Figure 14-6). Read the level of red blood cell packing; do not include the buffy coat (WBCs and platelets) when taking the reading (Figure 14-7).
6. The values of the duplicate hematocrits should agree within 1% (0.01 L/L).¹⁰General reference intervals according to sex and age can be found on the inside front cover of this text.

FIGURE 14-6 Microhematocrit reader.

FIGURE 14-7 Capillary tube with anticoagulated whole blood after it has been centrifuged. Notice the layers containing plasma, the buffy coat (white blood cells and platelets), and the red blood cells.

BOX 14-2

Determining Maximum Packing Time for Microhematocrit

The time to obtain maximum packing of red blood cells should be determined for each centrifuge. Duplicate microhematocrit determinations should be made using fresh, well-mixed blood anticoagulated with ethylenediaminetetraacetic acid (EDTA). Two specimens should be used, with one of the specimens having a known hematocrit of 50% or higher. Starting at 2 minutes, centrifuge duplicates at 30-second intervals and record results. When the hematocrit has remained at the same value for two consecutive readings, optimum packing has been achieved, and the second time interval should be used for microhematocrit determinations.¹⁰

Sources of error and comments

1. Improper sealing of the capillary tube causes a decreased hematocrit reading as a result of leakage of blood during centrifugation. A higher number of red blood cells are lost compared with plasma due to the packing of the cells in the lower part of the tube during centrifugation.
2. An increased concentration of anticoagulant (short draw in an evacuated tube) decreases the hematocrit reading as a result of red blood cell shrinkage.
3. A decreased or increased result may occur if the specimen was not mixed properly.
4. The time and speed of the centrifugation and the time when the results are read are important. Insufficient centrifugation or a delay in reading results after centrifugation causes hematocrit readings to increase. Time for complete packing should be determined for each centrifuge and rechecked at regular intervals. When the microhematocrit centrifuge is calibrated, one of the samples used must have a hematocrit of 50% or higher.¹⁰
5. The buffy coat of the sample should not be included in the hematocrit reading because this falsely elevates the result.
6. A decrease or increase in the readings may be seen if the microhematocrit reader is not used properly.
7. Many disorders, such as sickle cell anemia, macrocytic anemias, hypochromic anemias, spherocytosis, and thalassemia, may cause plasma to be trapped in the red blood cell layer even if the procedure is performed properly. The trapping of the plasma causes the microhematocrit to be 1% to 3% (0.01 to 0.03 L/L) higher than the value obtained using automated instruments that calculate or directly measure the hematocrit and are unaffected by the trapped plasma.
8. A temporarily low hematocrit reading may result immediately after a blood loss because plasma is replaced faster than are the red blood cells.
9. The fluid loss associated with dehydration causes a decrease in plasma volume and falsely increases the hematocrit reading.
10. Proper specimen collection is an important consideration. The introduction of interstitial fluid from a skin puncture or the improper flushing of an intravenous catheter causes decreased hematocrit readings.

The READACRIT centrifuge (Becton, Dickinson and Company, Franklin Lakes, NJ) uses precalibrated capillary tubes and has built-in hematocrit scales, which eliminates the need for separate reading devices (Figure 14-8). The use of SUREPREP Capillary Tubes (Becton, Dickinson) eliminates the use of sealants. They have a factory-inserted plug that seals automatically when the blood touches the plug.¹¹

FIGURE 14-8 READACRIT centrifuge with built-in capillary tube compartments and hematocrit scales. Source: (Courtesy and © Becton, Dickinson and Company, Franklin Lakes, NJ.)

Rule of three

When samples are analyzed by automated or manual methods, a quick visual check of the results of the hemoglobin and hematocrit can be done by applying the “rule of three.” This rule applies only to samples that have normocytic normochromic red blood cells. The value of the hematocrit should be three times the value of the hemoglobin plus or minus 3: HGB × 3 = HCT ± 3 (0.03 L/L). It should become habit for the analyst to multiply the hemoglobin by 3 mentally for every sample; a value discrepant with this rule may indicate abnormal red blood cells, or it may be the first indication of error.

For example, the following results are obtained from patients:

Case 1

HGB = 12 g/dL

HCT = 36% (0.36 L/L)

According to the rule of three,

An acceptable range for the hematocrit would be 33% to 39%. These values conform to the rule of three.

Case 2

HGB = 9 g/dL

HCT = 32%

According to the rule of three,

An acceptable range for hematocrit would be 24% to 30%, so these values do not conform to the rule of three.

Case 3

HGB = 15 g/dL

HCT = 36%

According to the rule of three,

An acceptable range for hematocrit would be 42% to 48%, so these values do not conform to the rule of three.

If values do not agree, the blood film should be examined for abnormal red blood cells; causes of false increases and decreases in the hemoglobin and/or hematocrit values should also be investigated. In the second example, the blood film reveals red blood cells that are low in hemoglobin concentration (hypochromic) and are smaller in volume (microcytic), so the rule of three cannot be applied. If red blood cells do appear normal, possible causes of a falsely low hemoglobin concentration or a falsely elevated hematocrit should be investigated. In the third example, the specimen is determined to have lipemic plasma causing a falsely elevated hemoglobin concentration, and a correction must be made to obtain an accurate hemoglobin value. (See Hemoglobin Determination in this chapter.)

When an unexplained discrepancy is found, the sample processed before and after the sample in question should be checked to determine whether they conform to the rule. If they do not conform, further investigation should be done to find the problem. A control sample should be run when such a discrepancy is found. If the instrument produces appropriate results for the control, random error may have occurred (Chapter 5).

Red blood cell indices

The mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) are the RBC indices. These are calculated to determine the average volume and hemoglobin content and concentration of the red blood cells in the sample. In addition to serving as a quality control check, the indices may be used for initial classification of anemias. Table 14-2 provides a summary of the RBC indices, morphology, and correlation with various anemias. The morphologic classification of anemia on the basis of MCV is discussed in detail in Chapter 19.

TABLE 14-2

Red Blood Cell Indices, Red Blood Cell Morphology, and Disease States

Hb, Hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume.

Mean cell volume

The MCV is the average volume of the red blood cell, expressed in femtoliters (fL), or 10⁻¹⁵ L:

The reference interval for MCV is 80 to 100 fL. RBCs with an MCV of less than 80 fL are microcytic; those with an MCV of more than 100 fL are macrocytic.

Mean cell hemoglobin

The MCH is the average weight of hemoglobin in a red blood cell, expressed in picograms (pg), or 10⁻¹² g:

For example, if the hemoglobin = 16 g/dL and the RBC count = 5 × 10¹²/L, the MCH = 32 pg.

The reference interval for adults is 26 to 32 pg. The MCH generally is not considered in the classification of anemias.

Mean cell hemoglobin concentration

The MCHC is the average concentration of hemoglobin in each individual red blood cell. The units used are grams per deciliter (formerly given as a percentage):

For example, if the HGB = 16 g/dL and the HCT = 48%, the MCHC = 33.3 g/dL.

Values of normochromic red blood cells range from 32 to 36 g/dL; values of hypochromic cells are less than 32 g/dL, and values of “hyperchromic” cells are greater than 36 g/dL. Hypochromic red blood cells occur in thalassemias, iron deficiency, and other conditions listed in Table 14-2. The term hyperchromic is a misnomer: a cell does not really contain more than 36 g/dL of hemoglobin, but its shape may have become spherocytic, which makes the cell appear full. An MCHC between 36 and 38 g/dL should be checked for spherocytes. An MCHCgreater than 38 g/dL should be investigated for an error in hemoglobin value (see Sources of Error and Comments in the section on hemoglobin determination). Another cause for a markedly increased MCHC could be the presence of a cold agglutinin. Incubating the specimen at 37° C for 15 minutes before analysis usually produces accurate results. Cold agglutinin disease is discussed in more detail in Chapter 26.

Reticulocyte count

The reticulocyte is the last immature red blood cell stage. Normally, a reticulocyte spends 2 days in the bone marrow and 1 day in the peripheral blood before developing into a mature red blood cell. The reticulocyte contains remnant cytoplasmic ribonucleic acid (RNA) and organelles such as the mitochondria and ribosomes (Chapter 8). The reticulocyte count is used to assess the erythropoietic activity of the bone marrow.

Principle

Whole blood, anticoagulated with EDTA, is stained with a supravital stain, such as new methylene blue. Any nonnucleated red blood cell that contains two or more particles of blue-stained granulofilamentous material after new methylene blue staining is defined as a reticulocyte (Figure 14-9).

FIGURE 14-9 Reticulocytes with new methylene blue vital stain (peripheral blood ×1000). Reticulocytes are nonnucleated red blood cells with two or more blue-stained filaments or particles.

Procedure

1. Mix equal amounts of blood and new methylene blue stain (2 to 3 drops, or approximately 50 μL each), and allow to incubate at room temperature for 3 to 10 minutes.¹²
2. Remix the preparation.
3. Prepare two wedge films (Chapter 16).
4. In an area in which cells are close together but not touching, count 1000 RBCs under the oil immersion objective lens (1000× total magnification). Reticulocytes are included in the total RBC count (i.e., a reticulocyte counts as both an RBC and a reticulocyte).
5. To improve accuracy, have another laboratorian count the other film; counts should agree within 20%.
6. Calculate the % reticulocyte count:

For example, if 15 reticulocytes are counted,

Or the number of reticulocytes counted can be multiplied by 0.1 (100/1000) to obtain the result.

Miller disc

Because large numbers of red blood cells should be counted to obtain a more precise reticulocyte count, the Miller disc was designed to reduce this labor-intensive process. The disc is composed of two squares, with the area of the smaller square measuring ¹/₉ the area of the larger square. The disc is inserted into the eyepiece of the microscope and the grid in Figure 14-10 is seen. RBCs are counted in the smaller square, and reticulocytes are counted in the larger square. Selection of the counting area is the same as described earlier. A minimum of 112 cells should be counted in the small square, because this is equivalent to 1008 red cells in the large square and satisfies the College of American Pathologists (CAP) hematology standard for a manual reticulocyte count based on at least 1000 red cells.¹³ The calculation formula for percent reticulocytes is

FIGURE 14-10 Miller ocular disc counting grid as viewed through a microscope. The area of square B is 1/9 the area of square A. Alternatively, square B may be in the center of square A.

For example, if 15 reticulocytes are counted in the large square and 112 red blood cells are counted in the small square,

Equation reference interval

General reference intervals can be found on the inside front cover of this text.

Sources of error and comments

1. If a patient is very anemic or polycythemic, the proportion of dye to blood should be adjusted accordingly.
2. An error may occur if the blood and stain are not mixed before the films are made. The specific gravity of the reticulocytes is lower than that of mature red blood cells, and reticulocytes settle at the top of the mixture during incubation.
3. Moisture in the air, poor drying of the slide, or both may cause areas of the slide to appear refractile, and these areas could be confused with reticulocytes. The RNA remnants in a reticulocyte are not refractile.
4. Other red blood cell inclusions that stain supravitally include Heinz, Howell-Jolly, and Pappenheimer bodies (Table 19-3). Heinz bodies are precipitated hemoglobin, usually appear round or oval, and tend to adhere to the cell membrane (Figure 14-11). Howell-Jolly bodies are round nuclear fragments and are usually singular. Pappenheimer bodies are iron in the mitochondria whose presence can be confirmed with an iron stain, such as Prussian blue. This stain is discussed in Chapter 17.
5. If a Miller disc is used, it is important to heed the “edge rule” as described in the WBC count procedure and illustrated in Figure 14-2. A significant bias is observed if the rule is ignored.¹²

FIGURE 14-11 Reticulocytes (A) and Heinz bodies (B) stained with supravital stain (peripheral blood ×1000).

Absolute reticulocyte count

Principle

The absolute reticulocyte count (ARC) is the actual number of reticulocytes in 1 liter (L) or 1 microliter (μL) of blood.

Calculations

For example, if a patient’s reticulocyte count is 2% and the RBC count is 2.20 × 10¹²/L, the ARC is calculated as follows (note that the calculated result has to be converted from 10¹²/L to 10⁹/L):

The absolute reticulocyte count can also be reported as the number of cells per μL. Using the example above, the RBC count in μL (2.20 × 10⁶/μL) is used in the formula, and the ARC result is 44 × 10³/μL.

Reference interval

Values between 20 × 10⁹/L and 115 × 10⁹/L are within the reference interval for most populations.¹⁴

Corrected reticulocyte count

Principle

In specimens with a low hematocrit, the percentage of reticulocytes may be falsely elevated because the whole blood contains fewer red blood cells. A correction factor is used, with the average normal hematocrit considered to be 45%.

Calculation

Reference interval

Patients with a hematocrit of 35% should have an elevated corrected reticulocyte count of 2% to 3% to compensate for the mild anemia. In patients with a hematocrit of less than 25%, the count should increase to 3% to 5% to compensate for the moderate anemia. The corrected reticulocyte count depends on the degree of anemia.

Reticulocyte production index

Principle

Reticulocytes that are released from the marrow prematurely are called shift reticulocytes. These reticulocytes are “shifted” from the bone marrow to the peripheral blood earlier than usual to compensate for anemia. Instead of losing their reticulum in 1 day, as do most normal circulating reticulocytes, these cells take 2 to 3 days to lose their reticula. When erythropoiesis is evaluated, a correction should be made for the presence of shift reticulocytes if polychromasia is reported in the red blood cell morphology. Most normal (nonshift) reticulocytes become mature red blood cells within 1 day after entering the bloodstream and thus represent 1 day’s production of red blood cells in the bone marrow. Cells shifted to the peripheral blood prematurely stay longer as reticulocytes and contribute to the reticulocyte count for more than 1 day. For this reason, the reticulocyte count is falsely increased when polychromasia is present, because the count no longer represents the cells maturing in just 1 day. On many automated instruments, this mathematical adjustment of the reticulocyte count has been replaced by the measurement of immature reticulocyte fraction (Chapter 15).¹²

The patient’s hematocrit is used to determine the appropriate correction factor (reticulocyte maturation time in days):

Patient’s Hematocrit Value (%)	Correction Factor (Maturation Time, Days)
40–45	1
35–39	1.5
25–34	2
15–24	2.5
< 15	3

Calculation

The reticulocyte production index (RPI) is calculated as follows:

Or

For example, for a patient with a reticulocyte count of 7.8% and a HCT of 30%, and with polychromasia noted, the previous table indicates a maturation time of 2 days. Thus

Reference interval

An adequate bone marrow response usually is indicated by an RPI that is greater than 3. An inadequate erythropoietic response is seen when the RPI is less than 2.¹⁴

Reticulocyte control

Several commercial controls are now available for monitoring manual and automated reticulocyte counts [e.g., Retic-Chex II, Streck Laboratories, Omaha, NE; Liquichek Reticulocyte Control (A), Bio-Rad Laboratories, Hercules, CA]. Most of the controls are available at three levels. The control samples are treated in the same manner as the patient samples. The control can be used to verify the laboratorian’s accuracy and precision when manual counts are performed.

Automated reticulocyte counts

The major instrument manufacturers offer are analyzers that perform automated reticulocyte counts. All of the analyzers evaluate reticulocytes using optical scatter or fluorescence after the red blood cells are treated with fluorescent dyes or nucleic acid stains to stain residual RNA in the reticulocytes. The percentage and the absolute count are provided. These results are statistically more valid because of the large number of cells counted. Other reticulocyte parameters that are offered on some automated instruments include a maturation index/immature reticulocyte fraction or IRF (reflecting the proportion of the more immature reticulocytes in the sample), the reticulocyte hemoglobin concentration, and reticulocyte indices (such as the mean reticulocyte volume and distribution width). The IRF may be especially useful in detecting early erythropoietic activity after chemotherapy or hematopoietic stem cell transplantation. The reticulocyte hemoglobin is useful to detect early iron deficiency (Chapter 20). Automated reticulocyte counting is discussed in Chapter 15.

Erythrocyte sedimentation rate

The erythrocyte sedimentation rate (ESR) is ordered with other tests to detect and monitor the course of inflammatory conditions such as, rheumatoid arthritis, infections, or certain malignancies. It is also useful in the diagnosis of temporal arteritis and polymyalgia rheumatica.¹⁵ The ESR, however, is not a specific test for inflammatory diseases and is elevated in many other conditions such as plasma cell myeloma, pregnancy, anemia, and older age. It is also prone to technical errors that can falsely elevate or decrease the sedimentation rate. Because of its low specificity and sensitivity, the ESR is not recommended as a screening test to detect inflammatory conditions in asymptomatic individuals.¹⁵ Other tests for inflammation, such as the C-reactive protein level, may be a more predictable and reliable alternative to monitor inflammation.¹⁶

Principle

When anticoagulated blood is allowed to stand at room temperature undisturbed for a period of time, the red blood cells settle toward the bottom of the tube. The ESR is the distance in millimeters that the red blood cells fall in 1 hour. The ESR is affected by red blood cell, plasma, and mechanical and technical factors. Red blood cells have a net negative surface charge and tend to repel one another. The repulsive forces are partially or totally counteracted if there are increased quantities of positively charged plasma proteins. Under these conditions the red blood cells settle more rapidly as a result of the formation of rouleaux (stacking of red blood cells). Examples of macromolecules that can produce this reaction are fibrinogen, β-globulins, and pathologic immunoglobulins.^{17, 18}

Normal red blood cells have a relatively small mass and settle slowly. Certain diseases can cause rouleaux formation, in which the plasma fibrinogen and globulins are altered. This alteration changes the red blood cell surface, which leads to stacking of the red blood cells, increased red blood cell mass, and a more rapid ESR. The ESR is directly proportional to the red blood cell mass and inversely proportional to plasma viscosity. Several methods, both manual and automated, are available for measuring the ESR. Only the most commonly used methods are discussed here.

Modified westergren erythrocyte sedimentation rate

The most commonly used method today is the modified Westergren method. One advantage of this method is that the taller column height allows the detection of highly elevated ESRs. It is the method recommended by the International Council for Standardization in Hematology and the Clinical and Laboratory Standards Institute.^{15, 19}

Procedure

1. Use well-mixed blood collected in EDTA and dilute at four parts blood to one part 3.8% sodium citrate or 0.85% sodium chloride (e.g., 2 mL blood and 0.5 mL diluent). Alternatively, blood can be collected directly into special sedimentation test tubes containing sodium citrate. Standard coagulation test tubes are not acceptable, because the dilution is nine parts blood to one part sodium citrate.¹⁵
2. Place the diluted sample in a 200-mm column with an internal diameter of 2.55 mm or more.
3. Place the column into the rack and allow to stand undisturbed for 60 minutes at room temperature (18 to 25° C). Ensure that the rack is level.
4. Record the number of millimeters the red blood cells have fallen in 1 hour. The buffy coat should not be included in the reading. Read the tube from the bottom of the plasma layer to the top of the sedimented red blood cells (Figure 14-12). Report the result as the ESR, 1 hour = x mm.¹⁵

FIGURE 14-12 Erythrocyte sedimentation rate (ESR), 1 hour = 93 mm, which is elevated above the reference intervals.

Wintrobe erythrocyte sedimentation rate

When the Wintrobe method was first introduced, the specimen used was oxalate-anticoagulated whole blood. This was placed in a 100-mm column. Today, EDTA-treated or citrated whole blood is used with the shorter column. The shorter column height allows a somewhat increased sensitivity in detecting mildly elevated ESRs.

Procedure

1. Use fresh blood collected in EDTA anticoagulant. A minimum of 2 mL of whole blood is needed.
2. After mixing the blood thoroughly, fill a Pasteur pipette using a rubber pipette bulb.
3. Place the filled pipette into the Wintrobe tube until the tip reaches the bottom of the tube.
4. Carefully squeeze the bulb and expel the blood into the Wintrobe tube while pulling the Pasteur pipette up from the bottom of the tube. There must be steady, even pressure on the bulb to expel blood into the tube as well as continuous movement of the pipette up the tube to prevent the introduction of air bubbles into the column of blood.
5. Fill the Wintrobe tube to the 0 mark.
6. Place the tube into a Wintrobe rack (tube holder) and allow to stand undisturbed for 1 hour at room temperature. The rack must be perfectly level and placed in a draft-free room.
7. Record the number of millimeters the red blood cells have fallen. Read the tube from the bottom of the plasma meniscus to the top of the sedimented red cells. The result is reported in millimeters per hour.

Reference interval

Reference intervals according to sex and age can be found on the inside front cover of this text. Table 14-3 lists some of the factors that influence the ESR.

TABLE 14-3

Factors Affecting the Erythrocyte Sedimentation Rate (ESR)

Category	Increased ESR	Decreased ESR
Blood proteins and lipids	Hypercholesterolemia Hyperfibrinogenemia Hypergammaglobulinemia Hypoalbuminemia	Hyperalbuminemia Hyperglycemia Hypofibrinogenemia Hypogammaglobulinemia Increased bile salts Increased phospholipids
Red blood cells	Anemia Macrocytosis	Acanthocytosis Anisocytosis (marked) Hemoglobin C Microcytosis Polycythemia Sickle cells Spherocytosis Thalassemia
White blood cells	Leukemia	Leukocytosis (marked)
Drugs	Dextran Heparin Penicillamine Procainamide Theophylline Vitamin A	Adrenocorticotropic hormone (corticotropin) Cortisone Ethambutol Quinine Salicylates
Clinical conditions	Acute heavy metal poisoning Acute bacterial infections Collagen vascular diseases Diabetes mellitus End-stage renal failure Gout Malignancy Menstruation Multiple myeloma Myocardial infarction Pregnancy Rheumatic fever Rheumatoid arthritis Syphilis Temporal arteritis	Cachexia Congestive heart failure Newborn status
Specimen handling	Refrigerated sample not returned to room temperature	Clotted blood sample Delay in testing
Technique	High room temperature Tilted ESR tube Vibration	Bubbles in ESR column Low room temperature Narrow ESR column diameter

From American Society for Clinical Pathology/American Proficiency Institute: 2006 2nd Test Event—Educational Commentary—The Erythrocyte Sedimentation Rate and Its Clinical Utility. API is the proficiency testing group that provides testing materials to the American Society for Clinical Pathology. The educational commentary itself is written by ASCP. This reference can also be accessed at: http://www.api-pt.com/Reference/Commentary/2006Bcoag.pdf. Accessed November 5, 2014.

Sources of error and comments

1. If the concentration of anticoagulant is increased, the ESR will be falsely low as a result of sphering of the RBCs, which inhibits rouleaux formation.
2. The anticoagulants sodium or potassium oxalate and heparin cause the red blood cells to shrink and falsely elevate the ESR.
3. A significant change in the temperature of the room alters the ESR.
4. Even a slight tilt of the pipette causes the ESR to increase.
5. Blood specimens must be analyzed within 4 hours of collection if kept at room temperature (18 to 25° C).¹⁵If the specimen is allowed to sit at room temperature for more than 4 hours, the red blood cells start to become spherical, which may inhibit the formation of rouleaux. Blood specimens may be stored at 4° C up to 24 hours prior to testing, but must be rewarmed by holding the specimen at ambient room temperature for at least 15 minutes prior to testing.¹⁵
6. Bubbles in the column of blood invalidate the test results.
7. The blood must be filled properly to the zero mark at the beginning of the test.
8. A clotted specimen cannot be used.
9. The tubes must not be subjected to vibrations on the lab bench which can falsely increase the ESR.
10. Hematologic disorders that prevent the formation of rouleaux (e.g., the presence of sickle cells and spherocytes) decrease the ESR.
11. The ESR of patients with severe anemia is of little diagnostic value, because it will be falsely elevated.

Disposable kits

Disposable commercial kits are available for ESR testing (Figure 14-13). Several kits include safety caps for the columns that allow the blood to fill precisely to the zero mark. This safety cap makes the column a closed system and eliminates the error involved in manually setting the blood to the zero mark.

FIGURE 14-13 Sediplast (Polymedco) disposable sedimentation rate system. Source: (Courtesy Polymedco, Cortlandt Manor, NY.)

Automated erythrocyte sedimentation rate

There are several automated ESR systems available using the traditional Westergren and Wintrobe methods, as well as alternate methods such as centrifugation. The Ves-Matic system (Diesse, Inc., Hialeah, FL) is a bench-top analyzer designed to determine ESR by use of an optoelectronic sensor, which measures the change in opacity of a column of blood as sedimentation of blood progresses. Blood is collected in special Ves-Tec or Vacu-Tec tubes, which contain sodium citrate and are compatible with the Vacutainer system. These tubes are used directly in the instrument (Figure 14-14). Acceleration of sedimentation is achieved by positioning the tubes at an 18-degree angle in relation to the vertical axis. Results comparable with Westergren 1-hour values are obtained in 20 minutes.²⁰

FIGURE 14-14 Two models of the Ves-Matic instruments for sedimentation rates: The Ves-Matic Easy (A) for up to 10 specimens (requiring special tubes) and the Ves-Matic Cube 30 (B) for up to 30 specimens, determining the ESR directly from EDTA tubes. Products with up to 190-specimen capacity are also available. Source: (Courtesy Diesse Inc., Hialeah, FL.)

Another automated ESR analyzer is the Sedimat 15 (Polymedco, Cortlandt Manor, NY), which uses the principle of infrared measurement. It is capable of testing one to eight samples randomly or simultaneously and provides results in 15 minutes (Figure 14-15).

FIGURE 14-15 Sedimat 15 (Polymedco) automated sedimentation rate system. Source: (Courtesy Polymedco, Cortlandt Manor, NY.)

The ESR STAT PLUS system (HemaTechnologies, Lebanon, NJ) is based on centrifugation. The advantages of this method are a smaller required sample volume and shorter testing time, which makes it more suitable for a pediatric patient population. The disadvantage of this method is the number of exacting preanalytical steps that must be strictly followed to prevent erroneous results. Compliance with these steps may be difficult to achieve consistently in a busy hematology laboratory.²¹

Additional methods

Additional manual and semi-automated methods are included in other chapters that are relevant to their clinical application. Examples include: Chapter 24 for the osmotic fragility test and qualitative and quantitative assays for glucose-6-phosphate dehydrogenase and pyruvate kinase activity; Chapter 27 for the solubility test for Hb S, hemoglobin electrophoresis (alkaline and acid pH), and unstable hemoglobin test; and Chapter 28 for the vital stain for hemoglobin H and the Kleihauer-Betke acid elution test for Hb F distribution in the RBCs.

Point-of-care testing

Point-of-care testing offers the ability to produce rapid and accurate results that help facilitate faster treatment, which can decrease patient length of stay. This testing is rarely performed by trained laboratory personnel; most often, it is carried out by nurses. Manufacturers have created analyzers with nonlaboratory operators in mind, but results obtained using these systems are still affected by preanalytic and analytical variables. The laboratory’s partnership with nursing is the key to success in any hospital’s point-of-care program.

Point-of-care testing is defined as diagnostic testing at or near the site of patient care. The Clinical Laboratory Improvement Amendments of 1988 (CLIA) introduced the concept of “testing site neutrality,” which means that regardless of where the diagnostic testing is performed or who performs the test, all testing sites must follow the same regulatory requirements based on the “complexity” of the test. Under CLIA, point-of-care testing (including physician-performed microscopy) is classified as “waived” or “moderately complex.” Tests are classified as waived if they are determined to be “simple tests with an insignificant risk of an erroneous result.” Point-of-care testing is commonly performed in hospital inpatient units, outpatient clinics, surgery centers, emergency departments, long-term care facilities, and dialysis units. For waived point-of-care testing, facilities are required to obtain a certificate of waiver, pay the appropriate fees, and follow the manufacturers’ testing instructions.²² For any point-of-care program to be successful, certain key elements must be present. Clear administrative responsibility, well-written procedures, a training program, quality control, proficiency testing, and equipment maintenance are essential for success. The first step is appointing a laboratory point-of-care testing coordinator. This person not only is the “go-to” person but is also an important liaison between the laboratory and nursing staff. The second step to ensuring a successful program is to create a multidisciplinary team with authority to impact all aspects of the POC program. This committee would have the authority to oversee the integrity and quality of the existing POC program and institute changes or new testing as needed. It is also important to have administrative support to help remove barriers.

A point-of-care testing program must incorporate all of the following. A written policy should be developed that defines the program. This policy should outline who is responsible for each part of the program. The policy should also indicate where the testing is to be performed and who is going to perform the testing. Testing procedures should be written that clearly state how to perform the tests and that address how to handle critical values and/or any discrepant results. The program must be monitored. An ongoing evaluation of the point-of-care testing is vital for success.

When the instrument to be used in the point-of-care testing program is being selected, it is helpful to invite the vendors to demonstrate their equipment. An equipment display that is available for hands-on use by the operators can be very helpful in selection of the appropriate instrumentation. Patient correlation studies are very useful in choosing equipment that best covers the patient population for that particular institution. Point-of-care operators need handheld analyzers that are lightweight, accurate, fast, and that require little specimen material. The point-of-care testing system should also address the following laboratory concerns:

• What is the range of measurement?
• How well does the test system correlate with laboratory instrumentation?
• Can it be interfaced to the laboratory information system?
• Does it give reliable results?
• Does the company supply excellent technical support?
• Is it affordable?

Paramount to point-of-care testing is patient safety. It is important to maintain good practices, and with waived testing, this often comes down to the basics. Such basics include proper and appropriate specimen collection, proper identification of the patient and specimen, proper storage of reagents, and good documentation of patient test results (use of point-of-care interfaces is beneficial), as well as proper performance of any necessary instrument maintenance. Laboratory oversight is sometimes absent, and basic safety precautions necessary for waived tests can be easily overlooked, often due to a lack of understanding, lack of training, and high personnel turnover rates.²³ Patient safety, risk management, and error reduction are primary goals of all health care facilities. All testing personnel should be properly trained in best practices to avoid exposure. The individual responsible for oversight—whether laboratory or nonlaboratory—must avoid taking safety for granted. All applicable standards (including those of the Occupation Safety and Health Administration, Centers for Disease Control and Prevention, The Joint Commission, CAP, CLIA, and so forth) should be implemented and easily accessible. Because the number of waived tests has grown significantly since waived tests were first defined by CLIA, it is paramount that standard safety precautions and the basic steps outlined earlier be implemented to ensure that patient safety is not sacrificed in the unique situation of CLIA-waived testing.

Point-of-care tests

Various point-of-care instruments are available to measure parameters such as hemoglobin level and hematocrit, and some perform a complete blood count.

Hematocrit

The most common methods for determining the hematocrit include the microhematocrit centrifuge, conductometric methods, and calculation by automated cell counters (Chapter 15).

Centrifuge-based microhematocrit systems have been available for years, and the results obtained correlate well with the results produced by standard cell counters. Nonlaboratorians and inexperienced operators, however, may be unaware of the error that can be introduced by insufficient centrifugation time and inaccurate reading of the microhematocrit tube (see comments in the Microhematocrit section). Examples of centrifuge-based devices are the Hematastat II (Separation Technology, Inc., Altamonte Springs, FL) and STAT Crit (Wampole Laboratories, Cranbury, NJ).

The i-STAT 1 (Abbott Laboratories, Abbott Park, IL)²⁴ (Figure 14-16) and the Epoc (Epocal, Inc., Ottawa, ON) (Figure 14-17)²⁵ use the conductivity method to determine the hematocrit. Plasma conducts electrical current, whereas WBCs act as insulators. In the i-STAT system, before the measured sample conductance is converted into the hematocrit value, corrections are applied for the temperature of the sample, the size of the fluid segment being measured, and the relative conductivity of the plasma component. The first two corrections are determined from the measured value of the calibrant conductance and the last correction from the measured concentrations of sodium and potassium in the sample.²⁴

FIGURE 14-16 i-STAT instrument for measuring hematocrit. Source: (Courtesy Abbott Laboratories, Abbott Park, IL.)

FIGURE 14-17 Epoc device for measuring hematocrit. Source: (Courtesy Epocal, Inc., Ottawa, Ontario, Canada.)

Sources of error and comments.

Conductivity of a whole blood sample is dependent on the amount of electrolytes in the plasma portion. Conductivity does not distinguish red blood cells from other nonconductive elements such as proteins, lipids, and WBCs that may be present in the sample.

A low total protein level will falsely decrease the hematocrit. The presence of lipids can interfere with the hematocrit measurement. An increased WBC count will falsely increase the hematocrit. The presence of cold agglutinins can falsely decrease the hematocrit.²⁴

Other instruments.

Other instruments that measure the hematocrit include the following:

• ABL 77 (Radiometer, Westlake, OH)
• IRMA (ITC, a subsidiary of Thoratec Corporation, Edison, NJ)
• Gem Premier (Instrumentation Laboratory Company, Lexington, MA) (Figure 14-18)

FIGURE 14-18 Gem Premier instrument for measuring hematocrit. Source: (Courtesy Instrumentation Laboratory Company, Lexington, MA.)

Hemoglobin concentration

In point-of-care testing, hemoglobin concentration is measured by modified hemoglobinometers or by oximeters integrated with a blood gas analyzer. The HemoCue hemoglobinometer (HemoCue, Inc., Brea, CA) uses a small cuvette that contains a lysing agent and reagents to form a hemoglobin azide, which is measured by a photometer at two wavelengths (570 nm and 880 nm) (Figure 14-19).⁶ This eliminates interference from turbidity in the sample. Results obtained with the instrument compare well with those produced by reference methods, but a major source of error is mixture of blood with tissue fluid during skin puncture collection. The AVOX 1000E (ITC) measures total hemoglobin by a spectrophotometric method. The STAT-Site MHbg Meter (Stanbio Laboratory, Boerne, TX) uses the azidemethemoglobin principle and reflectance photometry to measure reflected light in the test area. The test card is composed of molded plastic with a fluid well that contains numerous pads impregnated with specific chemical reagents. A drop of whole blood is applied to the center of the well and reacts with the chemicals in the pad to produce a specific color that is measured from the bottom of the card.²⁶

Cell and platelet counts

Traditional cell-counting methods can be employed at the point of care for the analysis of WBCs, RBCs, and platelets. The Ichor Hematology Analyzer (Helena Laboratories, Beaumont, TX) performs a complete blood count along with platelet aggregation. Another option for cell quantitation and differentiation employs a buffy coat analysis method. Quantitative buffy coat analysis (QBC STAR, manufactured by QBC Diagnostics, Inc., Philipsburg, PA) involves centrifugation in specialized capillary tubes designed to expand the buffy coat layer. The components (platelets, mononuclear cells, and granulocytes) can be measured with the assistance of fluorescent dyes and a measuring device.²⁷

Summary

• Although most laboratories are highly automated, the manual tests discussed in this chapter, such as the cyanmethemoglobin method of hemoglobin determination and centrifuge-based measurement of the microhematocrit, are used as a part of many laboratories’ quality control and backup methods of analysis.
• The hemacytometer allows counts of any type of cell or particle (e.g., WBCs or platelets) to be performed.
• The reference method for hemoglobin determination is based on the absorbance of cyanmethemoglobin at 540 nm. When a spectrophotometer is used, a standard curve is employed to obtain the results.
• The microhematocrit is a measure of packed red blood cell volume.
• The rule of three specifies that the value of the hematocrit should be three times the value of the hemoglobin plus or minus 3 (%) or 0.03 (L/L). A value discrepant with this rule may indicate abnormal red blood cells or it may be the first indication of error.
• RBC indices—the mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC)—are calculated to determine the average volume, hemoglobin content, and hemoglobin concentration of red blood cells. The indices give an indication of possible causes of an anemia.
• The reticulocyte count, which is used to assess the erythropoietic activity of the bone marrow, is accomplished through the use of supravital stains (e.g., new methylene blue) or by flow cytometric methods.
• The erythrocyte sedimentation rate (ESR), a measure of the settling of red blood cells in a 1-hour period, depends on the red blood cells’ ability to form rouleaux. It is used to detect and monitor conditions with inflammation such as rheumatoid arthritis, infections, and some malignancies. It is subject to many physiologic and technical errors.
• Point-of-care testing is often performed by nonlaboratory personnel. It is defined as diagnostic laboratory testing at or near the site of patient care.
• CLIA introduced the concept of “testing site neutrality,” which means that it does not matter where diagnostic testing is performed or who performs the test; all testing sites must follow the same regulatory requirements based on the “complexity” of the test.
• Tests are classified as waived if they are determined to be “simple tests with an insignificant risk of an erroneous result.” Most, but not all, point-of-care testing is waived.
• For a point-of-care testing program to be successful, key elements such as clear administrative responsibility, well-written procedures, quality control, proficiency testing, and equipment maintenance must be present.
• Paramount to point-of-care testing is patient safety.

Now that you have completed this chapter, read again the case studies at the beginning and respond to the questions presented.

Review questions

1. A 1:20 dilution of blood is made with 3% glacial acetic acid as the diluent. The four large corner squares on both sides of the hemacytometer are counted, for a total of 100 cells. What is the total WBC count (×10⁹/L)?
2. 0.25
3. 2.5
4. 5
5. 10
6. The total WBC count is 20 × 10⁹/L. Twenty-five NRBCs per 100 WBCs are observed on the peripheral blood film. What is the corrected WBC count (×10⁹/L)?
7. 0.8
8. 8
9. 16
10. 19
11. If potassium cyanide and potassium ferricyanide are used in the manual method for hemoglobin determination, the final product is:
12. Methemoglobin
13. Azide methemoglobin
14. Cyanmethemoglobin
15. Myoglobin
16. Which of the following would notinterfere with the result when hemoglobin determination is performed by the cyanmethemoglobin method?
17. Increased lipids
18. Elevated WBC count
19. Lyse-resistant RBCs
20. Fetal hemoglobin
21. A patient has a hemoglobin level of 8.0 g/dL. According to the rule of three, what is the expected range for the hematocrit?
22. 21% to 24%
23. 23.7% to 24.3%
24. 24% to 27%
25. 21% to 27%
26. Calculate the MCV and MCHC for the following values:

RBCs = 5.00 × 10¹²/L

HGB = 9 g/dL

HCT = 30%

MCV (fL)	MCHC (g/dL)
a. 30	18
b. 60	30
c. 65	33
d. 85	35

7. What does the reticulocyte count assess?
8. Inflammation
9. Response to infection
10. Erythropoietic activity of the bone marrow
11. Ability of red blood cells to form rouleaux
12. For a patient with the following test results, which measure of bone marrow red blood cell production provides the most accurate information?

Observed reticulocyte count = 5.3%

HCT = 35%

Morphology—moderate polychromasia

1. Observed reticulocyte count
2. Corrected reticulocyte count
3. RPI
4. ARC
5. Given the following values, calculate the RPI:

Observed reticulocyte count = 6%

HCT = 30%

1. 2
2. 3
3. 4
4. 5
5. Which of the following would be associated with an elevated ESR value?
6. Microcytosis
7. Polycythemia
8. Decreased globulins
9. Inflammation

References

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Additional resources

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31. The Joint Commission. Available at: http://www.jcaho.org
32. Point of Care.net. Available at: http://www.pointofcare.net
33. Available at: http://us.instrumentationlaboratory.com
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35. Available at: www.hemocue.com
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38. Available at: www.stanbio.com
39. Available at: www.Helena.com