Procedures

PART III Laboratory Evaluation of Blood Cells
Manual, Semiautomated, and 14

Point-of-Care Testing in Hematology
Karen S. Clark and Teresa G. Hippel
OUTLINE OBJECTIVES
Manual Cell Counts After completion of this chapter, the reader will be able to:
Equipment
Calculations 1. State the dimensions of the counting area of a 11. Calculate red blood cell indices (mean cell volume,
White Blood Cell Count Neubauer ruled hemacytometer. mean cell hemoglobin, and mean cell hemoglobin
Platelet Count 2. Describe the performance of manual cell counts concentration) when given appropriate data, and
Red Blood Cell Count for white blood cells, red blood cells, and platelets, interpret the results relative to the volume and
Disposable Blood Cell Count including types of diluting fluids, typical dilutions, and hemoglobin content and concentration in the red
Dilution Systems typical areas counted in the hemacytometer. blood cells.
Body Fluid Cell Counts 3. Calculate dilutions for cell counts when given 12. Describe the principle and procedure for performing
Hemoglobin Determination appropriate data. a manual reticulocyte count and the clinical value of
Principle 4. Calculate hemacytometer cell counts when given the test.
Microhematocrit
numbers of cells, area counted, and dilution. 13. Given the appropriate data, calculate the relative,
Rule of Three
5. Correct white blood cell counts for the presence of absolute, and corrected reticulocyte counts and the
Red Blood Cell Indices
Mean Cell Volume nucleated red blood cells. reticulocyte production index; interpret results to
Mean Cell Hemoglobin 6. Describe the principle of the cyanmethemoglobin determine the adequacy of the bone marrow eryth-
Mean Cell Hemoglobin Con- assay for determination of hemoglobin. ropoietic response in an anemia.
centration 7. Calculate the values for a standard curve for cyan- 14. Describe the procedure for performing the Wester-
Reticulocyte Count methemoglobin determination when given the ap- gren erythrocyte sedimentation rate and state its
Principle propriate data, describe how the standard curve is clinical utility.
Absolute Reticulocyte Count constructed, and use the standard curve to deter- 15. Describe the aspects of establishing a point-of-care
Corrected Reticulocyte mine hemoglobin values. testing program, including quality management and
Count 8. Describe the procedure for performing a microhematocrit. selection of instrumentation.
Reticulocyte Production In-
9. Identify sources of error in routine manual procedures 16. Discuss the advantages and disadvantages of point-
dex
discussed in this chapter and recognize written sce- of-care testing as they apply to hematology tests.
Reticulocyte Control
Automated Reticulocyte narios describing such errors. 17. Describe the principles of common instruments used
Count 10. Compare red blood cell count, hemoglobin, and for point-of-care testing for hemoglobin level, he-
Erythrocyte Sedimentation hematocrit values using the rule of three. matocrit, white blood cell counts, and platelet counts.
Rate
Principle
Modified Westergren Eryth-
CASE STUDIES
rocyte Sedimentation Rate After studying the material in this chapter, the reader should be able to respond to the following
Wintrobe Erythrocyte Sedi- case studies:
mentation Rate
Disposable Kits Case 1
Automated Erythrocyte Sed- The following results are obtained for a patient with normocytic, normochromic red blood cells on
imentation Rate a peripheral blood film:
Point-of-Care Testing RBC count ! 4.63 " 1012/L
Point-of-Care Tests HGB ! 15 g/dL
HCT ! 40% (0.40 L/L)
Continued
187
188 PART III Laboratory Evaluation of Blood Cells
CASE STUDIES—cont’d
After studying the material in this chapter, the reader should be able to respond to the following case studies:
1. Using the rule of three, given the hemoglobin concentra- Case 3

tion above, what is the expected value for the hematoctrit? The following results are obtained for a patient using a point-
2. What could cause the hemoglobin to be falsely elevated or of-care device that employs the conductivity method to
the hematocrit to be falsely low? measure the hematocrit:
3. What would you do to correct for the interferences you Sodium ! 160 mmol/L (Reference interval: 135 to
listed in question 2? 145 mmol/L)
Potassium ! 3.6 mmol/L (Reference interval: 3.5 to
Case 2 5.5 mmol/L)
For another patient, the following results are obtained: HCT ! 17.0% (0.17 L/L)
RBC count ! 3.20 " 1012/L HGB ! 6.0 g/dL
HGB ! 5.8 g/dL 1. Which electrolyte concentration could affect the hematocrit?
HCT ! 18.9% (0.19 L/L) 2. Would this electrolyte concentration falsely decrease or
1. Calculate the red blood cell indices. increase the hematocrit value?
2. How would you describe the red blood cell volume and 3. What other factors can decrease the hematocrit value
hemoglobin concentration based on these indices? using this point-of-care device?
3. How should you verify this?
C
linical laboratory hematology has evolved from simple Equipment
observation and description of blood and its compo- Hemacytometer
nents to a highly automated, extremely technical sci- The manual cell count uses a hemacytometer, or counting cham-
ence, including examination at the molecular level. However, ber. The most common one is the Levy chamber with improved
some of the more basic tests have not changed dramatically Neubauer ruling. It is composed of two raised surfaces, each with
over the years. This chapter provides an overview of these basic a 3 mm " 3 mm square counting area or grid (total area 9 mm2),
tests and presents the manual and semiautomated methods separated by an H-shaped moat. As shown in Figure 14-1, this grid
that can be used in lieu of automated instrumentation. In- is made up of nine 1 mm " 1 mm squares. Each of the four corner
cluded in this chapter is a discussion of point-of-care testing in (WBC) squares is subdivided further into 16 squares, and the cen-
hematology. ter square subdivided into 25 smaller squares. Each of these small-
est 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.
MANUAL CELL COUNTS
The distance between each counting surface and the coverslip is
Although most routine cell-counting procedures in the 0.1 mm; thus the total volume of one entire grid or counting area
hematology laboratory are automated, it may be necessary on one side of the hemacytometer is 0.9 mm3. Hemacytometers
to use manual methods when counts exceed the linearity of and coverslips must meet the specifications of the National Bureau
an instrument, when an instrument is nonfunctional and of Standards, as indicated by the initials “NBS” on the chamber.
there is no backup, in remote laboratories in Third World When the dimensions of the hemacytometer are thoroughly un-
countries, or in a disaster situation when testing is done in derstood, the area counted can be changed to facilitate the count-
the field. Although the discussion in this chapter concerns ing of samples with extremely low or high counts.
whole blood, body fluid cell counts are also often performed
using manual methods. Chapter 18 discusses the specific Calculations
diluents and dilutions used for body fluid cell counts. The general formula for manual cell counts is as follows and
Chapter 15 discusses automated cell-counting instrumenta- can be used to calculate any type of cell count:
tion in detail.
cells counted " dilution factor
Manual cell counts are performed using a hemacytometer, Total count !
or counting chamber, and manual dilutions made with cali- area (mm 2 ) " depth (0.1)
brated, automated pipettes and diluents (commercially avail- Or
able or laboratory prepared). The principle for the perfor-
cells counted " dilution factor " 10*
mance of cell counts is essentially the same for white blood Total count !
area (mm 2 )
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. *Reciprocal of depth.
CHAPTER 14 Manual, Semiautomated, and Point-of-Care Testing in Hematology 189
1 mm
1 mm
R R
R R
side view
coverslip 0.1 mm
moat moat
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.
The calculation yields the number of cells per mm3. One mm3 is 5. Mix again by inversion and fill a plain microhematocrit tube.
equivalent to one microliter (#L). The count per #L is converted 6. Charge both sides of the hemacytometer by holding the
to the count per liter (L) by multiplying by a factor of 106. microhematocrit tube at a 45-degree angle and touching the
tip to the coverslip edge where it meets the chamber floor.
White Blood Cell Count 7. After charging the hemacytometer, place it in a moist
The WBC or leukocyte count is the number of WBCs in 1 liter chamber (Box 14-1) for 10 minutes before counting the
(L) or 1 microliter (#L) of blood. Whole blood anticoagu- cells to give them time to settle. Care should be taken not
lated with ethylenediaminetetraacetic acid (EDTA) or blood to disturb the coverslip.
from a skin puncture is diluted with 1% buffered ammo- 8. While keeping the hemacytometer in a horizontal posi-
nium oxalate or a weak acid solution (3% acetic acid or tion, place it on the microscope stage.
1% hydrochloric acid). The diluting fluid lyses the nonnucle- 9. Lower the condenser on the microscope and focus by using
ated red blood cells in the sample to prevent their interfer- the low-power (10") objective lens (100" total magnifica-
ence in the count. The typical dilution of blood for the WBC tion). The cells should be distributed evenly in all of the
count is 1:20. A hemacytometer is charged (filled) with the squares.
well-mixed dilution and placed under a microscope and the 10. For a 1:20 dilution, count all of the cells in the four corner
number of cells in the 4 large corner squares (4 mm2) is squares, starting with the square in the upper left-hand
counted. corner (Figure 14-1). Cells that touch the top and left lines
should be counted; cells that touch the bottom and right
PR O C E DU R E lines should be ignored (Figure 14-2). See Figure 14-3 for
1. Clean the hemacytometer and coverslip with alcohol and the appearance of WBCs in the hemacytometer using the
dry thoroughly with a lint-free tissue. Place the coverslip on low-power objective lens of a microscope.
the hemacytometer.
2. Make a 1:20 dilution by placing 25 #L of well-mixed blood
into 475 #L of WBC diluting fluid in a small test tube.
3. Cover the tube and mix by inversion.
BOX 14-1 How to Make a Moist Chamber
4. Allow the dilution to sit for 10 minutes to ensure that the
A moist chamber may be made by placing a piece of damp filter
red blood cells have lysed. The solution will be clear once
paper in the bottom of a Petri dish. An applicator stick broken in half
lysis has occurred. WBC counts should be performed within
can serve as a support for the hemacytometer.
3 hours of dilution.
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:

WBC count !
area counted (mm 2 ) " depth
96 " 20
!
4 " 0. 1
! 4800/mm3 or 4800/# L or
4.8 " 103/# L or 4.8 " 109/L
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:

WBC count !
area counted (mm 2 ) " depth
Figure 14-2 One large corner square of a hemacytometer indicating
54 " 100
which cells to count. Cells touching the left and top lines (solid circles) are !
9 " 0. 1
counted. Cells touching bottom and right (open circles) are not counted.
! 6000/mm3 or 6000/# L or
6. 0 " 103/# L or 6. 0 " 109/L
WBC
General reference intervals for males and females in differ-
ent age groups can be found on the inside front cover of this
text. Reference intervals may vary slightly according to the popula-
tion tested and should be established for each laboratory.
Sources of Error and Comments

1. The hemacytometer and coverslip should be cleaned prop-
erly before they are used. Dust and fingerprints may cause
difficulty in distinguishing the cells.
2. The diluting fluid should be free of contaminants.
Figure 14-3 White blood cells as seen in the hemacytometer under low TABLE 14-1 Manual Cell Counts with Most Common
power (10" objective) 100" total magnification. Dilutions, Counting Areas
Cells
Counted Diluting Fluid Dilution Objective Area Counted
11. Repeat the count on the other side of the counting cham- White blood 1% ammonium 1:20 10" 4 mm2
ber. The difference between the total cells counted on each cells oxalate 1:100 10" 9 mm2
side should be less than 10%. A greater variation could or
indicate an uneven distribution, which requires that the 3% acetic acid
procedure be repeated. or
12. Average the number of WBCs counted on the two sides. 1% hydrochloric
Using the average, calculate the WBC count using one of acid
the equations given earlier. Red blood Isotonic saline 1:100 40" 0.2 mm2 (5 small
cells squares of
Example Using the First Equation center square)
When a 1:20 dilution is used, the four large squares on one Platelets 1% ammonium 1:100 40" 1 mm2
side of the chamber yield counts of 23, 26, 22, and 21. The oxalate phase
total count is 92. The four large squares on the other side of
3. If the count is low, a greater area may be counted (e.g., 5. Count the number of platelets in the 25 small squares in the
9 mm2) to improve accuracy. center square of the grid (Figure 14-1). The area of this cen-
4. The chamber must be charged properly to ensure an accu- ter square is 1 mm2. Platelets should be counted on each
rate count. Uneven flow of the diluted blood into the cham- side of the hemacytometer, and the difference between the
ber results in an irregular distribution of cells. If the cham- totals should be less than 10%.
ber is overfilled or underfilled, the chamber must be cleaned 6. Calculate the platelet count by using one of the equations
and recharged. given earlier. Using the first equation as an example, if 200
5. After the chamber is filled, allow the cells to settle for platelets were counted in the entire center square,
10 minutes before counting.
6. Any nucleated red blood cells (NRBCs) present in the sam- 200 ! 100
ple are not lysed by the diluting fluid. The NRBCs are 1 ! 0. 1
counted as WBCs because they are indistinguishable when " 200, 000/ mm3 or 200, 000 /# L
seen on the hemacytometer. If five or more NRBCs per 100 or 200 ! 103/# L or 200 ! 109/L
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 7. The accuracy of the manual platelet count should be veri-
formula: fied by performing a platelet estimate on a Wright-stained
peripheral blood film made from the same specimen
Uncorrected WBC count ! 100 (Chapter 16).
General reference intervals for males and females according
Number of NRBCs per 100 WBCs " 100
to age groups can be found on the inside front cover of
this text.
Report the result as the “corrected” WBC count.
7. The accuracy of the manual WBC count can be assessed Sources of Error and Comments
by performing a WBC estimate on a Wright-stained 1. Inadequate mixing and poor collection of the specimen
peripheral blood film made from the same specimen can cause the platelets to clump on the hemacytometer. If
(Chapter 16). the problem persists after redilution, a new specimen is
needed. A skin puncture specimen is less desirable because
Platelet Count of the tendency of the platelets to aggregate or form
A platelet count is the number of platelets in 1 liter (L) or 1 clumps.
microliter (#L) of whole blood. Platelets adhere to foreign 2. Dirt in the pipette, hemacytometer, or diluting fluid may
objects and to each other, which makes them difficult to count. cause the counts to be inaccurate.
They also are small and can be confused easily with dirt or 3. If fewer than 50 platelets are counted on each side, the pro-
debris. In this procedure, whole blood, with EDTA as the anti- cedure should be repeated by diluting the blood to 1:20. If
coagulant, is diluted 1:100 with 1% ammonium oxalate to lyse more than 500 platelets are counted on each side, a 1:200
the nonnucleated red blood cells. The platelets are counted in dilution should be made. The appropriate dilution factor
the 25 small squares in the large center square (1 mm2) of the should be used in calculating the results.
hemacytometer using a phase-contrast microscope in the refer- 4. If the patient has a normal platelet count, the 5 small, red
ence method described by Brecher and Cronkite.1 A light blood cell squares (Figure 14-1) may be counted. Then, the
microscope can also be used, but visualizing the platelets may area is 0.2 mm2 on each side.
be more difficult. 5. The phenomenon of “platelet satellitosis” may occur
when EDTA anticoagulant is used. This refers to the ad-
PR O C E DU R E herence of platelets around neutrophils, producing a ring
1. Make a 1:100 dilution by placing 20 #L of well-mixed blood or satellite effect (Figure 16-1). Using sodium citrate as
into 1980 #L of 1% ammonium oxalate in a small test tube. the anticoagulant should correct this problem. Because of
2. Mix the dilution thoroughly and charge the chamber. the dilution in the citrate evacuated tubes, it is necessary
(NOTE: A special thin, flat-bottomed counting chamber is to multiply the obtained platelet count by 1.1 for accuracy
used for phase-microscopy platelet counts.) (Chapter 16).
3. Place the charged hemacytometer in a moist chamber
(Box 14-1) for 15 minutes to allow the platelets to settle. Red Blood Cell Count
4. Platelets are counted using the 40" objective lens (400" Manual RBC counts are rarely performed because of the inac-
total magnification). The platelets have a diameter of 2 to curacy of the count and questionable necessity. Use of other,
4 #m and appear round or oval, displaying a light purple more accurate manual RBC procedures, such as the microhe-
sheen when phase-contrast microscopy is used. The shape matocrit and hemoglobin concentration, is desirable when
and color help distinguish the platelets from highly refrac- automation is not available.
tile dirt and debris. “Ghost” RBCs often are seen in the Table 14-1 contains information on performing manual
background. WBC, platelet, and RBC counts.
Disposable Blood Cell Count Dilution Systems Body Fluid Cell Counts
Capillary pipette and diluent reservoir systems are commer- Body fluid cell counts are discussed in detail in Chapter 18.
cially available for WBC and platelet counts. One such system
is LeukoChek™ (Biomedical Polymers, Inc., Gardner, MA). It
HEMOGLOBIN DETERMINATION
consists of a capillary pipette (calibrated to accept 20 #L of
blood) that fits into a plastic reservoir containing 1.98 mL of The primary function of hemoglobin within the red blood cell
1% buffered ammonium oxalate (Figure 14-4). Blood from a is to carry oxygen to and carbon dioxide from the tissues. The
well-mixed EDTA-anticoagulated specimen or from a skin cyanmethemoglobin (hemoglobincyanide) method for hemo-
puncture is allowed to enter the pipette by capillary action to globin determination is the reference method approved by the
the fill volume. The blood is added to the reservoir making a Clinical and Laboratory Standards Institute.2
1:100 dilution. After mixing the reservoir and allowing 10 min-
utes for lysis of the red blood cells, the reverse end of the capil- Principle
lary pipette is placed in the reservoir cap making a dropper. The In the cyanmethemoglobin method, blood is diluted in an
first 3 or 4 drops of the diluted sample is discarded, and the alkaline Drabkin solution of potassium ferricyanide, potas-
capillary pipette is used to charge the hemacytometer. sium cyanide, sodium bicarbonate, and a surfactant. The
Both WBC and platelet counts can be done from the same hemoglobin is oxidized to methemoglobin (Fe3$) by the po-
diluted sample. WBCs are counted in all 9 large squares tassium ferricyanide, K3Fe(CN)6. The potassium cyanide (KCN)
(9 mm2) using low power (100" total magnification). Platelets then converts the methemoglobin to cyanmethemoglobin:
are counted in the 25 small squares in the center square
(1 mm2) using high power (400" total magnification). The Hemoglobin (Fe2$) $ K3Fe (CN)6 n methemoglobin (Fe3$)
standard formula is used to calculate the cell counts. $ KCN n cyanmethemoglobin
The absorbance of the cyanmethemoglobin at 540 nm is

directly proportional to the hemoglobin concentration. Sulfhe-
moglobin 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
PR OC E DUR E
1. Create a standard curve, using a commercially available
cyanmethemoglobin standard.
a. When a standard containing 80 mg/dL of hemoglobin is
used, the following dilutions should be made:
Hemoglobin Blank 5 10 15 20
Concentration (g/dL)
Cyanmethemoglobin 0 1.5 3 4.5 6

standard (mL)
Cyanmethemoglobin 6 4.5 3 1.5 0
reagent (mL)
b. Transfer the dilutions to cuvettes. Set the wavelength on

the spectrophotometer to 540 nm and use the blank to
set to 100% transmittance.
c. 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).
d. A standard curve should be set up with each new lot of
Figure 14-4 LeukoChek™ blood diluting system for manual white blood cell reagents. It also should be checked when alterations are
and platelet counts. It consists of a 20 µL capillary pipette and plastic reservoir made to the spectrophotometer (e.g., bulb change).
containing 1.98 mL of 1% buffered ammonium oxalate that makes a 1:100 2. Controls should be run with each batch of samples. Com-
dilution of whole blood. (Courtesy Biomedical Polymers, Inc., Gardner, MA.) mercial controls are available.
20
25
30
35
Percent transmittance
40
45
50
55
60
65
70
75
80
85
90
95
100
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Hemoglobin concentration (g/dL)
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).
3. Using the patient’s whole blood anticoagulated with EDTA 4. Cells containing Hb S and Hb C may be resistant to he-
or heparin or blood from a capillary puncture, make a 1:251 molysis, causing turbidity; this can be corrected by making
dilution by adding 0.02 mL (20 #L) of blood to 5 mL of a 1:2 dilution with distilled water (1 part diluted sample
cyanmethemoglobin reagent. The pipette should be rinsed plus 1 part water) and multiplying the results from the stan-
thoroughly with the reagent to ensure that no blood re- dard curve by 2.
mains. Follow the same procedure for the control samples. 5. Abnormal globulins, such as those found in patients with
4. Cover and mix well by inversion or use a vortex mixer. Let plasma cell myeloma or Waldenström macroglobulinemia,
stand for 10 minutes at room temperature to allow full con- may precipitate in the reagent. If this occurs, add 0.1 g of
version of hemoglobin to cyanmethemoglobin. potassium carbonate to the cyanmethemoglobin reagent.
5. Transfer all of the solutions to cuvettes. Set the spectropho- Commercially available cyanmethemoglobin reagent has
tometer to 100% transmittance at the wavelength of been modified to contain KH2PO4 salt, so this problem is
540 nm, using cyanmethemoglobin reagent as a blank. not likely to occur.
6. Using a matched cuvette, continue reading the % transmit- 6. Carboxyhemoglobin takes 1 hour to convert to cyanmethe-
tance of the patient samples and record the values. moglobin and theoretically could cause erroneous results in
7. Determine the hemoglobin concentration of the control samples from heavy smokers. The degree of error is proba-
samples and the patient samples from the standard curve. bly not clinically significant, however.
General reference intervals can be found on the inside cover 7. Because the hemoglobin reagent contains cyanide, it is
of this text. highly toxic and must be used cautiously. Consult the safety
data sheet (Chapter 2) supplied by the manufacturer. Acidi-
Sources of Error and Comments fication of cyanide in the reagent releases highly toxic hy-
1. Cyanmethemoglobin reagent is sensitive to light. It should drogen cyanide gas. A licensed waste disposal service should
be stored in a brown bottle or in a dark place. be contracted to discard the reagent; reagent-sample solu-
2. A high WBC count (greater than 20 " 109/L) or a high plate- tions should not be discarded into sinks.
let count (greater than 700 " 109/L) can cause turbidity and 8. Commercial absorbance standards kits are available to cali-
a falsely high result. In this case, the reagent-sample solu- brate spectrophotometers.
tion can be centrifuged and the supernatant measured. 9. Handheld systems are commercially available to measure the
3. Lipemia also can cause turbidity and a falsely high result. It hemoglobin concentration. An example is the HemoCue4,5
can be corrected by adding 0.01 mL of the patient’s plasma (HemoCue, Inc., Brea, CA) (Figure 14-19) in which hemo-
to 5 mL of the cyanmethemoglobin reagent and using this globin is converted to azidemethemoglobin and is read
solution as the reagent blank. 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.6-9
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 per-
centage (e.g., 36%) or in liters per liter (0.36 L/L).
PR O C E D U R E
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 Figure 14-6 Microhematocrit reader.
risk of capillary tube injuries.10 Alternatively, 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 nonab-
sorbent 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 g to 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 read- Plasma
ing 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).10
BOX 14-2 Determining Maximum Packing Time

for Microhematocrit Buffy coat
The time to obtain maximum packing of red blood cells should be (white blood cells
and platelets)
determined for each centrifuge. Duplicate microhematocrit determi-
nations should be made using fresh, well-mixed blood anticoagulated Red blood cells
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 Clay
remained at the same value for two consecutive readings, optimum
packing has been achieved, and the second time interval should be Figure 14-7 Capillary tube with anticoagulated whole blood after it has
used for microhematocrit determinations.10 been centrifuged. Notice the layers containing plasma, the buffy coat (white
blood cells and platelets), and the red blood cells.
General reference intervals according to sex and age can be

found on the inside front cover of this text.

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 cen-
trifugation or a delay in reading results after centrifugation
causes hematocrit readings to increase. Time for complete
packing should be determined for each centrifuge and re-
checked at regular intervals. When the microhematocrit
centrifuge is calibrated, one of the samples used must have
a hematocrit of 50% or higher.10
Figure 14-8 READACRIT centrifuge with built-in capillary tube compart-
5. The buffy coat of the sample should not be included in the ments and hematocrit scales. (Courtesy and © Becton, Dickinson and
hematocrit reading because this falsely elevates the result. Company, Franklin Lakes, NJ.)
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 thalas- hematocrit can be done by applying the “rule of three.” This
semia, may cause plasma to be trapped in the red blood rule applies only to samples that have normocytic normo-
cell layer even if the procedure is performed properly. The chromic red blood cells. The value of the hematocrit should
trapping of the plasma causes the microhematocrit to be three times the value of the hemoglobin plus or minus 3:
be 1% to 3% (0.01 to 0.03 L/L) higher than the value HGB " 3 ! HCT % 3 (0.03 L/L). It should become habit for
obtained using automated instruments that calculate or the analyst to multiply the hemoglobin by 3 mentally for
directly measure the hematocrit and are unaffected by the every sample; a value discrepant with this rule may indicate
trapped plasma. abnormal red blood cells, or it may be the first indication of
8. A temporarily low hematocrit reading may result immedi- error.
ately after a blood loss because plasma is replaced faster For example, the following results are obtained from
than are the red blood cells. patients:
9. The fluid loss associated with dehydration causes a de-
Case 1
crease in plasma volume and falsely increases the hemato-
HGB ! 12 g/dL
crit reading.
HCT ! 36% (0.36 L/L)
10. Proper specimen collection is an important consideration.
According to the rule of three,
The introduction of interstitial fluid from a skin puncture
or the improper flushing of an intravenous catheter causes
HGB (12) " 3 ! HCT (36)
decreased hematocrit readings.
The READACRIT centrifuge (Becton, Dickinson and Com-
An acceptable range for the hematocrit would be 33% to
pany, Franklin Lakes, NJ) uses precalibrated capillary tubes and
39%. These values conform to the rule of three.
has built-in hematocrit scales, which eliminates the need for
separate reading devices (Figure 14-8). The use of SUREPREP Case 2
Capillary Tubes (Becton, Dickinson) eliminates the use of seal- HGB ! 9 g/dL
ants. They have a factory-inserted plug that seals automatically HCT ! 32%
when the blood touches the plug.11 According to the rule of three,
HGB (9.0) " 3 ! HCT (27 versus actual value of 32)

RULE OF THREE
When samples are analyzed by automated or manual meth- An acceptable range for hematocrit would be 24% to 30%,
ods, a quick visual check of the results of the hemoglobin and so these values do not conform to the rule of three.
Case 3 Mean Cell Volume

HGB ! 15 g/dL The MCV is the average volume of the red blood cell, expressed
HCT ! 36% in femtoliters (fL), or 10&15 L:
According to the rule of three,
HCT (%) " 10
MCV !
HGB (15) " 3 ! HCT (45 versus obtained value of 36) RBC count ( " 1012/L)
An acceptable range for hematocrit would be 42% to 48%, For example, if the HCT ! 45% and the RBC count ! 5 " 1012/L,
so these values do not conform to the rule of three. the MCV ! 90 fL.
If values do not agree, the blood film should be examined
for abnormal red blood cells; causes of false increases and de- The reference interval for MCV is 80 to 100 fL. RBCs with an
creases in the hemoglobin and/or hematocrit values should MCV of less than 80 fL are microcytic; those with an MCV of
also be investigated. In the second example, the blood film more than 100 fL are macrocytic.
reveals red blood cells that are low in hemoglobin concentra-
tion (hypochromic) and are smaller in volume (microcytic), so Mean Cell Hemoglobin
the rule of three cannot be applied. If red blood cells do appear The MCH is the average weight of hemoglobin in a red blood
normal, possible causes of a falsely low hemoglobin concen- cell, expressed in picograms (pg), or 10&12 g:
tration or a falsely elevated hematocrit should be investigated.
HGB (g/dL) " 10
In the third example, the specimen is determined to have lipe- MCH !
mic plasma causing a falsely elevated hemoglobin concentra- RBC count ( " 1012/L)
tion, and a correction must be made to obtain an accurate
hemoglobin value. (See Hemoglobin Determination in this For example, if the hemoglobin ! 16 g/dL and the RBC
chapter.) count ! 5 " 1012/L, the MCH ! 32 pg.
When an unexplained discrepancy is found, the sample The reference interval for adults is 26 to 32 pg. The MCH
processed before and after the sample in question should generally is not considered in the classification of anemias.
be checked to determine whether they conform to the rule. If
they do not conform, further investigation should be done Mean Cell Hemoglobin Concentration
to find the problem. A control sample should be run when The MCHC is the average concentration of hemoglobin in each
such a discrepancy is found. If the instrument produces appro- individual red blood cell. The units used are grams per deciliter
priate results for the control, random error may have occurred (formerly given as a percentage):
(Chapter 5).
HGB (g/dL) " 100
MCHC !
HCT (%)
RED BLOOD CELL INDICES

For example, if the HGB ! 16 g/dL and the HCT ! 48%, the
The mean cell volume (MCV), mean cell hemoglobin (MCH), MCHC ! 33.3 g/dL.
and mean cell hemoglobin concentration (MCHC) are the Values of normochromic red blood cells range from 32 to
RBC indices. These are calculated to determine the average 36 g/dL; values of hypochromic cells are less than 32 g/dL, and
volume and hemoglobin content and concentration of the red values of “hyperchromic” cells are greater than 36 g/dL. Hypo-
blood cells in the sample. In addition to serving as a quality chromic red blood cells occur in thalassemias, iron deficiency,
control check, the indices may be used for initial classification and other conditions listed in Table 14-2. The term hyperchro-
of anemias. Table 14-2 provides a summary of the RBC indices, mic is a misnomer: a cell does not really contain more than
morphology, and correlation with various anemias. The mor- 36 g/dL of hemoglobin, but its shape may have become sphe-
phologic classification of anemia on the basis of MCV is dis- rocytic, which makes the cell appear full. An MCHC between
cussed in detail in Chapter 19. 36 and 38 g/dL should be checked for spherocytes. An MCHC
TABLE 14-2 Red Blood Cell Indices, Red Blood Cell Morphology, and Disease States
MCHC Red Blood Cell
MCV (fL) (g/dL) Morphology Found in
'80 '32 Microcytic; hypochromic Iron deficiency anemia, anemia of inflammation, thalassemia, Hb E disease and trait,
sideroblastic anemia
80–100 32–36 Normocytic; normochromic Hemolytic anemia, myelophthisic anemia, bone marrow failure, chronic renal disease
(100 32–36 Macrocytic; normochromic Megaloblastic anemia, chronic liver disease, bone marrow failure, myelodysplastic
syndrome
Hb, Hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume.
greater than 38 g/dL should be investigated for an error in 5. To improve accuracy, have another laboratorian count the
hemoglobin value (see Sources of Error and Comments other film; counts should agree within 20%.
in the section on hemoglobin determination). Another cause 6. Calculate the % reticulocyte count:
for a markedly increased MCHC could be the presence of
a cold agglutinin. Incubating the specimen at 37° C for number of reticulocytes " 100
Reticulocytes (%) !
15 minutes before analysis usually produces accurate 1000 (RBCs counted)
results. Cold agglutinin disease is discussed in more detail in
Chapter 26. For example, if 15 reticulocytes are counted,
15 " 100
Reticulocytes (%) ! ! 1.5%
RETICULOCYTE COUNT 1000
The reticulocyte is the last immature red blood cell stage. Or the number of reticulocytes counted can be multiplied by
Normally, a reticulocyte spends 2 days in the bone marrow 0.1 (100/1000) to obtain the result.
and 1 day in the peripheral blood before developing into a
mature red blood cell. The reticulocyte contains remnant Miller Disc
cytoplasmic ribonucleic acid (RNA) and organelles such as Because large numbers of red blood cells should be counted to
the mitochondria and ribosomes (Chapter 8). The reticulo- obtain a more precise reticulocyte count, the Miller disc was
cyte count is used to assess the erythropoietic activity of the designed to reduce this labor-intensive process. The disc is com-
bone marrow. posed of two squares, with the area of the smaller square mea-
suring 1/9 the area of the larger square. The disc is inserted into
Principle the eyepiece of the microscope and the grid in Figure 14-10 is
Whole blood, anticoagulated with EDTA, is stained with a su- seen. RBCs are counted in the smaller square, and reticulocytes
pravital stain, such as new methylene blue. Any nonnucleated are counted in the larger square. Selection of the counting area
red blood cell that contains two or more particles of blue- is the same as described earlier. A minimum of 112 cells should
stained granulofilamentous material after new methylene blue be counted in the small square, because this is equivalent to
staining is defined as a reticulocyte (Figure 14-9). 1008 red cells in the large square and satisfies the College of
American Pathologists (CAP) hematology standard for a manual
PR O C E D U R E reticulocyte count based on at least 1000 red cells.13 The calcula-
1. Mix equal amounts of blood and new methylene blue stain tion formula for percent reticulocytes is
(2 to 3 drops, or approximately 50 #L each), and allow to
incubate at room temperature for 3 to 10 minutes.12
no. reticulocytes in squar e A
2. Remix the preparation.
(large square) " 100
3. Prepare two wedge films (Chapter 16). Reticulocytes % !
no. RBCs in square B (small square) " 9
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).
Figure 14-9 Reticulocytes with new methylene blue vital stain (peripheral Figure 14-10 Miller ocular disc counting grid as viewed through a micro-
blood "1000). Reticulocytes are nonnucleated red blood cells with two or scope. The area of square B is 1/9 the area of square A. Alternatively, square
more blue-stained filaments or particles. 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,
15 " 100 B
Reticulocytes % ! ! 1.5%
112 " 9
B
Equation Reference Interval
General reference intervals can be found on the inside front
cover of this text. A

1. If a patient is very anemic or polycythemic, the proportion A
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 reticu-
locytes is lower than that of mature red blood cells, and re-
ticulocytes settle at the top of the mixture during incubation. Figure 14-11 Reticulocytes (A) and Heinz bodies (B) stained with supra-
3. Moisture in the air, poor drying of the slide, or both may vital stain (peripheral blood "1000).
cause areas of the slide to appear refractile, and these areas
could be confused with reticulocytes. The RNA remnants in Reference Interval
a reticulocyte are not refractile. Values between 20 " 109/L and 115 " 109/L are within the
4. Other red blood cell inclusions that stain supravitally reference interval for most populations.14
include Heinz, Howell-Jolly, and Pappenheimer bodies
(Table 19-3). Heinz bodies are precipitated hemoglobin, Corrected Reticulocyte Count
usually appear round or oval, and tend to adhere to the cell Principle
membrane (Figure 14-11). Howell-Jolly bodies are round In specimens with a low hematocrit, the percentage of reticu-
nuclear fragments and are usually singular. Pappenheimer locytes may be falsely elevated because the whole blood con-
bodies are iron in the mitochondria whose presence can be tains fewer red blood cells. A correction factor is used, with the
confirmed with an iron stain, such as Prussian blue. This average normal hematocrit considered to be 45%.
stain is discussed in Chapter 17.
5. If a Miller disc is used, it is important to heed the “edge Calculation
rule” as described in the WBC count procedure and illus- Corrected reticulocyte count (%) !
trated in Figure 14-2. A significant bias is observed if the patient HCT (%)
rule is ignored.12 reticuloc yte (%) "
45
Absolute Reticulocyte Count Reference Interval

Principle Patients with a hematocrit of 35% should have an elevated cor-
The absolute reticulocyte count (ARC) is the actual number of rected reticulocyte count of 2% to 3% to compensate for the
reticulocytes in 1 liter (L) or 1 microliter (#L) of blood. mild anemia. In patients with a hematocrit of less than 25%,
the count should increase to 3% to 5% to compensate for the
Calculations moderate anemia. The corrected reticulocyte count depends on
the degree of anemia.
reticulocytes (%) " RBC count ( " 1012/L)
ARC !
100 Reticulocyte Production Index
Principle
For example, if a patient’s reticulocyte count is 2% and the RBC Reticulocytes that are released from the marrow prematurely are
count is 2.20 " 1012/L, the ARC is calculated as follows (note that called shift reticulocytes. These reticulocytes are “shifted” from the
the calculated result has to be converted from 1012/L to 109/L): bone marrow to the peripheral blood earlier than usual to com-
pensate for anemia. Instead of losing their reticulum in 1 day, as
2 " (2.20 " 1012/L) do most normal circulating reticulocytes, these cells take 2 to
ARC ! ! 44 " 109/L
100 3 days to lose their reticula. When erythropoiesis is evaluated, a
correction should be made for the presence of shift reticulocytes
The absolute reticulocyte count can also be reported as the if polychromasia is reported in the red blood cell morphology.
number of cells per #L. Using the example above, the RBC Most normal (nonshift) reticulocytes become mature red blood
count in #L (2.20 " 106/#L) is used in the formula, and the cells within 1 day after entering the bloodstream and thus repre-
ARC result is 44 " 103/#L. sent 1 day’s production of red blood cells in the bone marrow.
Cells shifted to the peripheral blood prematurely stay longer as the red blood cells are treated with fluorescent dyes or nucleic
reticulocytes and contribute to the reticulocyte count for more acid stains to stain residual RNA in the reticulocytes. The per-
than 1 day. For this reason, the reticulocyte count is falsely in- centage and the absolute count are provided. These results are
creased when polychromasia is present, because the count no statistically more valid because of the large number of cells
longer represents the cells maturing in just 1 day. On many counted. Other reticulocyte parameters that are offered on
automated instruments, this mathematical adjustment of the some automated instruments include a maturation index/im-
reticulocyte count has been replaced by the measurement of mature reticulocyte fraction or IRF (reflecting the proportion of
immature reticulocyte fraction (Chapter 15).12 the more immature reticulocytes in the sample), the reticulo-
The patient’s hematocrit is used to determine the appropri- cyte hemoglobin concentration, and reticulocyte indices (such
ate correction factor (reticulocyte maturation time in days): 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 trans-
Patient’s Hematocrit Correction Factor
plantation. The reticulocyte hemoglobin is useful to detect
Value (%) (Maturation Time, Days)
early iron deficiency (Chapter 20). Automated reticulocyte
40–45 1 counting is discussed in Chapter 15.
35–39 1.5
25–34 2
ERYTHROCYTE SEDIMENTATION RATE
15–24 2.5
'15 3 The erythrocyte sedimentation rate (ESR) is ordered with other
tests to detect and monitor the course of inflammatory condi-
Calculation tions such as, rheumatoid arthritis, infections, or certain malig-
The reticulocyte production index (RPI) is calculated as nancies. It is also useful in the diagnosis of temporal arteritis
follows: and polymyalgia rheumatica.15 The ESR, however, is not a spe-
reticulocyte (%) " [HCT (%)/45]
RPI ! cific test for inflammatory diseases and is elevated in many
maturation time
other conditions such as plasma cell myeloma, pregnancy,
anemia, and older age. It is also prone to technical errors that
Or can falsely elevate or decrease the sedimentation rate. Because
corrected reticulocyte count
RPI ! of its low specificity and sensitivity, the ESR is not recom-
maturation time
mended as a screening test to detect inflammatory conditions
in asymptomatic individuals.15 Other tests for inflammation,
For example, for a patient with a reticulocyte count of 7.8% such as the C-reactive protein level, may be a more predictable
and a HCT of 30%, and with polychromasia noted, the previ- and reliable alternative to monitor inflammation.16
ous table indicates a maturation time of 2 days. Thus
Principle
7. 8 " [30/45] When anticoagulated blood is allowed to stand at room tem-
RPI !
2 perature undisturbed for a period of time, the red blood cells
RPI ! 2. 6 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
Reference Interval affected by red blood cell, plasma, and mechanical and techni-
An adequate bone marrow response usually is indicated by cal factors. Red blood cells have a net negative surface charge
an RPI that is greater than 3. An inadequate erythropoietic and tend to repel one another. The repulsive forces are partially
response is seen when the RPI is less than 2.14 or totally counteracted if there are increased quantities of
positively charged plasma proteins. Under these conditions
Reticulocyte Control the red blood cells settle more rapidly as a result of the forma-
Several commercial controls are now available for monitoring tion of rouleaux (stacking of red blood cells). Examples of
manual and automated reticulocyte counts [e.g., Retic-Chex II, macromolecules that can produce this reaction are fibrinogen,
Streck Laboratories, Omaha, NE; Liquichek Reticulocyte Con- )-globulins, and pathologic immunoglobulins.17,18
trol (A), Bio-Rad Laboratories, Hercules, CA]. Most of the con- Normal red blood cells have a relatively small mass and
trols are available at three levels. The control samples are settle slowly. Certain diseases can cause rouleaux formation, in
treated in the same manner as the patient samples. The control which the plasma fibrinogen and globulins are altered. This
can be used to verify the laboratorian’s accuracy and precision alteration changes the red blood cell surface, which leads to
when manual counts are performed. stacking of the red blood cells, increased red blood cell mass,
and a more rapid ESR. The ESR is directly proportional to
Automated Reticulocyte Counts the red blood cell mass and inversely proportional to plasma
The major instrument manufacturers offer are analyzers that viscosity. Several methods, both manual and automated, are
perform automated reticulocyte counts. All of the analyzers available for measuring the ESR. Only the most commonly
evaluate reticulocytes using optical scatter or fluorescence after 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
P R O C E DU R E
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 undis-
turbed 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
Wintrobe Erythrocyte Sedimentation Rate

When the Wintrobe method was first introduced, the specimen Figure 14-12 Erythrocyte sedimentation rate (ESR), 1 hour ! 93 mm,
used was oxalate-anticoagulated whole blood. This was placed which is elevated above the reference intervals.
in a 100-mm column. Today, EDTA-treated or citrated whole
blood is used with the shorter column. The shorter column to the top of the sedimented red cells. The result is reported in
height allows a somewhat increased sensitivity in detecting millimeters per hour.
mildly elevated ESRs.
Reference Interval
PR O C E D U R E Reference intervals according to sex and age can be found on
1. Use fresh blood collected in EDTA anticoagulant. A mini- the inside front cover of this text. Table 14-3 lists some of the
mum of 2 mL of whole blood is needed. factors that influence the ESR.
2. After mixing the blood thoroughly, fill a Pasteur pipette
using a rubber pipette bulb. Sources of Error and Comments
3. Place the filled pipette into the Wintrobe tube until the tip 1. If the concentration of anticoagulant is increased, the ESR
reaches the bottom of the tube. will be falsely low as a result of sphering of the RBCs, which
4. Carefully squeeze the bulb and expel the blood into the inhibits rouleaux formation.
Wintrobe tube while pulling the Pasteur pipette up from the 2. The anticoagulants sodium or potassium oxalate and
bottom of the tube. There must be steady, even pressure on heparin cause the red blood cells to shrink and falsely
the bulb to expel blood into the tube as well as continuous elevate the ESR.
movement of the pipette up the tube to prevent the intro- 3. A significant change in the temperature of the room alters
duction of air bubbles into the column of blood. the ESR.
5. Fill the Wintrobe tube to the 0 mark. 4. Even a slight tilt of the pipette causes the ESR to
6. Place the tube into a Wintrobe rack (tube holder) and allow increase.
to stand undisturbed for 1 hour at room temperature. The 5. Blood specimens must be analyzed within 4 hours of
rack must be perfectly level and placed in a draft-free room. collection if kept at room temperature (18 to 25° C).15 If
7. Record the number of millimeters the red blood cells have the specimen is allowed to sit at room temperature for
fallen. Read the tube from the bottom of the plasma meniscus more than 4 hours, the red blood cells start to become
TABLE 14-3 Factors Affecting the Erythrocyte Sedimentation Rate (ESR)

Category Increased ESR Decreased ESR
Blood proteins and lipids Hypercholesterolemia Hyperalbuminemia
Hyperfibrinogenemia Hyperglycemia
Hypergammaglobulinemia Hypofibrinogenemia
Hypoalbuminemia Hypogammaglobulinemia
Increased bile salts
Increased phospholipids
Red blood cells Anemia Acanthocytosis
Macrocytosis Anisocytosis (marked)
Hemoglobin C
Microcytosis
Polycythemia
Sickle cells
Spherocytosis
Thalassemia
White blood cells Leukemia Leukocytosis (marked)
Drugs Dextran Adrenocorticotropic hormone (corticotropin)
Heparin Cortisone
Penicillamine Ethambutol
Procainamide Quinine
Theophylline Salicylates
Vitamin A
Clinical conditions Acute heavy metal poisoning Cachexia
Acute bacterial infections Congestive heart failure
Collagen vascular diseases Newborn status
Diabetes mellitus
End-stage renal failure
Gout
Malignancy
Menstruation
Multiple myeloma
Myocardial infarction
Pregnancy
Rheumatic fever
Rheumatoid arthritis
Syphilis
Temporal arteritis
Specimen handling Refrigerated sample not returned to room temperature Clotted blood sample
Delay in testing
Technique High room temperature Bubbles in ESR column
Tilted ESR tube Low room temperature
Vibration 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 Clini-
cal 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.
spherical, which may inhibit the formation of rouleaux. 8. A clotted specimen cannot be used.
Blood specimens may be stored at 4° C up to 24 hours 9. The tubes must not be subjected to vibrations on the lab
prior to testing, but must be rewarmed by holding bench which can falsely increase the ESR.
the specimen at ambient room temperature for at least 10. Hematologic disorders that prevent the formation of rou-
15 minutes prior to testing.15 leaux (e.g., the presence of sickle cells and spherocytes)
6. Bubbles in the column of blood invalidate the test results. decrease the ESR.
7. The blood must be filled properly to the zero mark at the 11. The ESR of patients with severe anemia is of little diagnostic
beginning of the test. value, because it will be falsely elevated.
Disposable Kits The ESR STAT PLUS system (HemaTechnologies, Lebanon,

Disposable commercial kits are available for ESR testing NJ) is based on centrifugation. The advantages of this method
(Figure 14-13). Several kits include safety caps for the columns are a smaller required sample volume and shorter testing
that allow the blood to fill precisely to the zero mark. This time, which makes it more suitable for a pediatric patient
safety cap makes the column a closed system and eliminates population. The disadvantage of this method is the number
the error involved in manually setting the blood to the of exacting preanalytical steps that must be strictly followed
zero mark. to prevent erroneous results. Compliance with these steps
may be difficult to achieve consistently in a busy hematology
Automated Erythrocyte Sedimentation Rate laboratory.21
There are several automated ESR systems available using
the traditional Westergren and Wintrobe methods, as well
ADDITIONAL METHODS
as alternate methods such as centrifugation. The Ves-Matic
system (Diesse, Inc., Hialeah, FL) is a bench-top analyzer Additional manual and semi-automated methods are in-
designed to determine ESR by use of an optoelectronic cluded in other chapters that are relevant to their clinical
sensor, which measures the change in opacity of a column application. Examples include: Chapter 24 for the osmotic
of blood as sedimentation of blood progresses. Blood is fragility test and qualitative and quantitative assays for
collected in special Ves-Tec or Vacu-Tec tubes, which contain glucose-6-phosphate dehydrogenase and pyruvate kinase
sodium citrate and are compatible with the Vacutainer activity; Chapter 27 for the solubility test for Hb S, hemoglo-
system. These tubes are used directly in the instrument bin electrophoresis (alkaline and acid pH), and unstable
(Figure 14-14). Acceleration of sedimentation is achieved by hemoglobin test; and Chapter 28 for the vital stain for he-
positioning the tubes at an 18-degree angle in relation to moglobin H and the Kleihauer-Betke acid elution test for Hb
the vertical axis. Results comparable with Westergren 1-hour F distribution in the RBCs.
values are obtained in 20 minutes.20
Another automated ESR analyzer is the Sedimat 15
POINT-OF-CARE TESTING
(Polymedco, Cortlandt Manor, NY), which uses the principle
of infrared measurement. It is capable of testing one to eight Point-of-care testing offers the ability to produce rapid
samples randomly or simultaneously and provides results in and accurate results that help facilitate faster treatment,
15 minutes (Figure 14-15). 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 “test-
ing 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 clas-
sified 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 admin-
istrative responsibility, well-written procedures, a training
program, quality control, proficiency testing, and equipment
Figure 14-13 Sediplast (Polymedco) disposable sedimentation rate maintenance are essential for success. The first step is appoint-
system. (Courtesy Polymedco, Cortlandt Manor, NY.) ing a laboratory point-of-care testing coordinator. This person
A B
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. (Courtesy Diesse Inc., Hialeah, FL.)
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 per-
form 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 pro-
gram 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 corre-
lation 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 light-
weight, accurate, fast, and that require little specimen material.
The point-of-care testing system should also address the fol-
lowing laboratory concerns:
• What is the range of measurement?
Figure 14-15 Sedimat 15 (Polymedco) automated sedimentation rate • How well does the test system correlate with laboratory
system. (Courtesy Polymedco, Cortlandt Manor, NY.) instrumentation?
• Can it be interfaced to the laboratory information system?
• Does it give reliable results?
not only is the “go-to” person but is also an important liaison • Does the company supply excellent technical support?
between the laboratory and nursing staff. The second step to • Is it affordable?
ensuring a successful program is to create a multidisciplinary Paramount to point-of-care testing is patient safety. It is
team with authority to impact all aspects of the POC program. important to maintain good practices, and with waived testing,
This committee would have the authority to oversee the integ- this often comes down to the basics. Such basics include
rity and quality of the existing POC program and institute proper and appropriate specimen collection, proper identifica-
changes or new testing as needed. It is also important to have tion of the patient and specimen, proper storage of reagents,
administrative support to help remove barriers. and good documentation of patient test results (use of point-
A point-of-care testing program must incorporate all of the of-care interfaces is beneficial), as well as proper performance
following. A written policy should be developed that defines 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 turn-
over rates.23 Patient safety, risk management, and error reduc-
tion 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 Dis-
ease Control and Prevention, The Joint Commission, CAP,
CLIA, and so forth) should be implemented and easily accessi-
ble. 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 sac-
rificed 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.
Figure 14-16 i-STAT instrument for measuring hematocrit. (Courtesy
Hematocrit Abbott Laboratories, Abbott Park, IL.)
The most common methods for determining the hematocrit
include the microhematocrit centrifuge, conductometric meth-
ods, and calculation by automated cell counters (Chapter 15).
Centrifuge-based microhematocrit systems have been avail-
able 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 com-
ments 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 Figure 14-17 Epoc device for measuring hematocrit. (Courtesy Epocal,
act as insulators. In the i-STAT system, before the measured Inc., Ottawa, Ontario, Canada.)
sample conductance is converted into the hematocrit value,
corrections are applied for the temperature of the sample, the measurement. An increased WBC count will falsely increase
size of the fluid segment being measured, and the relative con- the hematocrit. The presence of cold agglutinins can falsely
ductivity of the plasma component. The first two corrections decrease the hematocrit.24
are determined from the measured value of the calibrant con-
ductance and the last correction from the measured concentra- Other Instruments. Other instruments that measure the
tions of sodium and potassium in the sample.24 hematocrit include the following:
• ABL 77 (Radiometer, Westlake, OH)
Sources of Error and Comments. Conductivity of a • IRMA (ITC, a subsidiary of Thoratec Corporation, Edison, NJ)
whole blood sample is dependent on the amount of electro- • Gem Premier (Instrumentation Laboratory Company,
lytes in the plasma portion. Conductivity does not distinguish Lexington, MA) (Figure 14-18)
red blood cells from other nonconductive elements such as
proteins, lipids, and WBCs that may be present in the sample. Hemoglobin Concentration
A low total protein level will falsely decrease the hematocrit. In point-of-care testing, hemoglobin concentration is measured
The presence of lipids can interfere with the hematocrit by modified hemoglobinometers or by oximeters integrated
Figure 14-19 The HemoCue ® Hb 201+ System for measuring hemo-

globin. (Courtesy HemoCue, Inc., Brea, CA.)
composed of molded plastic with a fluid well that contains

Figure 14-18 Gem Premier instrument for measuring hematocrit. (Courtesy
Instrumentation Laboratory Company, Lexington, MA.) 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
with a blood gas analyzer. The HemoCue hemoglobinometer
(HemoCue, Inc., Brea, CA) uses a small cuvette that contains a Cell and Platelet Counts
lysing agent and reagents to form a hemoglobin azide, which is Traditional cell-counting methods can be employed at the point
measured by a photometer at two wavelengths (570 nm and of care for the analysis of WBCs, RBCs, and platelets. The Ichor
880 nm) (Figure 14-19).6 This eliminates interference from Hematology Analyzer (Helena Laboratories, Beaumont, TX) per-
turbidity in the sample. Results obtained with the instrument forms a complete blood count along with platelet aggregation.
compare well with those produced by reference methods, but a Another option for cell quantitation and differentiation employs
major source of error is mixture of blood with tissue fluid dur- a buffy coat analysis method. Quantitative buffy coat analysis
ing skin puncture collection. The AVOX 1000E (ITC) measures (QBC STAR, manufactured by QBC Diagnostics, Inc., Philips-
total hemoglobin by a spectrophotometric method. The STAT- burg, PA) involves centrifugation in specialized capillary tubes
Site MHbg Meter (Stanbio Laboratory, Boerne, TX) uses the designed to expand the buffy coat layer. The components (plate-
azidemethemoglobin principle and reflectance photometry to lets, mononuclear cells, and granulocytes) can be measured with
measure reflected light in the test area. The test card is the assistance of fluorescent dyes and a measuring device.27
SU M M A RY
• Although most laboratories are highly automated, the manual tests 0.03 (L/L). A value discrepant with this rule may indicate abnormal
discussed in this chapter, such as the cyanmethemoglobin method red blood cells or it may be the first indication of error.
of hemoglobin determination and centrifuge-based measurement • RBC indices—the mean cell volume (MCV), mean cell hemoglobin
of the microhematocrit, are used as a part of many laboratories’ (MCH), and mean cell hemoglobin concentration (MCHC)—are
quality control and backup methods of analysis. calculated to determine the average volume, hemoglobin content,
• The hemacytometer allows counts of any type of cell or particle and hemoglobin concentration of red blood cells. The indices give
(e.g., WBCs or platelets) to be performed. an indication of possible causes of an anemia.
• The reference method for hemoglobin determination is based on • The reticulocyte count, which is used to assess the erythropoietic
the absorbance of cyanmethemoglobin at 540 nm. When a spec- activity of the bone marrow, is accomplished through the use of
trophotometer is used, a standard curve is employed to obtain the supravital stains (e.g., new methylene blue) or by flow cytometric
results. methods.
• The microhematocrit is a measure of packed red blood cell volume. • The erythrocyte sedimentation rate (ESR), a measure of the settling
• The rule of three specifies that the value of the hematocrit should of red blood cells in a 1-hour period, depends on the red blood cells’
be three times the value of the hemoglobin plus or minus 3 (%) or ability to form rouleaux. It is used to detect and monitor conditions
with inflammation such as rheumatoid arthritis, infections, and some • For a point-of-care testing program to be successful, key elements
malignancies. It is subject to many physiologic and technical errors. such as clear administrative responsibility, well-written procedures,
• Point-of-care testing is often performed by nonlaboratory person- quality control, proficiency testing, and equipment maintenance
nel. It is defined as diagnostic laboratory testing at or near the site must be present.
of patient care. • Paramount to point-of-care testing is patient safety.
• CLIA introduced the concept of “testing site neutrality,” which
means that it does not matter where diagnostic testing is performed Now that you have completed this chapter, read again
or who performs the test; all testing sites must follow the same the case studies at the beginning and respond to the
regulatory requirements based on the “complexity” of the test. questions presented.
• 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.
RE V I E W Q U ES T I ONS
1. A 1:20 dilution of blood is made with 3% glacial acetic acid 6. Calculate the MCV and MCHC for the following values:
as the diluent. The four large corner squares on both sides RBCs ! 5.00 " 1012/L
of the hemacytometer are counted, for a total of 100 cells. HGB ! 9 g/dL
What is the total WBC count ("109/L)? HCT ! 30%
a. 0.25
MCV (fL) MCHC (g/dL)
b. 2.5
c. 5 a. 30 18
d. 10 b. 60 30
c. 65 33
2. The total WBC count is 20 " 109/L. Twenty-five NRBCs per d. 85 35
100 WBCs are observed on the peripheral blood film. What
is the corrected WBC count ("109/L)? 7. What does the reticulocyte count assess?
a. 0.8 a. Inflammation
b. 8 b. Response to infection
c. 16 c. Erythropoietic activity of the bone marrow
d. 19 d. Ability of red blood cells to form rouleaux
3. If potassium cyanide and potassium ferricyanide are used 8. For a patient with the following test results, which measure
in the manual method for hemoglobin determination, the of bone marrow red blood cell production provides the
final product is: most accurate information?
a. Methemoglobin Observed reticulocyte count ! 5.3%
b. Azide methemoglobin HCT ! 35%
c. Cyanmethemoglobin Morphology—moderate polychromasia
d. Myoglobin a. Observed reticulocyte count
b. Corrected reticulocyte count
4. Which of the following would not interfere with the result c. RPI
when hemoglobin determination is performed by the cyan- d. ARC
methemoglobin method?
a. Increased lipids 9. Given the following values, calculate the RPI:
b. Elevated WBC count Observed reticulocyte count ! 6%
c. Lyse-resistant RBCs HCT ! 30%
d. Fetal hemoglobin a. 2
b. 3
5. A patient has a hemoglobin level of 8.0 g/dL. According to the c. 4
rule of three, what is the expected range for the hematocrit? d. 5
a. 21% to 24%
b. 23.7% to 24.3%
c. 24% to 27%
d. 21% to 27%
10. Which of the following would be associated with an c. Decreased globulins

elevated ESR value? d. Inflammation
a. Microcytosis
b. Polycythemia
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ADDITIONAL RESOURCES
College of American Pathologists, http://www.cap.org. Point of Care.net, http://www.pointofcare.net.
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PART III Laboratory Evaluation of Blood Cells

Manual, Semiautomated, and 14

1. Using the rule of three, given the hemoglobin concentra- Case 3

cells counted " dilution factor

Alternately, a 1:100 dilution may be used counting the number

cells counted " dilution factor

Sources of Error and Comments

The absorbance of the cyanmethemoglobin at 540 nm is

Cyanmethemoglobin 0 1.5 3 4.5 6

b. Transfer the dilutions to cuvettes. Set the wavelength on

This method avoids the necessity of sample dilution and

BOX 14-2 Determining Maximum Packing Time

General reference intervals according to sex and age can be

Sources of Error and Comments

HGB (9.0) " 3 ! HCT (27 versus actual value of 32)

Case 3 Mean Cell Volume

RED BLOOD CELL INDICES

For example, if 15 reticulocytes are counted in the large square

Sources of Error and Comments

Absolute Reticulocyte Count Reference Interval

Modified Westergren Erythrocyte

Wintrobe Erythrocyte Sedimentation Rate

TABLE 14-3 Factors Affecting the Erythrocyte Sedimentation Rate (ESR)

Disposable Kits The ESR STAT PLUS system (HemaTechnologies, Lebanon,

the program. This policy should outline who is responsible for

is sometimes absent, and basic safety precautions necessary

Figure 14-19 The HemoCue ® Hb 201+ System for measuring hemo-

composed of molded plastic with a fluid well that contains

10. Which of the following would be associated with an c. Decreased globulins

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