Osmotic fragility.

The osmotic fragility test demonstrates increased RBC fragility in blood specimens in which the RBCs have
decreased surface area–to–volume ratios. Blood is added to a series of tubes with increasingly hypotonic
sodium chloride (NaCl) solutions. In each tube, water enters and leaves the RBCs until equilibrium is
achieved.

In 0.85% NaCl, the amount of water entering the cell is equivalent to the water. leaving the cell because
the intracellular and extracellular osmolarity is the same. In a hypotonic solution, more water will enter
the cell to dilute the intracellular contents until equilibrium is reached between the cytoplasm and the
hypotonic extracellular solution. As this phenomenon occurs, the cells swell. As the RBCs are subjected to
increasingly hypotonic solutions, even more water will enter the RBCs until the internal volume is too
great and lysis occurs.

Because spherocytes already have a decreased surface area–to–volume ratio, they lyse in less hypotonic
solutions than normal-shaped, biconcave RBCs and thus have increased osmotic fragility. In the
procedure, a standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from 0.85%
(isotonic saline) to 0.0% (distilled water) in 0.05% to 0.1% increments. After a 30-minute incubation at
room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured
spectrophotometrically at 540 nm. The percent hemolysis is calculated for each tube as follows

Ax% is the absorbance in the tube being measured,

A0.85% is the absorbance in the 0.85% NaCl tube (representing 0% hemolysis), and
A0.0% is the absorbance in the 0.0% NaCl tube (representing 100% hemolysis).

The % hemolysis for each % NaCl concentration is plotted, and an osmotic fragility curve is drawn
. Normal biconcave RBCs show initial hemolysis at 0.45% NaCl, and 100% or complete hemolysis generally
occurs between 0.35% and 0.30% NaCl. If the curve is shifted to the left, the patient’s RBCs have
increased osmotic fragility, and in this case, initial hemolysis begins at an NaCl concentration greater than
0.5%. Conversely, if the curve is shifted to the right, the RBCs have decreased osmotic fragility. Decreased
osmotic fragility is found in conditions characterized by numerous target cells, such as thalassemia.

The existence of a distinct subpopulation of the most fragile cells, those most conditioned by the spleen,
is reflected by the presence of a “tail” on the osmotic fragility curve (Figure 24-4).13 After splenectomy is
performed, the osmotic fragility improves, and this subpopulation of conditioned cells disappears.

Incubating the blood at 37° C for 24 hours before performing the test (called the incubated osmotic
fragility test) allows HS cells to become more spherical and is often needed to detect mild cases. Patients
who have increased osmotic fragility only when their blood is incubated tend to have mild disease and a
low number of spherocytes in the total RBC population.

The osmotic fragility test is time-consuming, and it requires a fresh heparinized blood specimen collected
without trauma (to avoid hemolysis) and accurately made NaCl solutions. Specimens are stable for 2 hours
at room temperature or 6 hours if the specimen is refrigerated.23 A major drawback of the osmotic
fragility test is its lack of sensitivity.16 In a 2011 study of 150 HS patients by Bianchi and colleagues, the
sensitivity for the unincubated test was 68%, with only a modest increase in sensitivity to 81% with the
incubated test.24 In non-splenectomized HS patients with compensated anemia, those sensitivity figures
dropped to only 53% and 64%, respectively.24 The osmotic fragility test is also nonspecific. A result
indicating increased fragility does not differentiate between HS and spherocytosis caused by other
conditions, such as burns, immune hemolytic anemias, and other acquired disorders.13,16 These
disadvantages have led some not to recommend this test for routine use.

Osmotic Fragility Test Red cells are suspended in a series of tubes containing hypotonic solutions of NaCl,
varying from 0.9%–0.0%, incubated at room temperature for 30 minutes, and centrifuged. The percent
hemolysis in the supernatant solutions is measured and plotted for each NaCl concentration.

Cells that are more spherical, with a decreased surface/volume ratio, have a limited capacity to expand
in hypotonic solutions and lyse at a higher concentration of NaCl than do normal biconcave red cells. They
are said to have increased osmotic fragility.

Conversely, cells that are hypochromic and flatter have a greater capacity to expand in hypotonic
solutions, lyse at a lower concentration than normal cells, and are said to have decreased osmotic fragility
(Fig. 32-11).

The osmotic fragility of freshly drawn blood is usually increased in HS but may be normal in mildly affected
patients. In blood that is incubated at 37° C for 24 hours before the test is performed, the osmotic fragility
is almost always increased (Fig. 32-12).

The increased osmotic fragility of freshly drawn blood is characteristic but not specific; it may occur in
acquired spherocytic anemias, such as immune-mediated hemolytic anemia. A greater difference in
median fragility (after incubation from before incubation) occurs in HS cells than in control normal cells;
this is an important diagnostic feature in HS. A tail of very fragile cells conditioned by the spleen is usually
seen. The tail usually disappears after splenectomy. Cells with increased surface/volume ratio are osmotic
resistant. These cells are seen in iron deficiency, thalassemia, liver disease, and reticulocytosis.

Reticulocyte Count
The reticulocyte count is elevated, which indicates that the bone marrow is responding to a
hemolytic process. In Hb H disease, the typical reticulocyte count is 5% to 10%. In homozygous b-
thalassemia, it is typically 2% to 8%, disproportionately low relative to the degree of anemia. An
inadequate reticulocytosis reflects the ineffective erythropoiesis

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 bluestained
granulofilamentous material after new methylene blue staining is defined as a reticulocyte

PROCEDURE
1. Mix equal amounts of blood and new methylene blue stain (2 to 3 drops, or approximately 50 mL 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 (10003 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:

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. The calculation
formula for percent reticulocytes is 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.1

Absolute Reticulocyte Count

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

For example, if a patient’s reticulocyte count is 2% and the RBC count is 2.20 3 10^12/L, the ARC is
calculated as follows (note that the calculated result has to be converted from 10^12/L to 10^9/L):

The absolute reticulocyte count can also be reported as the number of cells per uL. Using the example
above, the RBC count in uL (2.20 x 10^6/uL) is used in the formula, and the ARC result is 44 x 10^3/uL.

Reference Interval Values

between 20 x 10^9/L and 115x 10^9/L are within the reference interval for most populations.

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%.

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 (non-shift) 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):

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

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.

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. 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. Other tests for
inflammation, such as the C-reactive protein level, may be a more predictable and reliable alternative to
monitor inflammation.

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, b-globulins, and pathologic immunoglobulins.
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.

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 5 x mm.15

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.

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).15 If 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.
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. 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.

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
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

Factors Affecting the Erythrocyte Sedimentation Rate (ESR)

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
Heparin (corticotropin)
Penicillamine Cortisone
Procainamide Ethambutol
Theophylline Quinine
Vitamin A Salicylates
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 Clotted blood sample
returned to room temperature Delay in testing
Technique High room temperature Bubbles in ESR column
Tilted ESR tube Low room temperature
 Vibration Narrow ESR column diameter

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.

Once the red cells are lysed by the saponin allowing the hemoglobin to escape.
Deoxygenation will happen
Hemoglobin escapes as red cells are lysed by the saponin. Deoxygenation will
happen via the binding of sodium dithionite in the test environment. A
deoxygenated state will result to the polymerization of Hb S forming a precipitate.
Turbidity of the solution is seen as tactoids present in the solution deflects and
refracts light.