CHAPTER 11: Manual, Semiautomated, and Point of Care

Testing in Hematology EQUIPMENT

Hemacytometer / Counting Chamber
SPECIMEN COLLECTION - Most common: Levy Chamber with improved Neubauer
- Specimen for most routine hematology tests: Whole
ruling
blood
- Composed of two raised surfaces → each with a 3 mm x
o collected by venipuncture or skin puncture
3 mm square counting area or grid (total of 9mm 2)
o into tubes with anticoagulant to prevent clotting
separated by an H-shaped moat
- Ethylenediaminetetraacetic acid (EDTA, usually
- Grid → made up of nine 1 mm x 1 mm squares.
K2EDTA) - most common anticoagulant for routine
- Four corner squares (WBC) → subdivided further into 16
hematology testing, including the complete blood count
squares
(CBC) and differential count because of its minimal
- Center square → subdivided into 25 smaller squares
effects on blood cell morphology
- Smallest squares → 0.2 mm x 0.2 mm, which is 1 ⁄ 25 of
o Mechanism of Action: prevents clotting in the
the center square or 0.04 mm
specimen by binding (chelating) calcium required for
- Cover slip → placed on top of the counting surface
fibrin clot formation
- 0.1 mm → distance between each counting surface and
o Lavender or pink stoppers/top
the coverslip
- Established protocols must be followed: (1) accurate
- 0.9mm3→ total volume of one entire grid or counting area
patient identification, (2) proper patient preparation, and
on one side of the hemacytometer
(3) use of the appropriate body sites for venipuncture and
- National Bureau of Standards or “NBS” → provides the
skin puncture.
specifications that should be meet by hemacytometers
- Ideally specimens for CBC testing should be analyzed
and coverslips; initials are usually found on the chamber
within 6 hours of collection (if stored at room
NOTE:
temperature), and 24 hours (if stored at 4°C) Primay Squares – there are two; each 3mm x 3mm [0.9 mm3 ]
- Peripheral blood films prepared within 3 hours of Secondary Squares – there are nine (9); each 1mm x 1mm
collection reduce cell deterioration and morphology [0.2 mm2]
artifacts. Tertiary Squares – 64 for WBC (16 x 4); 25 for center square
- BEFORE TESTING, Specimen must be well mixed and
inspected to ensure it is free of clots.
- Following published protocols for specimen collection → a
critically important step in providing accurate laboratory
test information for patients
- Procedures published by Clinical and Laboratory
Standards Institute (CLSI) - describe state of the art
methods for collecting and processing blood specimens
and should be followed by all health care professionals
who collect blood specimens.
- COMPONENTS OF SPECIMEN COLLECTION THAT
INFLUENCE TESTING:
o equipment employed to obtain the specimen
o site used and technique for venipuncture or skin CALCULATIONS
puncture - General Formula:
o handling of the specimen after collection
MANUAL CELL COUNTS
- Most routine cell-counting procedures in the hematology
laboratory are AUTOMATED, it may be necessary tuse
manual methods when:
(1) count exceed the linearity of an instrument,
(2) an instrument is nonfunctional and there is no
backup - Calculation yields number of cells per mm3
(3) in remote laboratories in Third World countries o One mm3 = 1 microliter (uL)
(4) disaster situation when testing is done on field o Count per uL x 106 = count per liter
- Manual cell counts - performed using a hemacytometer, o Conversion factor: 106
or counting chamber, and manual dilutions made with WHITE BLOOD CELL COUNT
calibrated, automated pipettes and diluents (commercially - Defined as: the number of WBCs in 1 liter (L) or 1 mL of
available or laboratory prepared) blood
- PRINCIPLE OF PERFORMING CELL COUNTS: Same - Whole blood anticoagulated with EDTA or blood from a
for WBC, RBC and platelets; only the dilution, diluting skin puncture → diluted with 1% ammonium oxalate or
fluid, and area counted vary. weak acid solution (3% acetic acid or 1% hydrochloric
acid)
Dearroz, John Rhenire P. | 3MT02
- Diluting fluid - lyses the nonnucleated RBCs in the Example Using the First Equation
specimen to prevent their interference in the count When a 1:20 dilution is used, the four large squares on one
WHITE BLOOD CELL DILUTING FLUID – ideal is “hypotonic” side of the chamber yield counts of 23, 26, 22, and 21. The
- Will hemolyze the RBCs except metarubricyte total count is 92. The four large squares on the other side of
- Must be capable of lysing erythrocytes without destroying the chamber yield counts of 28, 24, 22, and 26. The total count
leukocytes: is 100. The difference between sides is less than 10%.
- Glacial Acetic Acid: 2% (v/v)
- HCl: 1% (v/v)
- Turk’s Solution: enhances leukocyte nuclear definition
- THINGS TO REMEMBER:
o Overcharging will lead to decrease in cell count since cells
will fall into the moat of the counting chamber.
o It is important that we allow the counting chamber to stand
for at least 3 minutes (“Poisson’s Law of Distribution”)
o The allowable difference between 2 chambers is:
▪ RBC: 15 to 16 counts
▪ WBC: 10 to 12 counts

- Typical dilution for WBC Count: 1:20

- Hemacytometer is charged/filled with well-mixed dilution
and placed under a microscope and number of cells are
counted in the FOUR LARGE CORNER SQUARES (4
Manual Cell Counts with Most Common Dilutions,
mm2) is counted. Counting Areas
- 1:20 dilution = 25 mL of well-mixed blood + 475 mL of Cells Diluting Dilutions Objective Area
WBC diluting fluid Counted Fluid Counted
- Examined using low power objective White ♦ 1% ♦ 1:20 ♦ 10 X ♦ 4 mm2
Blood ammonium ♦ 1:100 ♦ 10 X ♦ 9 mm2
Cells oxalate
(WBC) ♦ 3% acetic
acid
♦ 1%
hydrochloric
acid
Red Isotonic 1:100 40 X 0.2 mm2
Blood saline (5 small
NOTE: Cells that touch the top and left lines should Cells squares
be counted; cells that touch the bottom and right lines (RBC) of center
should be ignored square)
PROCEDURE: Platelets 1% 1:100 40X 1 mm2
ammonium phase
oxalate

- REMEMBER: Reference intervals may vary slightly

according to the population tested and should be
established for each laboratory
NOTE: A moist chamber may be made by placing a piece of Sources of Error and Comments
damp filter paper in the bottom of a Petri dish. An 1. The hemacytometer and coverslip should be cleaned
applicator stick broken in half can serve as a support for the properly before they are used. Dust and fingerprints may cause
hemacytometer difficulty in distinguishing the cells.
2. The diluting fluid should be free of contaminants.
Dearroz, John Rhenire P. | 3MT02
3. If the count is low, a greater area may be counted (e.g., 9 PROCEDURE:
mm2 ) to improve accuracy. 1. Make a 1:100 dilution by placing 20 mL of well-mixed
4. The chamber must be charged properly to ensure an blood into 1980 mL of 1% ammonium oxalate in a small test
tube.
accurate count. Uneven flow of the diluted blood into the
2. Mix the dilution thoroughly and charge the chamber. (Note:
chamber results in an irregular distribution of cells. If the A special thin, flat-bottomed counting chamber is used for
chamber is overfilled or underfilled, the chamber must be phase-microscopy platelet counts.)
cleaned and recharged. 3. Place the charged hemacytometer in a moist chamber (Box
5. After the chamber is filled, allow the cells to settle for 10 11.1) for 15 minutes to allow the platelets to settle.
minutes before counting. 4. Platelets are counted using the 40X objective lens (400X
6. Any nucleated red blood cells (NRBCs) present in the total magnification). The platelets have a diameter of 2 to 4
mm and appear round or oval, displaying a light purple sheen
specimen are not lysed by the diluting fluid. The NRBCs are
when phase-contrast microscopy is used. The shape and color
counted as WBCs because they are indistinguishable when help distinguish the platelets from highly refractile dirt and
seen on the hemacytometer. If five or more NRBCs per 100 debris. “Ghost” RBCs often are seen in the background.
WBCs are observed on the differential count on a stained 5. Count the number of platelets in the 25 small squares in the
peripheral blood film, the WBC count must be corrected for center square of the grid (Figure 11.1). The area of this center
these cells. This is accomplished by using the following square is 1 mm2. Platelets should be counted on each side of
formula: the hemacytometer, and the difference between the totals
should be less than 10%.
6. Calculate the platelet count by using one of the equations
given earlier. Using the first equation as an example, if 200
platelets were counted in the entire center square,
Report the result as the “corrected” WBC count.
7. The accuracy of the manual WBC count can be assessed by
performing a WBC estimate on a Wright-stained peripheral
blood film made from the same specimen
PLATELET COUNT
- Defined as: the number of platelets in 1 L or 1 mL of 7. The accuracy of the manual platelet count should be verified
whole blood by performing a platelet estimate on a Wright-stained
- Platelets - adhere to foreign objects and to each other, peripheral blood film made from the same specimen (Chapter
13).
which makes them difficult to count.
Sources of Error and Comments
o Also, they are small and can be confused easily with 1. Inadequate mixing and poor collection of the specimen can
dirt or debris. cause the platelets to clump on the hemacytometer. If the
- Specimen: Whole Blood problem persists after redilution, a new specimen is needed. A
- Anticoagulant: EDTA skin puncture specimen is less desirable because of the
- Dilution: 1:100 tendency of the platelets to aggregate or form clumps.
- Diluting Fluid: 1% Ammonium oxalate (to lyse the 2. Dirt in the pipette, hemacytometer, or diluting fluid may
nonnucleated RBCs cause the counts to be inaccurate.
- counted in the 25 small squares in the large center square 3. If fewer than 50 platelets are counted on each side, the
(1 mm2) of the hemacytometer using a phase-contrast procedure should be repeated by diluting the blood to 1:20. If
microscope (Brecher and Cronkite Method) more than 500 platelets are counted on each side, a 1:200
NOTE: A light microscope can also be used but visualizing the dilution should be made. The appropriate dilution factor should
platelets may be more difficult. be used in calculating the results.
PLATELET COUNT METHODS: INDIRECT OR DIRECT
INDIRECT 4. If the patient has a normal platelet count, the five small, RBC
- Platelets are counted in relation to 1000 RBCs in the blood smear squares (Figure 11.1) may be counted. Then the area is 0.2
- Not so reliable since results depends on the distribution of mm2 on each side.
platelets and on the RBC count
- Advantage: Allows study of plateletmorphology 5. The phenomenon of “platelet satellitosis” may occur when
- Indirect methods: EDTA anticoagulant is used.
o Dameshek’s o Platelet satelliteosis or satellitism - refers to the
o Fonio’s
adherence of platelets around neutrophils, producing a
o Olef’s – the best method in the indirect procedures but
somehow hard to do. ring or satellite effect
DIRECT o Using sodium citrate as the anticoagulant should
- Light Microscopy Method correct this problem. Because of the dilution in the
o Rees and Ecker’s – preserves RBC which may be counted
simultaneously; diluting fluid is composed of: citrate evacuated tubes, it is necessary to multiply the
❖ Brilliant Cresyl obtained platelet count by 1.1 for accuracy
❖ Blue Sodium RED BLOOD CELL COUNT
❖ Citrate Distilled Water - Rarely performed because of the inaccuracy of the count
o Guy and Leake’s - diluting fluid is composed of:
❖ Crystal Violet and questionable necessity.
❖ Sodium Citrate - Use of other, more accurate manual RBC procedures,
❖ Distilled Water such as the microhematocrit and hemoglobin
❖ Formalin (40%)
- Phase Contrast Microscopy: Brecher-Cronkite Method
Dearroz, John Rhenire P. | 3MT02
 concentration, is desirable when automation is not - Cyanmethemoglobin (hemiglobincyanide) method –
available. for hemoglobin determination is the reference method
Disposable Blood Cell Count Dilution Systems approved by CLSI
- Capillary pipette and diluent reservoir systems are o PRINCIPLE: blood is diluted in an alkaline Drabkin
commercially available for WBC and platelet counts. solution of potassium ferricyanide, potassium cyanide,
- Ex. Leuko Chek (Biomedical Polymers) - consists of a sodium bicarbonate and a surfactant.The hemoglobin
capillary pipette (calibrated to accept 20 mL of blood) that is oxidized to methemoglobin (Fe3+) by the potassium
fits into a plastic reservoir containing 1.98 mL of 1% ferricyanide, K3Fe(CN)6. The potassium cyanide (KCN)
buffered ammonium oxalate then converts the methemoglobin to
cyanmethemoglobin:

o ABSORBANCE: measured at 540nm, it is directly

proportional to the hemoglobin concentration
- NOTE: Sulfhemoglobin is not converted to
cyanmethemoglobin; it cannot be measured by this method.
However, sulfhemoglobin fractions of more than 0.05 g/dL are
seldom encountered in clinical practice
Sources of Error and Comments
1. Cyanmethemoglobin reagent is sensitive to light. It
should be stored in a brown bottle or in a dark place.
2. A high WBC count (greater than 20 X 109 /L) or a high
platelet count (greater than 700 X 109 /L) = turbidity and a
o Blood from a well-mixed EDTA-anticoagulated falsely high/elevated result.
specimen or from a skin puncture is allowed to enter SOLUTION: In this case the reagent specimen solution can be
the pipette by capillary action to the fill volume. centrifuged and the supernatant measured.
o Blood is added to the reservoir making a 1:100 dilution 3. Lipemia → can cause turbidity and a falsely high result.
o After mixing the reservoir and allowing 10 minutes for SOLUTION: It can be corrected by adding 0.01 mL of the
lysis of the RBCs, the reverse end of the capillary patient’s plasma to 5 mL of the cyanmethemoglobin reagent
pipette is placed in the reservoir cap making a dropper. and using this solution as the reagent blank.
o REMEMBER!! The first three or four drops of the 4. Cells containing Hemoglobin S (Hb S) and Hemoglobin C
diluted specimen are discarded and the capillary (Hb C) may be resistant to hemolysis, causing turbidity.
pipette is used to charge the hemacytometer !! SOLUTION: can be corrected by making a 1:2 dilution with
o Both WBC and platelet counts can be done from the distilled water (1 part diluted specimen plus 1 part water) and
same diluted specimen. multiplying the results from the standard curve by 2.
▪ WBCs are counted in all nine large squares (9 5. Abnormal globulins, such as those found in patients with
mm2) using low power (100X total magnification) plasma cell myeloma or Waldenström macroglobulinemia,
▪ Platelets are counted in the 25 small squares in may precipitate in the reagent.
the center square (1 mm2) using high power SOLUTION: Add 0.1 g of potassium carbonate to the
(400X total magnification) cyanmethemoglobin reagent. Commercially available
▪ The standard formula is used to calculate the cyanmethemoglobin reagent has been modified to contain KH2
cell counts. PO4 salt, so this problem is not likely to occur.
RED BLOOD CELL DILUTING FLUIDS – ideal is “isotonic” 6. Carboxyhemoglobin takes 1 hour to convert to
- Hayem’s – initiates mold formation and Rouleaux formation cyanmethemoglobin and theoretically could cause erroneous
- Gower’s – Prevents Rouleaux formation and precipitation of results in specimens from heavy smokers. The degree of error
proteins in cases of hyperglobulinemia and hemoglobinemia
- Toisson’s – Initiates mold formation so it should be filtered. is probably not clinically significant, however.
o With High Specific Gravity 7. Because the hemoglobin reagent contains cyanide, it is
o With stains so it is used by beginners (RBCs can be highly toxic and must be used cautiously. Consult the safety
easily differentiated from WBCS [Nuclei-Blue])
data sheet supplied by the manufacturer. Acidification of
o Stain used: Methyl Violet
- Bethel’s cyanide in the reagent releases highly toxic hydrogen cyanide
- Formol-Citrate (Dacie’s Fluid) gas. A licensed waste disposal service should be contracted to
o Best RBC diluting fluid discard the reagent; reagent-specimen solutions should not be
o With best preservative action so no mold formation discarded into sinks.
o The cell’s morphology is not changed.
8. Commercial absorbance standards kits are available to
- NSS – Ideal to use in case of excessive Rouleaux Formation and
Autoagglutination calibrate spectrophotometers.
- 3.8% Sodium Citrate 9. Handheld systems are commercially available to measure
the hemoglobin concentration.
HEMOGLOBIN DETERMINATION
- Hemoglobin – primary function is to carry oxygen to and
carbon dioxide from the tissues.
Dearroz, John Rhenire P. | 3MT02
 o An example is the
HemoCue8,9 (HemoCue) in
which hemoglobin is
converted to
azidemethemoglobin and is
read photometrically at two
wavelengths (570 nm and
880 nm). This method
avoids the necessity of specimen dilution and
interference from turbidity.
o Another method that has been used in some
automated instruments involves the use of sodium
lauryl sulfate (SLS) to convert hemoglobin to SLS-
methemoglobin. Sources of Error and Comments
▪ ADVANTAGE: does not generate toxic wastes. 1. Improper sealing of the capillary tube causes a decreased
MICROHEMATOCRIT hematocrit reading as a result of leakage of blood during
- Hematocrit / Packed Cell Volume (PCV) - the volume of centrifugation. A higher number of RBCs are lost compared
packed RBCs that occupies a given volume of whole with plasma because of the packing of the cells in the lower
blood. part of the tube during centrifugation.
o reported either as a percentage (e.g., 36%) or in liters 2. An increased concentration of anticoagulant (short draw in
per liter (0.36 L/L). an evacuated tube) decreases the hematocrit reading as a
PRCEDURE: result of RBC shrinkage.
1. Fill two plain capillary tubes approximately three-quarters full 3. A decreased or increased result may occur if the specimen
with blood anticoagulated with EDTA or heparin.
was not mixed properly.
Mylarwrapped tubes - are recommended by the National
Institute for Occupational Safety and Health (NIOSH) to 4. The time and speed of the centrifugation and the time when
reduce the risk of capillary tube injuries. the results are read are important. Insufficient centrifugation or
Alternatively, blood may be collected into heparinized capillary a delay in reading results after centrifugation causes
tubes by skin puncture. Wipe any excess blood from the hematocrit readings to increase. Time for complete packing
outside of the tube. should be determined for each centrifuge and rechecked at
2. Seal the end of the tube with the colored ring using regular intervals. When the microhematocrit centrifuge is
nonabsorbent clay. Hold the filled tube horizontally and seal by
calibrated, one of the specimens used must have a hematocrit
placing the dry end into the tray with sealing compound at a
90-degree angle. Rotate the tube slightly and remove it from of 50% or higher.
the tray. The plug should be at least 4 mm long. 5. The buffy coat of the specimen should not be included in the
3. Balance the tubes in a microhematocrit centrifuge with the hematocrit reading because this falsely elevates the result.
clay ends facing the outside away from the center, touching the 6. A decrease or increase in the readings may be seen if the
rubber gasket. microhematocrit reader is not used properly.
4. Tighten the head cover on the centrifuge and close the top. 7. Many disorders, such as sickle cell anemia, macrocytic
Centrifuge the tubes at 10,000 g to 15,000 g for the time that
anemias, hypochromic anemias, spherocytosis, and
has been determined to obtain maximum packing of RBCs. Do
not use the brake to stop the centrifuge thalassemia, may cause plasma to be trapped in the RBC layer
5. Determine the hematocrit by using a microhematocrit even if the procedure is performed properly. The trapping of
reading device (Figure 11.7). Read the level of RBC packing; the plasma causes the microhematocrit to be 1% to 3% (0.01
do not include the buffy coat (WBCs and platelets) when to 0.03 L/L) higher than the value obtained using automated
taking the reading. instruments that calculate or directly measure the hematocrit
6. The values of the duplicate hematocrits should agree within
and are unaffected by the trapped plasma. 8. A temporarily low
1% (0.01 L/L).
Determining Maximum Packing Time for Microhematocrit hematocrit reading may result immediately after a blood loss
The time to obtain maximum packing of red blood cells should because plasma is replaced faster than are the RBCs.
be determined for each centrifuge. Duplicate microhematocrit 9. The fluid loss associated with dehydration causes a
determinations should be made using fresh, well-mixed blood decrease in plasma volume and falsely increases the
anticoagulated with ethylenediaminetetraacetic acid (EDTA). hematocrit reading.
Two specimens should be used, with one of the specimens 10. Proper specimen collection is an important consideration.
having a known hematocrit of 50% or higher. Starting at 2
The introduction of interstitial fluid from a skin puncture or the
minutes, centrifuge duplicates at 30-second intervals and
record results. When the hematocrit has remained at the same improper flushing of an intravenous catheter causes decreased
value for two consecutive readings, optimum packing has been hematocrit readings.
achieved, and the second time interval should be used for READACRIT centrifuge (Becton, Dickinson)
microhematocrit determinations - uses precalibrated capillary tubes and has built-in
hematocrit scales, which eliminates the need for separate
reading devices
- use of SUREPREP Capillary Tubes (Becton, Dickinson)
eliminates the use of sealants as they have a factory-

Dearroz, John Rhenire P. | 3MT02

 inserted plug that seals automatically when the blood - served as quality control check and may be used for initial
touches the plug. classification of anemias
RULE OF THREE
- Specimens are analyzed by automated or manual
methods, a quick visual check of the results of the
hemoglobin and hematocrit can be done by applying the
“rule of three.”
- applies only to specimens that have normocytic
normochromic RBCs
- States that: the value of the hematocrit should be three
times the value of the hemoglobin plus or minus 3: Mean Cell Volume (MCV)
Hemoglobin (HGB) X 3 = Hematocrit (HCT) +/- 3 (0.03 - the average volume of the RBC, expressed in femtoliters
L/L). (fL), or 10-15 L:
- It should become habit for the analyst to multiply the
hemoglobin by 3 mentally for every specimen.
- Value discrepant with this rule may indicate abnormal
RBCs, or it may be the first indication of error.

- REFERENCE INTERVAL: 80 to 100 fL

- Less than 80 fL (<80 fL): Microcytic
- Greater than 100 fL(>100 fL) : Macrocytic
Mean Cell Hemoglobin (MCH)
- the average weight of hemoglobin in a RBC, expressed in
picograms (pg), or 10-12 g:

- REFERENCE INTERVAL: 26 – 32 pg
- Not considered in classification of anemias.
Mean Cell Hemoglobin Concentration (MCHC)
- the average concentration of hemoglobin in each
individual RBC.
- Unit: grams per deciliter (formerly percentage)

- If values do not agree, the blood film should be examined

for abnormal RBCs → causes of false increases and
decreases in the hemoglobin or hematocrit values should
- REFERENCE INTERVAL: 32 to 36 g/dL
also be investigated.
- Less than 32 g/dL (<32g/dL): hypochromic
- If RBCs do appear normal, possible causes of a falsely
- Greater than 36 g/dL (>36g/dL): hyperchromic
low hemoglobin concentration or a falsely elevated
- Hypochromic RBCs occur in: thalassemias, iron
hematocrit should be investigated.
deficiency, and other conditions
- When an unexplained discrepancy is found, the specimen
- Hyperchromic RBCs – “misnomer”; cell does not really
analyzed before and after the specimen in question should
contain more than 36 g/dL of hemoglobin, but its shape
be checked to determine whether their results conform to
may have become spherocytic, which makes the cell
the rule
appear full.
- A control specimen should be analyzed when such a
- 36 to 38 g/dL → should be checked for “spherocytes”
discrepancy is found.
- Presence of cell agglutinin → increased MCHC
o If appropriate results are obtained for the control,
random error may have occurred
RETICULOCYTE COUNT
RED BLOOD CELL INDICES - Reticuloyte - the last immature RBC stage, spends 2
- Includes: Mean Cell Volume (MCV), Mean Cell
days in the bone marrow and 1 day in the peripheral blood
Hemoglobin (MCH), Mean Cell Hemoglobin
before developing into a mature RBC (normally).
Concentration (MCHC)
- contains remnant cytoplasmic ribonucleic acid (RNA) and
- are calculated to determine the average volume and
organelles such as the mitochondria and ribosomes
hemoglobin content and concentration of the RBCs in the
- Reticulocyte count: used to assess the erythropoietic
specimen
activity of the bone marrow.

Dearroz, John Rhenire P. | 3MT02

- PRINCIPLE: Whole blood, anticoagulated with EDTA, is MILLER DISC
stained with a supravital stain, such as new methylene - Miller disc was designed to reduce labor intensive process
blue. Any nonnucleated RBC that contains two or more of large numbers of RBCs to be counted to obtain a more
particles of blue-stained granulofilamentous material after precise reticulocyte count
new methylene blue staining is defined as a reticulocyte - composed of two squares, with the area of the smaller
square measuring 1 ⁄ 9 the area of the larger square.
- inserted into the eyepiece of the microscope and the grid
is seen (Figure 11.11)
- RBCs are counted in the smaller square, and reticulocytes
are counted in the larger square. . 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
- CAP Standard: Reticuloyte count based on 1000 red cells
- Formula for calculation:

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 RBCs, 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 RBC inclusions that stain supravitally include Heinz,
Howell-Jolly, and Pappenheimer bodies (HHP).
o Heinz bodies - are
precipitated hemoglobin,
usually appear round or
oval, and tend o adhere to
the cell membrane.
o Howell-Jolly bodies - are
round nuclear fragments
and are usually singular.
o Pappenheimer bodies -
are iron in the mitochondria
whose presence can be
confirmed with an iron
stain, such as Prussian
blue.
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 11.2; significant bias is observed if the rule is ignored.
ABSOLUTE RETICULOCYTE COUNT
- PRINCIPLE: The absolute reticulocyte count (ARC) is the
actual number of reticulocytes in 1 L or 1 mL of blood.
Dearroz, John Rhenire P. | 3MT02
 - Calculated as follows:

- The ARC can also be reported as the number of cells per

mL.
- REFERENCE INTERVAL: 20 X 109 /L – 115 X 109 /L
- REFERENCE INTERVAL:
CORRECTED RETICULOCYTE COUNT
- PRINCIPLE: specimens with a low hematocrit, the o >3 RPI = adequate bone marrow response
percentage of reticulocytes may be falsely elevated o <2 RPI = inadequate erythropoietic response
because the whole blood contains fewer RBCs RETICULOCYTE CONTROL
- Commercial controls available for monitoring manual
o A correction factor is used, with the average normal
and automated reticulocyte counts: (e.g., Retic-Chex II,
hematocrit considered to be 45%.
Streck Laboratories; Liquichek Reticulocyte Control [A],
BioRad Laboratories).
- Most are available at THREE levels
- The control specimens are treated in the same manner as
the patient specimens.
- Used to verify the laboratorian’s accuracy and precision
when manual counts are performed.
- REFERENCE INTERVAL:
AUTOMATED RETICULOCYTE COUNT
o Hematocrit of 35% = elevated corrected reticulocyte - All the analyzers evaluate reticulocytes using optical
count of 2% to 3% to compensate for the mild anemia. scatter or fluorescence after the RBCs are treated with
o Hematocrit <25% = ount should increase to 3% to fluorescent dyes or nucleic acid stains to stain residual
5% to compensate for the moderate anemia. RNA in the reticulocytes.
o NOTE: corrected reticulocyte count depends on the - results are statistically more valid because of the large
degree of anemia number of cells counted.
RETICULOYTE PRODUCTION INDEX - percentage and the absolute count are provided.
- PRINCIPLE: Reticulocytes that are released from the
- a maturation index/immature reticulocyte fraction or
marrow prematurely are called shift reticulocytes.
IRF - reflecting the proportion of the more immature
- Shift reticulocytes - reticulocytes are “shifted” from the
reticulocytes in the specimen
bone marrow to the peripheral blood earlier than usual to
o may be especially useful in detecting early
compensate for anemia
erythropoietic activity after chemotherapy or
o these cells take 2 to 3 days to lose their reticula
hematopoietic stem cell transplantation
(normally 1 day, cell loses reticulum)
- Reticulocyte hemoglobin – useful to detect early iron
- When erythropoiesis is evaluated, a correction should be
deficiency
made for the presence of shift reticulocytes if
ERYTHROCYTE SEDIMENTATION RATE
polychromasia is reported in the RBC morphology. - ordered with other tests to detect and monitor the course
- Cells shifted to the peripheral blood prematurely stay of inflammatory conditions such as rheumatoid arthritis,
longer as reticulocytes and contribute to the reticulocyte infections, or certain malignancies
count for more than 1 day → reticulocyte count is falsely - useful in the diagnosis of temporal arteritis and
increased when polychromasia is present
polymyalgia rheumatica.
- The patient’s hematocrit is used to determine the - NOT a specific test for inflammatory diseases as it is
appropriate correction factor (reticulocyte maturation time elevated in other conditions such as plasma cell
in days): myeloma, pregnancy, anemia, and older age
- also prone to technical errors that can falsely elevate or
decrease the sedimentation rate.
- Low specificity and sensitivity → not recommended as a
screening test to detect inflammatory conditions in
asymptomatic individuals
- PRINCIPLE: Anticoagulated blood is allowed to stand at
room temperature undisturbed for a period of time, the
RBCs settle toward the bottom of the tube.
-
Dearroz, John Rhenire P. | 3MT02
 o ESR - is the distance in millimeters that the RBCs fall WINTHROBE ERYTHROCYTE SEDIMENTATION RATE
in 1 hour. - When it was first introduced, the specimen was an
o Affected by RBC, plasma, and mechanical and oxalate-anticoagulated whole blood placed in a 100-mm
technical factors. column
o RBCs have a net negative surface charge and tend to - Today, EDTA-treated or citrated whole blood is used with
repel one another; repulsive forces are partially or the shorter column
totally counteracted if there are increased quantities of o Shorter column → increased sensitivity in detecting
positively charged plasma proteins. mildly elevated ESRs
o ESR is directly proportional to the RBC mass and Sources of Error and Comments
inversely proportional to plasma viscosity. 1. If the concentration of anticoagulant is increased, the
MODIFIED WESTERGREN ERYTHROCYTE ESR will be falsely low as a result of sphering of the RBCs,
SEDIMENTATION RATE which inhibits rouleaux formation.
- Modified Westergren Method - most commonly used 2. The anticoagulants sodium or potassium oxalate and
method today heparin cause the RBCs to shrink and falsely elevate the
- ADVANTAGE: taller column height allows the detection of ESR.
highly elevated ESRs 3. A significant change in the temperature of the room alters
- Method recommended by the (1) International Council the ESR.
for Standardization in Hematology and the (2) CLSI. 4. Even a slight tilt of the pipette causes the ESR to
PROCEDURE: increase.
1. Use well-mixed blood collected in EDTA and dilute at four 5. Blood specimens must be analyzed within 4 hours of
parts blood to one part 3.8% sodium citrate or 0.85% collection if kept at room temperature (18° to 25° C). If the
sodium chloride (e.g., 2 mL blood and 0.5 mL diluent).
specimen is allowed to sit at room temperature for more than 4
Alternatively, blood can be collected directly into special
sedimentation test tubes containing sodium citrate. Standard hours, the RBCs start to become spherical, which may inhibit
coagulation test tubes are not acceptable because the dilution the formation of rouleaux. Blood specimens may be stored at
is nine parts blood to one part sodium citrate.19 4° C up to 24 hours before testing, but must be rewarmed by
2. Place the diluted specimen in a 200-mm column with an holding the specimen at ambient room temperature for at least
internal diameter of 2.55 mm or more. 15 minutes before testing.
3. Place the column into the rack and allow to stand 6. Bubbles in the column of blood invalidate the test
undisturbed for 60 minutes at room temperature (18° to 25°
results.
C). Ensure that the rack is level.
4. Record the number of millimeters the RBCs have fallen in 1 7. The blood must be filled properly to the zero mark at the
hour. The buffy coat should NOT be included in the reading. beginning of the test.
8. A clotted specimen cannot be used.
9. The tubes must not be subjected to vibrations on the
laboratory 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.
ELEVATED INCREASED
• Pregnancy (after 3rd • Polycythemia
month) • CHF hypofibrinogenemia
• Acute/chronic infection • Presence of RBC
Rheumatic fever abnormalities (poikilocytosis,
• MI spherocytes, sickle cells)
• Nephrosis
• Acute Hepa
• Menstruation
• Tuberculosis
• Macroglobulinemia
• Cryoglobulinemia
• Hypo/hyperthyroidism

FACTORS AFFECTING ESR

INCREASED / FASTER DECREASED /
SLOWER
RBCs • Macrocytes • Microcytes
• Anemia • Poikilocytes
• Anisocytes
• Polycythemia

Dearroz, John Rhenire P. | 3MT02

Plasma • Fibrinogen • Albumin • Provide on-board mixing and sampling of patient’s
Composition • Alpha 1 globulin • Lecithin specimen
• Alpha 2 globulin
• Minimize handling of blood specimens
• Cholesterol
• Incorporate a quality control (QC) system
Technical / • Tilting (3 degrees • Increase EDTA • Include capability of transmitting data to the laboratory
Extrinsic appx. 30% error) (shrinkage of cell) information system
Factors • Increase temperature • Delay (more than
• Provide results within a shorter turnaround time (TAT)
• Longer tube = faster 2 hoiurs) – RBCs
ESR become more - Features to consider:
• Wet glassware → spherical → ✓ Size of the analyzer
Hemolysis → faster slower ✓ Required sample volume
ESR
✓ specimen identification,
• Bubbles (not
accepted) ✓ walkaway and continuous process flow
✓ testing time
STAGES OF ERYTHROCYTE SEDIMENTATION: ✓ quality control program
INITIAL ROULEAUX 10 mins ✓ interface capabilities.
FORMATION ADDITIONAL TESTS
RAPID SETTLING OF RBCs 40 mins - osmotic fragility test
FINAL SEDIMENTATION OF 10 mins - qualitative and quantitative assays for glucose-6-
RBCs phosphate dehydrogenase and pyruvate kinase activity
TOTAL: 60 mins - solubility test for Hb S
- hemoglobin electrophoresis
METHODS:
- unstable hemoglobin test
1. Standard/Original Westergren
• Anticoagulant: Citrate (black) 1:4 - vital stain for hemoglobin H
• Length tube: 30 cm - Kleihauer-Betke acid elution test for Hb F distribution in
• Bore: 2.5 mm the RBCs
POINT-OF-CARE TESTING
2. Modified Westergren - Offers the ability to produce rapid and accurate results that
• Anticoagulant: EDTA (2mL EDTA + 0.5mL NSS/Citrate) facilitate faster treatment, which can decrease hospital
3. Wintrobe and Landsberg length of stay
• Anticoagulant: double oxalate or balance oxalate - rarely performed by trained laboratory personnel; most
• Length: 11.5 cm often, it is carried out by nurses
• Bore: 2.0 mm - Point-of-care testing: defined as diagnostic testing at or
near the site of patient care.
- Clinical Laboratory Improvement Amendments of 1988
INCREASED ESR NORMAL ESR (CLIA) → introduced the concept of “testing site
Inflammatory conditions Polycythemia neutrality,” which means that regardless of where the
Acute/chronic infections Spherocytes diagnostic testing is performed or who performs the test,
Rheumatic fever Sickle cells
all testing sites must follow the same regulatory
RA and MI Sickle cell anemia
Nephrosis Tuberculosis MM Hb CC disease requirements based on the “complexity” of the test.
Waldenstrom’s macro. SBE; Hereditary spherocytosis - POC testing is classified as “waived” or “moderately
Hepa complex.”
Menstruation o Classified as waived → if they are determined to be
Pregnancy “simple tests with an insignificant risk of an erroneous
result.
DISPOSABLE KITS - Commonly performed in hospital inpatient units, outpatient
- Several kits include safety caps for the columns that allow clinics, surgery centers, emergency departments, long-
the blood to fill precisely to the zero mark term care facilities, and dialysis units.
- Safety cap makes the column a closed system and - POC testing program must incorporate all of the following:
eliminates the error involved in manually setting the blood o written policy that defines the program, outlines who
to the zero mark. is responsible for each part of the program, policy
AUTOMATED ERYTHROCYTE SEDIMENTATION RATE indicates where testing is performed and who performs
- several automated ESR systems available using the the testing
traditional Westergren and Wintrobe methods, as well as o Testing procedures should clearly state how to
alternate methods such as centrifugation and capillary perform the tests and address how to handle critical
photometry. values and discrepant results.
- Automated ESR systems usually do the following: o Program should be monitored
• Include specimen identification capability o An ongoing evaluation of the POC testing is vital for
• Require a smaller sample volume success.

Dearroz, John Rhenire P. | 3MT02

- The POC testing system should also address the Hemoglobin Concentration
following laboratory concerns: - Hemoglobin concentration is measured by modified
✓ What is the range of measurement? hemoglobinometers or by oximeters integrated with a
✓ How well does the test system correlate with blood gas analyzer.
laboratory instrumentation? - HemoCue hemoglobinometer (HemoCue) - uses a
✓ Can it be interfaced to the laboratory information small cuvette that contains a lysing agent and reagents to
system? form a hemoglobin azide, which is measured by a
✓ Does it provide reliable results? photometer at two wavelengths (570 nm and 880 nm)
✓ Does the company supply excellent technical o This eliminates interference from turbidity in the
support? specimen.
✓ Is it affordable? o Results obtained with the instrument compare well with
- Paramount to POC testing is patient safety. those produced by reference methods
- It is important to maintain good laboratory practices, which o MAJOR SOURCE OF ERROR: mixing of blood with
include use of appropriate procedures for specimen tissue fluid during skin puncture collection
collection, identification of the patient and specimen, - AVOXimeter 1000E (Instrumentation Laboratory) -
storage of reagents, instrument maintenance, and measures total hemoglobin by a spectrophotometric
accurate documentation of patient test results (use of POC method.
interfaces to the laboratory information system and Cell and Platelet Counts
electronic medical record is beneficial) - Traditional cell-counting methods can be employed at the
POINT-OF-CARE TESTS point of care for the analysis of WBCs, RBCs, and
Hematocrit platelets.
- most common methods for determining the hematocrit - Ichor Hematology Analyzer (Helena Laboratories) -
include the microhematocrit centrifuge, conductometric performs a CBC along with platelet aggregation.
methods, and calculation by automated cell counters - Buffy coat analysis method → employs cell differentiation
- Centrifuge-based microhematocrit systems have been and quantification
available for years, and the results obtained correlate well - Quantitative buffy coat analysis (QBC STAR,
with the results produced by standard cell counters. manufactured by QBC Diagnostics, Inc., Philipsburg,
- One example of a centrifuge-based device is the PA) - involves centrifugation in specialized capillary tubes
Hematastat II Separation Technology designed to expand the buffy coat layer
- The i-STAT 1 (Abbott Laboratories) and the Epoc o Components (platelets, mononuclear cells, and
(Siemens Healthineers) use the conductivity method to granulocytes) can be measured with the assistance of
determine the hematocrit. Plasma conducts electrical fluorescent dyes and a measuring device.
current, whereas WBCs act as insulators
- In the i-STAT system, before the measured conductance
is converted into the hematocrit value, corrections are
applied for temperature of the specimen, size of the fluid
segment being measured, and relative conductivity of the
plasma component
- The first two corrections are determined from the (1)
measured value of the calibrant conductance and the
(2) last correction from the measured concentrations
of sodium and potassium in the specimen.
Sources of Error and Comments
- Conductivity of a whole blood specimen is dependent on
the amount of electrolytes in the plasma portion.
- Conductivity does not distinguish RBCs from other
nonconductive elements such as proteins, lipids, and
WBCs that may be present in the specimen.
- A low total protein level will falsely decrease the
hematocrit.
- The presence of lipids can interfere with the hematocrit
measurement.
- An increased WBC count will falsely increase the
hematocrit.
- The presence of cold agglutinins can falsely decrease
the hematocrit.
Other Instruments for Hematocrit measurement:
• ABL 77 (Radiometer)
• IRMA (Accriva, a subsidiary of LifeHealth)
• Gem Premier (Instrumentation Laboratory Company
Dearroz, John Rhenire P. | 3MT02