Kathleen Kelly B.S., MT(ASCP)
Kathleen Kelly B.S., MT(ASCP)
1. State the preferred anticoagulant for most hematologic testing.
2. Discuss the most common principles utilized in table top
hematology analyzers.
a. Cell counts
b. Hemoglobin
c. Hematocrit
3. Briefly discuss sources of error in CBC results.
a. Cell counts
b. Hemoglobin
c. Hematocrit
4. Correlate hemoglobin and hematocrit results.
5. Discuss the use and reliability of hemacytometer cell counts, centrifuged hematocrits, and alternative hemoglobin procedures.
6. Discuss the centrifuged hematocrit, including sources of error, preventive maintenance, and quality control.
7. Discuss the differences between automated and centrifuged hematocrits.
8. Discuss the derivation and use of red cell indices.
Kathleen Kelly B.S., MT(ASCP)
Simple equipment that requires minimal operator intervention has made a complete blood count (CBC) readily available. The through put of these instruments is fast enough to give cell counts, indices, hemoglobin, hematocrit, and even a differential in the same time it takes to perform a manual hematocrit. Most of the instruments currently marketed to physician office laboratories are specifically designed to be user friendly and can be purchased with ready to use charts and procedures for the required preventive maintenance and quality assurance.
This unit will discuss the hematology testing most often done in a physician office laboratory. The intent is to discuss general information on instrumentation and commonly performed tests. Specific manufacturer's information will not be included. This can be obtained from the individual manufacturer.
Kathleen Kelly B.S., MT(ASCP)
Bullet tubes, with EDTA, are available for collecting blood from skin punctures. The bullet tube must fill quickly and the blood mixed with the EDTA before coagulation begins. The quality of the skin puncture sample is very technique dependent.
Samples collected in EDTA must be well mixed at the time of draw and before sampling. The correct mixing time before sampling is five minutes on an aliquot mixer or, if they are mixed manually, a minimum of sixty inversions. However, avoid over mixing. Prolonged mixing on aliquot mixers causes hemolysis.
As a safety precaution, always open evacuated tubes under a
protective shield. 
Residual vacuum causes aerosols.
Brunson D, Smith D, Bak A, et al. Comparing hematology aK2EDTA vs. K3EDT. Laboratory Hematology 1995, 1:112-119.
Kathleen Kelly B.S., MT(ASCP)
These are examples of freestanding protective shields. They are available through laboratory supply and safety product distributors. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
A. Table Top Analyzers
These analyzers vary in the extent of the automation involved. The
simplest analyzers require the operator to mix the sample and present
an open sample tube to the instrument. Sampling units that pierce the
cap of the tube save time, are readily available, and are safer. The
more sophisticated systems read bar coded labels, mix, and sample
through the cap of the tube. Skin puncture samples may need to be
diluted and/or presented to a different sampling probe. The ease of
testing skin puncture samples and 2 or 3 ml tubes may be an important
consideration in choosing an instrument. The top of the line
hematology systems can make peripheral smears, store thousands of
results, and store and manipulate quality control data. Most of the
instruments can be interfaced with a laboratory information system.
The following URLs will connect to some of the manufacturers of table top hematology analyzers.
1. http://www.beckmancoulter.com/products/instrument/hematology/actseries.asp
Beckman Coulter
2. http://www.bayerdiag.com/
Bayer Diagnostics
3. http://www.sysmex.com/usa/ourbusiness/ourbusiness.cfm?dis_id=1
Dade Sysmex
Kathleen Kelly B.S., MT(ASCP)
Red cell histograms 
derived from these counts have a small tail to the right of the
curve. This represents coincidence, which is multiple cells passing
through the aperture at the same time. The correction for coincidence
takes place in the cell count calculation and is not a concern.
The other basic counting principle is based on light scatter. Here, a single cell passes through a beam of light from a laser or tungsten-halogen light source. The cell is counted as it breaks the beam of light and the light is scattered. The scattered light is measured and translated into cell size. The counts are reported in units per volume of whole blood.
Some hematology instrumentation prints the red cell and platelet histograms as part of the report. These histograms give a visual interpretation of the cell population and correlate with the indices.
A third type of instrument is advertised as a dry system that works by centrifugation. It generates the cell counts, hemoglobin, and hematocrit from a centrifuged specimen. These are light, portable instruments made by Becton Dickinson. The rest of this discussion does not apply to this type of instrument.
Sources of error in cell counts include:
1. Cold agglutinins - low red cell counts and high MCVs can be caused by a decreased number of large red cells or red cell
agglutinates. 
If agglutinated red cells are present, the automated hematocrits and
MCHCs are also incorrect. Cold agglutinins cause agglutination of the
red cells as the blood cools. Cold agglutinins can be present in a
number of disease states, including infectious mononucleosis and
mycoplasma pneumonia infections. If red cell agglutinates are seen on
the peripheral smear, warm the sample in a 37 degrees C heating block
and mix and test the sample while it is warm. Strong cold agglutinins
may not disperse and need to be redrawn in a pre-warmed tube and kept
at body temperature.
2. Fragmented or very microcytic red cells 
these may cause red cell counts to be decreased and may flag the
platelet count as the red cells become closer in size to the
platelets and cause an abnormal platelet histogram. The population is
visible at the left side of the red cell histogram and the right end
of the platelet histogram. 
3. Platelet clumps 
and platelet satellitosis 
- these cause falsely decreased platelet counts. Platelet clumps can
be seen on the right side of the platelet histogram. Decreased
platelet counts are confirmed by reviewing the peripheral smear.
Always scan the edge of the smear when checking low platelet counts.
4. Giant platelets 
- these are platelets that approach or exceed the size of the red
cells. They cause the right hand tail of the histogram to remain
elevated and may be seen at the left of the red cell histogram. 
5. Nucleated red blood cells 
-
these interfere with the WBC on some instruments by being counted as
white cells/lymphocytes
Kathleen Kelly B.S., MT(ASCP)
Red cell histograms are derived by plotting the size of each red cell on x axis and the relative number on the y axis. They are used to determine the average size, distribution of size, and to detect subpopulations. This histogram represents a normal red cell distribution. The small tail to the right of the curve represents coincidence, multiple cells passing through the aperture at the same time. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
The arrow points to a red cell agglutinate. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
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Kathleen Kelly B.S., MT(ASCP)
The black curves on the red cell and platelet histograms indicate ìexpectedî or normal cell distributions. The red curves demonstrate the effect of very microcytic red cells on the histograms. Since microcytic red cells only effect the right end of the platelet curve, the black and red lines are superimposed for most of the curve. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
There is a large clump of platelets in the center of the field. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
The neutrophils in the center of the field are surrounded by platelets. This is an uncommon phenomenon seen in EDTA samples. The platelets surround the neutrophils and "stick". This results in a false decrease in the platelet count. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
Platelets that are 6 microns in diameter or larger, are considered giant platelets. Multiple giant platelets are visible in this field. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
The black curves on the red cell and platelet histograms indicate "expected" or normal cell distributions. The red curves demonstrate the effect of giant platelets on the red cell and platelet histograms. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
The two nucleated cells are immature red cells that have been released from the bone marrow ahead of schedule. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
Red cells are lysed and potassium ferricyanide oxidizes hemoglobin to methemoglobin, which combines with potassium cyanide forming cyanmethemoglobin. The brown color is measured spectrophotometrically and the corresponding hemoglobin reported. The end point of the reaction is stable and the reaction is linear to 20 g/dL or higher. Reagents for cyanmethemoglobin are light sensitive and poisonous.
However, the Sysmex systems can use a sulfhemoglobin methodology to measure total hemoglobin. The reagent sodium lauryl sulfate disrupts the red cell membrane and binds to the globin chains, causing methemoglobin to be formed. This binds to the sulfate group producing a conjugate with an absorption peak at 535 nm. No hazardous waste is produced.
Common sources of error in measuring hemoglobin include anything that will cause turbidity and interfere with a spectophotometric method. Examples are a very high WBC or platelet count, lipemia and hemoglobins that are resistant to lysis, such as hemoglobins S and C. The hemoglobin will be falsely increased with turbidity. Manufacturer's guidelines address interferences.
A simple method for obtaining a hemoglobin value from a lipemic sample is to use plasma replacement.
Kathleen Kelly B.S., MT(ASCP)
Hematocrits calculated by automated instruments depend on correct red cell counts and red cell volumes to arrive at an accurate hematocrit. Hence, anything affecting the red cell count or volume measurement will affect the hematocrit. This method is not as sensitive to the ratio of blood to EDTA as the centrifuged hematocrit. Since the red cells are resuspended in isotonic saline, they regain their normal shape and size. The practical side of this is that a reliable automated hematocrit can frequently be obtained from a short sample.
It is very useful to have a hematocrit centrifuge for back up. It can be used when the main instrument is down and to trouble shoot or check hemoglobins that do not correlate with the other results.
Kathleen Kelly B.S., MT(ASCP)
Example:
14.8 x 3 = 44 (patient's hematocrit result is 45 L/L)
11.0 x 3 = 33 (patient's hematocrit result is 32 L/L)
The exception to this rule is in patients with hypochromic red
cells. 
These patients will have hematocrits that are more than three times
the hemoglobin.
Kathleen Kelly B.S., MT(ASCP)
Hypochromic red cells have an increased zone of pallor on Wright's stain. These cells contain decreased hemoglobin for their size and have a decreased MCHC. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
Understanding how the automated hematocrit is derived is very important when trouble shooting or using back up methods such as a centrifuged hematocrit. If the hematocrits appear incorrect on the automated equipment, it means that the MCV and/or red cell count are also suspect. If a centrifuged hematocrit is substituted for an automated value, the MCV must be manually calculated.
Kathleen Kelly B.S., MT(ASCP)
Errors occur in all three steps of the procedure; diluting, plating, and counting. Dilution errors occur when the pipettes are under-filled, not wiped correctly, or blood is left in the neck of the reservoir. Plating errors include errors in expelling well-mixed sample, filling technique, and handling the hemacytometer between filling and counting. Scanning each chamber for even cell distribution before it is counted can help identify some errors. Counting errors include choosing an inappropriate area to count based on the number of cells present, improper counting technique, missing cells, counting junk, and misidentifying cells. Manual WBC and platelet counts should be confirmed with an estimate performed on a Wright's stained peripheral smear.
Manual red counts are inaccurate and imprecise. The hemoglobin and/or hematocrit are usually adequate if a CBC is not available.
Using manual cell counts for back up is usually more trouble than it is worth. Besides requiring excellent technique, UNOPETTES® outdate and are expensive. The procedure is time consuming. In a pinch it may be preferable to look at a well prepared, stained peripheral smear. An increase in white cells, the presence of immature cells, or reactive lymphocytes may answer the immediate question.
Quality control of manual cell counts is time consuming. At least two levels of controls for all cell types counted must be counted on every shift and the diluent from the vials must be examined in the hemacytometer to ensure that there is nothing contaminating the diluent that might be counted as a cell. It is also necessary to do counts on proficiency samples and to ensure that everyone who is performing counts is getting equivalent results, i.e., competency testing.
Hemacytometers are necessary for body fluid counts. Laboratories that examine synovial and other body fluids may perform cell counts as well as a crystal analysis performed on a polarized microscope.
In summary, if you do not do manual cell counts on blood samples, do not start.
Kathleen Kelly B.S., MT(ASCP)
A centrifuged hematocrit is also called a packed cell volume
(PCV). As in the automated hematocrit, a hematocrit is the volume of
the red cells as compared to the volume of the whole blood sample and
is reported in L/L or as a %. The packed cell volume is determined by
centrifuging the specimen in capillary tubes and measuring the height
of the red cell column. 
The sample is venous blood drawn in EDTA or capillary blood
collected in heparinized (red banded) microhematocrit tubes. A short
EDTA sample will have an increased anticoagulant to red cell ratio,
which causes the red cells to shrink and the hematocrit to be falsely
decreased. EDTA must not exceed 2 mg/ml of whole blood. For EDTA
samples use blue banded, plain capillary tubes. Traditional tubes are
glass and unplugged. Mylar coated tubes, plastic tubes, and
pre-plugged tubes are available. Plastic pre-plugged tubes are a good
choice. They decrease the risk of breaking a tube and puncuturing the
finger of the person performing the test and avoid some sources of
error. 
To perform a microhematocrit:
1. Fill two red or blue banded capillary tubes, from the end without the colored band, 1/2 to 2/3 full. Tilt each tube so
that the blood is near the colored band. Hold the tube horizontally
and wipe all of the excess blood off of the tubes before it dries. Be
careful not to wipe across the end of the tube. Absorbent material
will pull out more plasma than cells.
2. For unplugged tubes, hold each tube horizontally and seal the
end with the colored band by inserting it into the clay. 
This is the fire-polished end. Add the sealant until it is just above
or below the colored band. Filling and sealing this way keeps the
clay from becoming contaminated with blood, helps prevent leakage, is
safer for the person testing, and keeps the gasket in the centrifuge
from being cut by the capillary tubes.
3. Insert the capillary tubes into the centrifuge with duplicate samples across from each other. Place the sealed end toward the outside, making sure that the tubes are seated in the groves and firmly against the gasket.
4. If there is an internal cover, make sure it is screwed down. The tubes will break if the cover is not on correctly. Centrifuge the specimens for five minutes at 11,000 to 12,000 rpms.
5. Open the centrifuge after it has come to a complete stop. Read the results immediately after the centrifuge stops. If this is not possible, place the tubes upright until they are read. The red cells will slide if the tubes are left in a horizontal position and the hematocrit will be falsely increased.
6. When reading hematocrits, make sure the clay red cell interface
is aligned with the 0% line and the bottom of the plasma meniscus is
at the 100% line. The reading that corresponds to the top of the red
cell column is the hematocrit. 
7. Duplicate readings should match within 1 L/L and must be within 2 L/L. Readings that match can be averaged and reported in 0.5 L/L. Centrifuged hematocrits are always reported in whole numbers or halves.
Effect of Error on the Hematocrit |
Error |
Solution |
Decreased |
Sample drawn above the IV line |
Redraw sample from another site |
Decreased |
Red cell leakage |
Check sealant. Hard sealant causes leaks. Check gasket for cuts. Do not fill and seal from the same end of the capillary tube. |
Decreased |
Short sample, red cells shrink |
Perform an automated hematocrit or redraw the sample. |
Decreased/Increased |
Mixing errors |
Mix sample well before each use. |
Increased/Decreased |
Reading errors |
Check reading device by reading the hematocrits on a card reader or with a ruler. Check employee competence.
|
Increased |
Time too short, timer not working Inadequate rpms |
Centrifuge longer, check timer. Check centrifuge with tachometer. |
Increased |
Sample allowed to stay in the centrifuge after it has stopped |
Remove samples immediately and store them upright. |
Increased |
Buffy coat included when reading the red cell column |
Carefully read the top of the red cell column below the layer of white cells and platelets. |
Increased |
Abnormal red cell morphology that results in increased trapped plasma |
Perform an automated hematocrit. |
Kathleen Kelly B.S., MT(ASCP)
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Filling and sealing with the clay perpendicular to the table keeps the clay from becoming contaminated with blood, helps prevent leakage and is safer. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
The 100 % line must bisect the bottom of the meniscus and the zero line must align with the red cell/clay interface. When both the 0% and 100% lines are positioned correctly, the hematocrit in per cent is the line that aligns with the top of the red cells. The buffy coat, which is comprised of white cells and platelets, is not included. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
Everyone performing hematocrits must demonstrate competency. An easy way to do this is to have everyone run the same sample or a split sample and compare the results. This should include samples with normal and abnormal hematocrits and be done on a quarterly basis. Quality assurance could also include documenting that multiple people read the same spun hematocrits and obtained the same results. Proficiency testing samples are available and may be combined with the proficiency testing done on a hematology multi parameter instrument.
Preventive maintenance
These are generic recommendations. Always check manufacturer's
guidelines for specific instructions.
Daily:
Clean the inside of the centrifuge and the gasket.
Monthly:
Check the reading device. Misuse and zeroing of the reading devices
can inject large errors. Always use a second, simple reading device
to check the fancier devices. Use a ruler or a flat plastic card. 
These
cards are available from laboratory vendors and are inexpensive.
Quarterly:
1. Check the brushes if they are present - this may need to be done
more often.
2. Check the gasket for cuts and breaks. Cut gaskets allow tubes to leak and need to be replaced.
3. Check the timer with a stopwatch.
4. Check the speed of the centrifuge with a tachometer. These can easily be shared among multiple laboratories.
5. Perform a maximum cell pack to verify the time required for
complete packing.
Centrifuge hematocrits (low, normal, high) for 2 minutes and then
repeat the procedure adding 30 seconds each time you centrifuge until
the results are the same for two consecutive centrifugations. The
required time is 30 seconds longer than the second time the
hematocrit results match.
Example: The required time is 5 minutes.
Time |
2 min. |
2.5 min. |
3 min. |
3.5 min. |
4 min. |
4.5 min. |
Result |
26% |
23% |
22% |
22% |
|
|
Result |
52% |
49% |
47% |
46% |
45% |
45% |
Kathleen Kelly B.S., MT(ASCP)
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Stand-alone hemoglobinometers are also available. These are usually based on the same spectophotometric principle as CBC analyzers and use cyanmethemoglobin as the end point.
With the inexpensive small multi parameter analyzers available, hemoglobin analyzers are no longer popular. The use of the HemoCue® for back up and lipemic specimens is an exception.
Kathleen Kelly B.S., MT(ASCP)
MCV is Mean Corpuscular Volume in femtoliters. On automated instruments, it is computed using the measurements of each red cell. With manual methods, it is calculated using the hematocrit and the red cell count. Hint: do not worry about the decimal points. This applies to the MCV, MCH and MCHC. Just divide the raw numbers and place the decimal where it makes sense. It is a physiologic impossibility to have an MCV of 9.0 or 900. It must be 90.
Manual MCV = |
spun hematocrit in L/L |
red cells in millions/L --> |
Automated Hematocrit = RBC x MCV
MCV correlates to the cell diameter on the peripheral smear.
Macrocytes have a high MCV 
and
microcytes have a low MCV. 
It is possible to have abnormal sized cells and a normal MCV because
the MCV is an average. Agglutinated
red cells also cause an increased MCV. These samples need to be
warmed in a 370 degrees C heating block and rerun warm.
MVC generic reference range: 82 - 99 femtoliters
MCH is Mean Corpuscular Hemoglobin weight in picograms. This is the average weight of the hemoglobin in picograms in a red cell. It is a calculated value.
MCH = |
hemoglobin in gm/L |
red cell count in millions/L |
MCH generic reference range: 27 - 32 picograms
MCHC is Mean Corpuscular Hemoglobin Content. This indicates the average weight of hemoglobin as compared to the cell size. It is traditionally a calculated value. How ever some instruments may measure the density of the cells as they are counted and use this value to compare to the calculated value. Bayer calls this measured value a CHCM (Cellular Hemoglobin Concentration Mean) on the Bayer/Technicon hematology instruments.
MCHC = |
Hemoglobin in g/mL |
Red cell count in millions/L |
or
MCHC = |
MCH in picograms |
MCV in femtoliters |
MCHC correlates with the degree of hemoglobinization of the red cells on the peripheral smear.
It is reported in gm/dL, picograms/100 femtoliter or in %. A
decreased MCHC corresponds to cells with increased zones of central
pallor on a Wright's stained peripheral smear. These cells are called
hypochromic red cells.
An increased MCHC is rarely a true value. MCHCs above the reference range are suspect. A large number of spherocytes, the most common physiologic reason, is not common. A more likely reason is an error in the hemoglobin or the hematocrit. Solutions include: checking the smear for spherocytes, retesting the sample, performing a spun microhematocrit, performing an alternate hemoglobin method, and checking the quality control and other patient results to identify shifts or trends in the hemoglobin or hematocrit determinations.
MCHC generic reference range: 32-36 g/dL or pg/fL
RDW is the Red cell Distribution Width. This value indicates the degree of red cell size
variation or how much difference exists between the largest and
smallest red cells. This value is derived from the MCV histogram. An
increased RDW corresponds with an increase in anisocytosis on
the peripheral smear. 
The
RDW is only available if it is included in the instrument menu.
Although different manufacturers use slightly different methods of
obtaining data the RDW is generally thought of as the coefficient of
variation of red cell volume distribution.
RDW = |
standard deviation x100 |
mean MCV |
RDW generic reference range: 9.0 - 14.5
The RDW, coupled with the MCV, gives more relevant information than an individual index. The following is an attempt to clarify the relationship of the MCV and RDW.
There are varying opinions on the clinical value of red cell indices. They are used to morphologically classify anemias and to select additional tests to determine the cause of an anemia. Indices also change in response to treatment of some anemias.
Platelet Indices: These include the platelet distribution width and average platelet volume. They have limited use, but do correlate to flags on the platelet histogram that indicate giant platelet or platelet clumps.
Anemia - A reduction in the oxygen carrying capacity of the blood. Almost always manifested by a decreased hemoglobin and frequently accompanied by a decreased hematocrit and red cell count.
Kathleen Kelly B.S., MT(ASCP)
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These red cells vary in size and have an average cell size that falls with in the reference range. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
Hypochromic red cells have an increased zone of pallor on Wright's stain. These cells contain decreased hemoglobin for their size and have a decreased MCHC. Previous Page |
Section Top | Title PageKathleen Kelly B.S., MT(ASCP)
These cells exhibit a large variation in size. The RDW (Red Cell Distribution width) is a numerical indication of this morphologic abnormality. Previous Page |
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