TOPIC OUTLINE

SUMMARY
INTRODUCTION
SAMPLE COLLECTION
THE COULTER IMPEDANCE APERTURE
Multichannel instruments
The red blood cell volume histogram
Red cell distribution width
Improvements enabling platelet counting
Platelet counting
- Mean platelet volume
CELL COUNTING BY LIGHT SCATTERING
The optical platelet count
- Immunologic counting of platelets
- Reticulated platelets
ALTERNATIVE MEASUREMENTS OF HEMOGLOBIN CONCENTRATION
AUTOMATED COUNTING OF RETICULOCYTES
AUTOMATION OF THE WHITE CELL DIFFERENTIAL
The three part differential
The five part differential
- Suspect flags
AUTOMATED SAMPLING MODES AND COMPUTER ADVANCES
CAUSES OF SPURIOUS RESULTS
Spurious increase in the MCHC
Spurious decrease in the MCHC
Platelet error flags
Leukocyte counting errors
Platelet clumping with pseudothrombocytopenia
SELECTED AUTOMATED HEMATOLOGY INSTRUMENTS
Beckman Coulter DxH 800, LH780, and other models
Siemens Advia (and H series)
Abbott Cell-Dyn 4000, Sapphire
Abbott CD 3500, CD3700
Sysmex XE Series
Sysmex NE/SE Series
SUMMARY
REFERENCES
GRAPHICS View All
FIGURES
RBC size sideroblastic anemia
Automated platelet counting low
Predicting platelet recovery
WBC differential Advia2120
WBC differential CD4000
PICTURES
Manual hematocrit
Microhematocrit determination
Cold agglutinin
Giant platelets
Platelet clumping in EDTA
RELATED TOPICS
Approach to the adult patient with anemia
Approach to the adult patient with thrombocytopenia
Causes and diagnosis of anemia due to iron deficiency
Clinical aspects, diagnosis, and treatment of the sideroblastic anemias
Clinical features and treatment of autoimmune hemolytic anemia: Cold agglutinins
Evaluation of the peripheral blood smear
Mean corpuscular volume
Megakaryocyte biology and the production of platelets
Myeloperoxidase deficiency and other enzymatic WBC defects causing immunodeficiency
Oxygen carriers as alternatives to red cell transfusion
Platelet function testing
Automated hematology instrumentation

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Automated hematology instrumentation
Author
Tracy I George, MD
Section Editor
Stanley L Schrier, MD
Deputy Editor
Jennifer S Tirnauer, MD
Disclosures
All topics are updated as new evidence becomes available and our peer review process is complete.
Literature review current through: Mar 2013. | This topic last updated: Jul 24, 2012.
INTRODUCTION During the first half of the twentieth century, the complete blood count (CBC), one of
the most commonly ordered laboratory tests, was performed using exclusively manual techniques:
Blood cell counts (red cells, white cells, platelets) were performed using appropriately diluted blood
samples and a ruled counting chamber (hemocytometer).
Hemoglobin concentration was analyzed colorimetrically by the cyanomethemoglobin method.
The hematocrit (packed cell volume) was measured by high speed centrifugation of a column of blood,
either in a specially designed tube (the Wintrobe tube) ( picture 1 ), or in sealed microcapillary tubes
(ie, the "spun" hematocrit, often obtained by fingerstick blood collection) ( picture 2 ).
The white blood cell differential was obtained by examining and enumerating by class (eg,
granulocytes, lymphocytes, monocytes) 100 to 200 individual white blood cells on a suitably stained
blood smear.
In 1932, Wintrobe developed a set of calculated indices that estimated erythrocyte size and hemoglobin
content based on the red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT).
These indices included:
Mean corpuscular volume (MCV) the volume (in femtoliters, fL) of the average circulating red
blood cell
Mean corpuscular hemoglobin (MCH) the hemoglobin content (in picograms) of the average
circulating red blood cell
Mean corpuscular hemoglobin concentration (MCHC) the hemoglobin concentration within
circulating red blood cells (grams of hemoglobin per 100 mL of packed red blood cells)
These three indices were calculated manually, as follows:
MCV (fL) = 10 x HCT (percent) RBC (millions/microL)
MCH (pg/red cell) = HGB (g/dL) x 10 RBC (millions/microL)
MCHC (g/dL) = HGB (g/dL) X 100 HCT (percent)
These early methods were laborious, and, for the hemocytometer counts, very imprecise. This topic review
will discuss the various methods and apparatus which have been employed in an attempt to automate all of
the above procedures [ 1-3 ].
SAMPLE COLLECTION Samples to be used for a complete blood count should be anticoagulated
with a suitable agent, such as heparin or EDTA. The use of EDTA may cause platelet clumping in some
patients (ie, pseudothrombocytopenia). (See "Approach to the adult patient with thrombocytopenia", section
on 'Pseudothrombocytopenia' .)
It is recommended that blood samples be kept at room temperature if analysis is to occur within 8 hours of
collection. Samples should be refrigerated if the analysis is to occur up to 24 hours after collection. We do
not recommend that samples greater than 36 hours old be used for CBC testing.
THE COULTER IMPEDANCE APERTURE In 1956, Walter and Joseph Coulter patented a device
developed in their basement that used an electric impedance aperture to count red cells and white cells. In the
impedance method, blood is diluted in a current-conducting solution, such as isotonic saline. The suspension
of cells is drawn by a vacuum through a small orifice or aperture positioned between two sensing electrodes
connected across a direct current potential. As each cell passes through the aperture, the resistance between
the electrodes increases. The non-conductive cell being counted produces a momentary increase in
impedance, resulting in an electrical pulse, with the number of pulses indicating the cell count, and the
amplitude of the pulse being proportional to the cell's volume. Upper and lower pulse height thresholds could
be selected such that only particles within a specific volume range would be counted.
Since typical red cell counts are three orders of magnitude greater than white cell counts, and since
appropriate settings of the lower pulse height threshold eliminate counting of the smaller platelets (platelet
volume is approximately 9 fL, or about one-tenth the volume of mature red cells), a cell count on
appropriately diluted whole blood samples served to yield a red cell count. On the other hand, red cell lysing
reagents need to be added to the sample before the white blood cells can be counted with accuracy.
In the first semiautomated systems, separate red blood cell and white blood cell dilutions were performed
manually or by using a separate dilutor. A combination lysing/potassium ferricyanide solution was added to
the white blood cell dilution, allowing enumeration of leukocytes as well as spectrophotometric measurement
of hemoglobin (as cyanmethemoglobin) from the same dilution. Red blood cell indices were manually
calculated, using the formulae shown above.
When the cell count is especially high, two (or more) cells can be so close together while passing through the
aperture that they are counted as one cell. To correct for this phenomenon, a "coincidence" correction was
applied to the raw impedance aperture count. This was accomplished manually, using statistical tables
developed for this purpose.
Multichannel instruments In the 1960s, the first multichannel automated hematology instruments appeared.
Anticoagulated whole blood samples were aspirated into the apparatus and automatically aliquoted and
diluted into RBC and WBC counting chambers (baths) for cell counting and sizing with impedance apertures,
and into a spectrophotometric cuvette for hemoglobin determination using the cyanmethemoglobin method.
Coincidence correction and calculations of red cell indices were automatically performed.
Since these machines measure the red cell count (RBC) directly, as well as the mean corpuscular volume
(MCV), the HCT could be electronically calculated by rearranging one of the original Wintrobe formulae
shown above:
HCT (percent) = RBC (in millions/microL) x MCV (fL) 10
These first multichannel instruments revolutionized the CBC as a laboratory test, enabling high throughput, low
cost, and fast turnaround time with the ability to produce immediate (STAT) results. However, the platelet
count was not a part of the first automated CBC, since the aperture resolution on the early instruments was
not good enough to adequately separate platelets from red cells. Early attempts to automate the platelet count
used platelet-rich plasma or whole blood lysing methods, but there were many interferences, particularly
when the platelet count was low.
The red blood cell volume histogram Modern cell counting and sizing apertures have sufficient precision
to function as "channelizers." As an example, Coulter instruments channel cells within the RBC/platelet
aperture into 256 bins in the size range from zero to 360 fL. RBCs are not only enumerated as those particles
with a cell volume within the range of 36 to 360 fL, but they are also classified into appropriate "channels" by
size (related to the pulse height). For instruments with pulse editing circuitry, aberrant pulses are excluded
from these channels. A plot of frequency versus channel size allows the development of a RBC volume
histogram, which can be smoothed into a RBC size distribution curve.
The red cell size frequency distribution curve typically has a symmetrical or Gaussian shape. The MCV (mean
red cell volume) and red cell distribution width (RDW, the coefficient of variation of the RBC volume
distribution) are directly calculated from this curve. A number of abnormalities of this curve can be seen in
disease settings:
A left "shoulder" extension to the curve, or failure of the curve to reach baseline on the left side (ie,
RBCs with smaller volumes) can indicate the presence of a population of very small RBCs (eg,