Prepared By: Ashraf Shaqalaih

Bsc(MT), MSc(MT), CLS(H), CLSp(H)

Automated Hematology Cell Counters

1- Impedance Technology Hematology Counters These counters are based on the Coulter principle, i.e.

electrical resistance principle, which depends on the fact that blood cells are nonconductive to electricity, so when they pass through an electrical field (resistance). Well mixed blood is greatly diluted/suspended in an isotonic electrolyte solution, so that cell sizes are not altered/changed in terms of cell shrinkage nor cell swelling, this isotonic electrolyte solution conduct electricity very well, while blood cells are nonconductive. A counting chamber consists of (1) a beaker, (2) two electrodes with a direct current pass through them, and an orifice/small opening the aperture with specified dimensions; when the suspended cells/particles passes through the aperture it will displace its own volume of isotonic electrolyte solution and they will increase the electrical impedance

increase the electrical resistance (impedance) because of their non-conductivity between the two electrodes which are located on each side of the aperture. This electrical resistance is manifested as a pulse, each pulse means a cell/a particle, sum of these pulses equals the total cell count. This pulse has a height which is directly proportional to the cell size (i.e. pulse height indicates cell volume/size). By this, the counter has counted and sized the cells/ particles. In fact the counter has two chambers/ baths, one for red blood cell and platelet counting and sizing, while

the other one is for WBC counting and sizing. Each counting chamber has 2 (+/-) electrodes, and an aperture with specified dimensions for each, the RBC aperture is narrower than the WBC aperture, why? Because the WBCs have larger sizes than red blood cells and platelets. From the above we can conclude that impedance hematology counters actually does not identify the cells which are considered here as particles, rather it counts and size them; but according to their size cells/particles they can be discriminated into cell types or populations.
Beaker Electrodes Aperture Cells Cell passing through aperture

Coulter Principle Counting Chamber

Coulter Principle The Coulter principle of counting and sizing particles/cells is based on measurable changes in electrical resistance produced/created by nonconductive particles/cells suspended in conductive isotonic electrolyte solution.

Let us now go step by step , as if we are inside the counter. As soon as patient well mixed blood sample is aspirated into the counter, blood is highly diluted with the isotonic solution (the isotonic solution is called the diluent reagent), then this dilution is divided into 2 portions/parts; first portion will be enforced through the tubing toward the RBC chamber, where it is further diluted with the isotonic solution, and then cells/particles are passed through the aperture, in which red blood cells and platelets are counted and sized according to the total number and heights

of created pulses. But your question here is, how the counter has discriminated between platelets and red blood cells? Good

question, the answer is , cells/particles which have a volume between 2-20 fl are considered and counted as platelets, while cells between 36 to 360 fl are considered and counted as red blood cells. You can assimilate from this point that the counter actually didnt identify the cells as platelets or red blood cells, but according to their size manifested by the generated pulse height they are discriminated, right or not? I want you to understand these points, so that you will feel that it is not strange when we have microcytic red cells (decreased volume), or fragmented red cells (schistocytes, which are characterized by greatly reduced volume/size) they may be counted as platelets, on the other hand, also you feel it is not strange that when we have large platelets they may be counted as red blood cells, causing erroneous counting results according to the defect. The second portion of diluted blood sample will be moved through tubing towards the WBC chamber, where it is further diluted with a lyse reagent; not the diluent; this lyse reagent has several functions: (1) will lyse the red blood cells, so that red blood cells will not be counted or interfere with white blood cells; whereas in RBC chamber WBCs are not lysed, but because of their low count (WBCs, are in thousands/cumm) in comparison to the very high RBC count which are in millions per cumm, they will not affect significantly the total red blood cell count, only in cases of very high WBC count as seen in leukemias, they may affect the RBC total count, (2) lyse reagent contains Drabkins solution which is provided for hemoglobin determination, when the red cells are lysed they release hemoglobin. (3) lyse reagent will puncture the

WBC membranes so that they will collapse around the nucleus with their granules. So, when the WBCs pass through the aperture they are counted and sized in the same manner as red blood cells and platelets are counted and sized, but the difference here that the counter in counting and sizing the WBC nucleuses because their membranes are punctured by the lyse reagent. Also, the counter can perform three part WBC differential, i.e. neutrophils, monocytes and lymphocytes, but how? Again through their sizes, the particles/collapsed cells can be discriminated into three zones, the first zone is considering cells with a volume of 35-90 fl - which are differentiated as lymphocytes, the second

zone with cell volumes between 90-160 fl, which are differentiated as monocytes, and the third last zone with cell volumes between 160-450 fl, are differentiated as neutrophils. By this the counter has counted the total WBC count, and performed three part differential. You may ask, that we know that the monocytes are bigger than neutrophils, but why the counter considered

neutrophils as bigger? Very good question; the answer is because the lyse reagent puncture the cell membranes, with subsequent collapse around the nucleus with the granules, the nucleus plus granules size of neutrophils is bigger than monocyte nucleus with its granules, the counter is not sizing the whole WBC cell rather it is sizing the collapsed punctured WBC cell in which the neutrophil collapsed cell is bigger than monocyte collapsed cell. Actually the counter provide us with three part differential as said before, but with the following sequence: (1) Lymphocytes, (2) MID (which means middle cells, which are monocytes in normal states, but may represent immature cells or variant lymphocytes in abnormal states, but because of simplicity I explain it above as

monocytes), and (3) Neutrophils. One point also should be considered here, that platelets are not lysed here, but because of there small size they are not counted as WBCs, but when platelet clumps or aggregated platelets are present they can be counted as one big particle, they may be counted as WBCs and interfere with WBC count. After the WBCs are counted, the WBC diluted blood portion is moved toward the spectrophotometer which is provided inside the counter for hemoglobin determination at 530540 nm, according to Drabkins method. You may ask, can two or more cells enter at the same time to the aperture and be sized and counted as one cell (as one high pulse), giving a high pulse indicating a very large cell ?the

answer is yes, and this is called the coincidence error. But this error is minimized by the huge dilution, by decreasing the aperture dimensions, and by the computer which statistically corrects for this error. Also, one may ask since we can reduce it by the great dilution, why we are not doing more and more dilutions to further reduce this error?, this is because as you do more dilution, the great error you have, because of the dilution error, right? All of the above is supplied to the counter computer, which do its calculations to give meaningful data. The counter is not only supplying us with (1)RBC count,(2)Platelets count, (3) Hemoglobin concentration, (4) WBC count, with its 3 part differential (5)

Neutrophils %, (6) Neutrophils absolute count, (7) MID %, (8)MID absolute count, (9) Lymphocytes %, (10) Lymphocyte absolute

count, but the counter has extended hematology menu. It also supply us with the (11) MCV, which is here is considered as a measured parameter, not calculated parameter, and it is derived from the RBC histogram (will be discussed later), (12) Hct, which

is a calculated parameter here; the counter does have a hematocrit centrifuge inside it; it is derived from RBC total count and the MCV, by applying the following formula (Hct = MCV x RBC/10), (13) MCH, which is calculated from Hb and RBC count, (14) MCHC, which is calculated from Hct , and Hb, (15) RDW, Red cell Distribution Width, which measures the degree of anisocytosis; RDW is a calculated parameter, RDW is helpful in many

instances, it is the first parameter to increase in cases of nutritional deficiencies, especially iron deficiency anemia, it is also used to differentiate between iron deficiency anemia (low MCV , high RDW), and uncomplicated thalassemia minor (low MCV, normal RDW). RDW has a normal range between 11.6 and 14.6, so when RDW is normal it is expected to have red cells which are homogeneous and exhibit very minimal anisocytosis on

peripheral blood films, but when RDW is high it is expected to find red cells with altered sizes. (16) MPV, Mean Platelets Volume, which analogues the MCV for red cells, MPV have a nonlinear inverse relationship with platelets count, i.e. when platelet count is low, MPV is high (platelets are large in size), a technical point should be mentioned here, that when blood is extracted from the patient, platelets change shape from discoid to spheroid, this alteration in shape, causes an increase in MPV, this is why it is advisable to analyze the blood specimens after 30 to 60 minutes of blood extraction. When platelets count is low, and MPV is low, it is expected that the bone marrow platelet production is defected, because when platelets are low in number (thrombocytopenia) the bone marrow will react by producing platelets with increased volume/size(large platelets functions more than small platelets), while if we have low platelets count with high MPV, the cause of

thrombocytopenia is expected to be peripheral not bone marrow in origin, because here the bone marrow responded to

thrombocytopenia by producing and releasing large platelets, (17) PDW, Platelet Distribution Width, is a measure of the uniformity of platelet sizes, normal PDW is less than 20%, increased PDW is associated with abnormal megakaryocytic development and maturation, (18) Pct, Plateletcrit, this parameter represents an

estimate of the percent platelet mass, this parameter is especially in Europe, to determine if the thrombocytopenic patient is of great need of platelet concentrate transfusion or not, because patients with reduced Pct may bleed spontaneously and are considered as candidates for platelet concentrate transfusions. The counters

also display 3 histograms (for WBC, RBC, and PLT histograms), these will be discussed in the following pages.
Hematology Parameter Menu Supplied By Impedance Technology Counters
No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Abbreviation WBC count Granulocyte % MID % Lymphocyte % Granulocyte Abs. MID Abs. Lymphocyte Abs RBC count Hb Hct MCV MCH MCHC RDW PLT MPV PDW Pct RBC Histogram WBC Histogram PLT Histogram Description Total white Blood Cell Count % Differential of Neutrophils % Differential of Middle Cells % differential of Lymphocytes Absolute Total Neutrophil Count Absolute Total Middle Cells Count Absolute Total Lymphocytes Count Total Red Blood Cell Count Hemoglobin Concentration Hematocrit Mean Cell Volume Mean Cell Hemoglobin Mean Cell Hemoglobin Concentration Red Cell Distribution Width Total Platelets Count Mean Platelet Volume Platelet Distribution Width Plateletcrit Red Blood Cell Histogram White Blood Cells Histogram Platelets Histogram

There are many brand name counters available in the market, they may use different terminology, for example some counters will use large cell instead of granulocytes, and use small cells instead of lymphocytes , you must always look and read the counter manual to learn the used terminology in your Automated Hematology Impedance Counter, but all of these counters have the same principles, described above.

As said during our discussion above, that erroneous results may be obtained by the Automated Hematology Counters, table below summarize the frequent causes of erroneous CBC results obtained with Automated Impedance Hematology Counters.
Erroneous Results That May Be Obtained With Automated Impedance Counters Parameter Cause-Increase
Nucleated RBC Unlysed Red Cells Platelet Clumping/ Aggregation Cryoglobulin Heinz Bodies Malaria Parasite High WBC Giant Platelets

Cause Decrease
Clotting Smudge Cells

WBC

RBC

Hemoglobin MCV

MCH

MCHC

Platelets

High WBC Hyperlipidemia High Protein Autoagglutination High WBC Hyperglycemia Autoagglutination High WBC High Hemoglobin Low RBC count Autoagglutination High Hemoglobin Low Hematocrit Microcytic Red Cells Red Cell Inclusions White Cell Fragments Dust Particles Electronic/Electrical Noises Hemolysis-Schistocytes

Clotting Microcytic cells Hemolysis Cold agglutinin Clotting Sulfhemoglobin Giant Platelets

Low Hemoglobin High RBC

High WBC Low Hemoglobin High Hematocrit Clotting Giant Platelets Heparin Platelet Clumping Platelet Satellitosis

Examples of corrective actions can be taken by medical technologists Most of these erroneous results can be corrected by competent medical technologists, as you. Erroneous increase in hemoglobin can be corrected with the preparation of sample blank Erroneous increase in MCV , MCH, and MCHC, combined with the erroneous decrease in RBC counts caused by cold agglutinins, can be corrected by warming the patient specimen in 37 C water bath, for a short time, and then repeat CBC while the specimen is worm, if problem still persists, you can extract blood and place it in a prewarmed EDTA blood collection tube. The above are examples of corrective actions that can be taken/performed by medical laboratory technologists to correct the error. Always, a blood film should be performed and visualized whenever an abnormal results are obtained either they are true or erroneous, and almost all erroneous results can be resolved and clarified by visualizing a well made/stained blood film, because the machine does not always detect what the eye does. We have to mention that the counter consumes/utilizes three types of reagents:(1)the diluent, (2) the lyse reagent, and (3) the detergent, the first two reagents we have already talked about above. The detergent has two functions:(1)to remove protein precipitates from the tubing and the counting chambers, (2) is the blank solution for the spectrophotometer (for hemoglobin

determination). Automated Hematology Counter Histograms Histograms are graphical representations of relative Three

cell/particle frequency versus cell/particle volume/size.

histograms are displayed; RBC, WBC, and platelets histogram. Not only the histograms supply us with information about RBCs, WBCs, and platelets frequency, their distribution, and average sizes, but also depict the presence of cell subpopulations. Shifting of the histogram in one direction or the other direction can be of diagnostic importance. X-axis represents cell size , and theY axis represents relative frequency of cells/particles. Red Blood Cell Histogram As discussed before, particles that are considered as RBC have a cell size range from 36 to 360 fl (i.e. the counting zone for red blood cells is between 36 and 360 fl). The RBC histogram displays cells that are as small as 24 fl. The scale from 24 to 36 fl allows us for the detection of RBC fragments, WBC fragments, giant platelets or microcytic red blood cells, all of these may shift the histogram to the left. The non lysed WBCs that are counted as RBCs does not affect the RBC histogram curve because of their low relative frequency, but the curve may be affected, when the WBC count is very high as occurs in leukemias and infectious leukemoid reactions. The curve may be shifted to the right whenever high frequency of macrocytes are present, as seen in cases of megaloblastic anemias, in cases of reticulocytosis and polychromasia especially if accompanied with shifted

reticulocytosis, in cases of very high WBC, especially if anemia complicates the case. The RBC histogram may be bimodal in various conditions, and this may indicate the presence of two cell populations. Bimodal curve may be seen in cold agglutinin disease, in iron deficiency anemia with recent blood transfusion, in sideroblastic anemia especially in the acquired forms, and in

megaloblastic anemia with recent blood transfusion.

Automated

Hematology Counters extract MCV from the area under the curve or may apply the following formula: Sum of pulse heights/ sum of pulses. White Blood Cell Histogram WBC histogram displays the classification of WBCs according to their sizes after cytoplasmic puncture by the lyse reagent, i.e. does not display the native WBC sizes. From the histogram the percent, absolute counts, and frequency distribution of the 3 part WBC differential, monocytes, myeloblasts, i.e. lymphocytes, middle cells (normally may be immature and cells such can as be

abnormally and

myelocytes),

granulocytes

determined. WBC histogram displays particles/cells as small as 30 fl, but only those cells which are greater than 35 fl are counted as WBCs. The counter differentiate between lymphocytes, middle cells (or you can use the term mononuclear cells), and granulocytes. Mononuclear cells includes blasts, and other

immature cells, however, in normal specimens, monocytes represents the mononuclear counting zone. On the histogram 4 regions are displayed at 35, 90, 160, and 450 fl. A valley should be seen between the lymphocytes and mononuclear cells, and between the mononuclear cells and granulocytes, the automated counter determines the percentage of each cell type/

subpopulation according to these depression regions, if one or the two valleys is/are absent this triggers an alert. The region below 35 fl zone should be clear, with no interfering cells, if this region is not clear, it is expected to have either NRBC, or clumped/

aggregated platelets, or Heinz bodies, or cryoglobulin1, or unlysed mature red blood cells, or malaria parasite, or any other causes, a blood film should be made to clarify the reason for interference. Cells that can trigger an alert (interfere) in the region between the lymphocytes, and mononuclear cells are certain blast cell forms, plasma cells, or in some cases eosinophilia and basophilia. Cells that can trigger an alert in the region between mononuclear cells and the granulocytes includes immature granulocytes, blasts, and eosinophils. Cells that interfere in the far right side of the curve usually indicates a high absolute granulocytic count and/or presence of toxic granulation. Multiple region alerts may be encountered , in one patient sample. Also, an alert may be triggered at exactly 35 fl region, which is usually seen in cases of chronic lymphocytic leukemia (CLL). Platelets Histogram Platelets histogram is useful in interpreting platelets sizes and abnormal platelet morphology. Particles/Platelets that are between 2 to 20 fl are counted as platelets by the automated counter. The platelets histogram x axis has a range from (0 to 35) fl. The lower region between 0 to 2 fl can be interfered by air bubbles, dust, electrical and electronic noises, whereas the upper region (over 20 fl) can be interfered by microcytic red cells, red blood cell fragments (schistocytes), WBC fragments, giant platelets, clumped platelets, and platelets satillitosis. So, platelet histogram flags whenever the inverse relationship between the MPV and platelets count is abnormal, which may be caused by the previously mentioned causes.
Cryoglobulins are altered immunoglobulins, in which they become insoluble and precipitate to varying degrees at temperatures below 37C. They are associated mostly with pathological conditions.

From the above discussion we can depict that the blood cell histograms supplied by the automated hematology analyzers are of great diagnostic and morphologic importance, also they alert us if a patient sample need blood film preparation and examination or not. Although practice and blood films examination will increase the knowledge of interpreting and analyzing these blood cell histograms.

Normal RBC Histogram

Curve to the left represents microcytic red cells, whereas curve to the right represents normocytic RBC curve.

The effect of microcytes on red cell and platelet histograms

The effect of giant platelets on RBC and PLT histograms