Transcription
Okay, good morning! This is our last session for the hematology lecture. We are now going to discuss the different laboratory procedures in hematology.
First, we have the improved Neubauer ruling on the hemocytometer. In the ASCP exam, there are still questions about cell counts. I even had one during my exam, so it's important that we know the dimensions of the hemocytometer and where certain cells are being counted.
First, we have our WBCs. WBCs are usually counted in the four corner squares; however, that depends on the dilution being employed. Routinely, the dilution for WBC count is 1:20, and if the dilution is 1:20, that is usually counted in the four corner squares. However, there is an option to employ a 1:100 dilution, and if this is the dilution, usually white blood cells are counted in all nine squares on both sides of the hemocytometer.
What is the volume of one corner square or one secondary square in the hemocytometer? We can solve for the volume by multiplying the dimensions of this particular square: 1 mm by 1 mm by 0.1 mm. The 0.1 mm is the standard depth of our hemocytometer. This makes one square 0.1 mm³ or 0.1 microliters in volume.
Next, we have the red blood cells. Red blood cells are usually counted in the five squares found in the central large square of the hemocytometer. They are the ones in blue.
What would be the volume of one RBC square? The volume is 0.2 mm by 0.2 mm by 0.1 mm, which makes the volume of one RBC square 0.004 cubic millimeters or 0.004 microliters. For red blood cells, we could employ the 1:100 or the 1:200 dilution.
Lastly, we have the platelets. The platelets are counted in all 25 squares in the central large square. Having said that, it's like you are counting the entire central large square. Typically, the dilution employed for platelets is 1:100. Since you are counting the entire central large square, the volume is similar to that of one WBC square: 1 mm by 1 mm by 0.1 mm. So, the volume is also 0.1 mm³ or 0.1 microliters.
For platelet count, the gold standard or reference method employs the use of the phase contrast microscope. We know that platelets are really small, and because of that, their refractive index is low, making them difficult to see under the usual bright field microscope. Based on the method specified by Brecker and Cronkite, this method uses 0.1 percent ammonium oxalate and employs the phase contrast microscope.
We have a general formula for cell count using the Neubauer ruling. It's safe to use this formula as long as you know the dimensions of the hemocytometer. You just need to place the cells counted on both sides, the dilution factor, which is automatically the denominator of the dilution, and the total volume in the chamber counted.
How do we derive the total volume in the chamber counted? We derive this from multiplying the number of squares counted by the volume of one square. We need to be careful about the values given to us because sometimes they provide the average count on both sides. If that happens, for example, in the case of the RBC count, they might give you the average of the RBC counted, say 215. When you say average, that is already the average of the counts on both sides.
If we are counting 10 squares in total (five squares on both sides), and the value given is already the average, you will only place five in the number of squares counted since the average is the one given to you.
Next, we go to the corrected WBC count. Remember that whenever there are nucleated red blood cells present in the peripheral blood smear, this entails the performance of the corrected WBC counts. You have to do the correction if at least five nucleated red blood cells are seen in the peripheral blood smear.
Why do we have to do the correction for the WBC count? This is because nucleated red blood cells are counted as white blood cells, which could falsely increase the WBC count.
In the ASCP exam, there was a question about doing the corrected WBC count, but instead of doing the 100-cell differential, we sometimes opt for the 50-cell differential if the WBC count is low.
For the corrected WBC count, we usually do the correction if at least five nucleated red blood cells are present in the peripheral blood smear.
Now, electronic impedance counters are one of the basic methods used in automated cell counting. We have electrical impedance and optical light scatter.
One thing to know about electronic impedance is that it can only differentiate cells based on their size. The basic principle of electrical impedance is that cells do not conduct electricity. When they pass through a certain aperture, they create electrical resistance, resulting in voltage pulses. The number of pulses corresponds with the cell number, and the amplitude of the pulse corresponds with cell size.
In electrical impedance counters, the WBC count is performed in the WBC aperture. These are particles that are 35 femtoliters and above. The WBCs and red blood cells are counted in different apertures.
For the WBC count, human lymphocytes or small mononuclear cells are counted between 35 to 90 femtoliters, large mononuclear cells or monocytes are 90 to 160 femtoliters, and neutrophils and other granulocytes are in the range of 160 to 450 femtoliters.
Red blood cells are counted in the RBC aperture. The specimen is mixed with a diluent and a lysing agent. The purpose of the lysing agent is to lyse the red blood cells because we do not want red blood cells counted in the WBC aperture.
Hemoglobin determination is usually based on the cyanmethemoglobin method. This is why we need to correct for the presence of nucleated red blood cells, as they resist lysis and are counted as WBCs.
Lastly, platelets are counted in the same aperture as red blood cells, and platelets range from 2 to 20 femtoliters. The electrical impedance instrument can only differentiate cells based on their size.
Now, how do we discriminate granulocytes? For that, we use optical or light scatter. Forward side scatter assesses cellular granularity or complexity, such as the number of lobes in the nucleus and cytoplasmic granules.
We have MCV, MCH, and MCHC. MCHC is not used for the morphological classification of anemia. We usually only use MCV and MCHC.
For MCV, the formula is hematocrit in percent divided by the red blood cell count in times 10 to the 12 per liter times 10. The normal value for MCV is 80 to 100 femtoliters. Red blood cells below 80 femtoliters are referred to as microcytic, and those above 100 are macrocytic.
For MCHC, the formula is hemoglobin in grams per deciliter divided by the RBC count times 10. The normal value for MCH is 26 to 32 picograms.
Lastly, we have the mean corpuscular hemoglobin concentration (MCHC), which represents the average concentration of hemoglobin per 100 ml of red blood cells. The formula for this is hemoglobin in grams per deciliter divided by hematocrit in percent times 100. The normal value for MCHC is 32 to 36 grams per deciliter.
If MCHC is below 32 grams per deciliter, it indicates hypochromic RBCs, while those above 36 are referred to as hyperchromic RBCs. However, hyperchromic is a misnomer because technically, one RBC cannot contain more than 36 grams per deciliter of hemoglobin.
If the MCHC value is greater than 38 grams per deciliter, we need to check for possible errors in hemoglobin determination.
Now, let's go to histograms. A histogram utilizes impedance technology and represents cell number versus one measured property, usually cell size.
We have different histograms for different cellular populations. The WBC histogram corresponds to the volume of red blood cells with a mean cell volume of 35 to 450 femtoliters. The peaks in the WBC histogram represent different cellular populations: the first peak represents lymphocytes, the second peak represents monocytes, and the third peak represents granulocytes.
For the RBC histogram, the reference size range for red blood cells is 36 femtoliters and above. The RBC histogram will typically show one peak between 70 to 110 femtoliters, correlating with the usual range of red blood cell mean cell volume.
If the histogram shows a wider range of RBC sizes, it indicates an increased red cell distribution width, which could indicate the presence of anisocytosis.
Next, we have the platelet histogram, which occurs before the RBC histogram for particles ranging from 2 to 20 femtoliters.
Now, let's discuss abnormal histograms and their possible indications. For the WBC histogram, a population before 35 femtoliters usually indicates the presence of nucleated red blood cells or giant platelets. A peak overlap at 90 femtoliters usually indicates reactive lymphocytes or blast cells. A peak overlap at 160 femtoliters indicates an increased number of bands or immature granulocyte forms. A population after 450 femtoliters usually indicates a high granulocyte count.
For the abnormal RBC histogram, two peaks could indicate a dimorphic RBC population, possibly due to two deficiencies occurring in the body. For example, iron deficiency anemia could result in microcytic red blood cells, while vitamin B12 deficiency could result in macrocytic red blood cells.
If the curve width goes beyond the normal range of 70 to 110, it indicates an increased red cell distribution width, suggesting anisocytosis. A shift to the right or left in relation to the mean cell volume indicates the presence of macrocytes or microcytes, respectively.
Now, let's proceed to the conditions that cause interferences in most hematology analyzers. I only included the common ones here.
First, we have the presence of cold agglutinins, such as anti-small eye and anti-big iron, which are usually produced due to certain infections. The presence of cold agglutinins will decrease the RBC count and increase the mean cell volume.
The rationale is that when red blood cells agglutinate, they are counted as one big RBC, which decreases the count and increases the size. The indicator for this will be a shift to the right in the RBC histogram. The remedy is to warm the specimen to inactivate the cold agglutinins and rerun the sample.
Next, we have the presence of lipemia and chylomicrons, which can increase hemoglobin determination and the hemoglobin-dependent indices. The indicator here is quality control means. If there are technical errors, the rule of three will not hold. The remedy is to replace the plasma.
We also have the presence of lysis-resistant red blood cells with abnormal hemoglobin, such as hemoglobin S, C, and F. These cells resist lysis and are counted as WBCs, increasing the WBC count and hemoglobin value. The indicator is a flagging or interference in the histogram. The remedy is to make a manual dilution and adjust the tonicity of the lysing agent.
Next, we have the presence of platelet clumps. If platelets clump, they are not counted individually, leading to a decreased platelet count and an increased WBC count. The indicator is interference on the histogram. The remedy is to collect a new specimen using citrate as the anticoagulant and multiply the result by 1.1.
We also have the presence of hemolysis, which decreases the RBC count and increases the hematocrit. The indicator is the rule of three, where hemoglobin times three will not equal the hematocrit value. The remedy is to request a new specimen.
The presence of microcytes or schistocytes can lead to a left shift in the RBC histogram. The remedy is to review the blood film.
Lastly, we have nucleated red blood cells and megakaryocyte fragments, which are present in older hematology analyzers. These will result in an increased WBC count because they are counted as white blood cells. Newer analyzers can count nucleated red blood cells and perform the corrected WBC count themselves.
Now, let's proceed to the reticulocyte count. The reticulocyte count is an indicator of the bone marrow response whenever we have anemia. If your bone marrow is functioning properly, it will respond to anemia by releasing a lot of reticulocytes.
The reticulocyte count is the first test to perform to determine if anemia is due to defective production of RBCs or increased destruction or loss of RBCs. If the problem is defective production, the reticulocyte count will be low. If the cause is increased blood loss or destruction, the reticulocyte count will be high.
Reticulocytes can be seen under the Wright-stained smear as polychromatic macrocytes. The standard for counting reticulocytes is supravital staining, using new methylene blue or brilliant cresyl blue.
There are different formulas for reticulocyte counts, each with its diagnostic usefulness. The relative count formula is the number of reticulocytes counted divided by 1,000 red blood cells times 100 percent. The normal value for the relative reticulocyte count is 0.5 to 1.5 percent, which is higher in newborns.
The absolute reticulocyte count represents the actual number of reticulocytes per liter of whole blood. The formula is the relative count times the RBC count divided by 100. The normal value for the absolute reticulocyte count is 25 to 75 times 10 to the ninth per liter.
We also have the corrected reticulocyte count, which is necessary when there are conditions that could result in a falsely high reticulocyte count. The formula is the reticulocyte count in percent times the hematocrit in liters divided by 0.45. The normal value depends on the hematocrit level of the patient.
Lastly, we have the reticulocyte production index (RPI), which gives us information about the bone marrow response. The formula is the corrected reticulocyte count divided by the maturation time in days. If the RPI is greater than 3 percent, the bone marrow is adequately responding to anemia. If it is less than 2 percent, the response is inadequate.
Now, let's proceed to the erythrocyte sedimentation rate (ESR). ESR is a non-specific measurement used to detect and monitor an inflammatory response. It is based on the distance settled by red blood cells in milliliters within one hour.
Factors that affect the ESR result can be classified into those that decrease and those that increase the settling of red blood cells.
For RBC factors, larger red blood cells settle faster. In anemia, the ESR is increased due to a lower hematocrit value, while in polycythemia, the red cell mass is high, creating a taller red blood cell column.
Plasma factors are the most important in determining ESR. Albumin decreases ESR because it prevents rouleaux formation, while fibrinogens and globulins increase ESR by promoting rouleaux formation.
Technical factors can also affect ESR. Delay in testing can decrease ESR, while delay in reading can increase it. Other factors that increase ESR include tilting of the tube, increase in temperature, mechanical vibration, and the length of the tube.
Now, let's go to lab staining, which is important for differentiating certain conditions. This technique is commonly used to differentiate chronic myelogenous leukemia from leukemoid reactions.
In this technique, we stain for alkaline phosphatase present in neutrophils. The specimen of choice is fresh capillary blood, but we can also use blood collected with heparin as an anticoagulant.
The procedure involves counting 100 neutrophils and scoring their staining intensity from 0 to 4. To solve the LAP score, multiply the grade by the number of cells counted and total the scores.
The LAP score is commonly used to differentiate leukemia reactions from chronic myelogenous leukemia, where CML will have a decreased LAP score and leukemoid reactions will have an increased LAP score.
Lastly, we have immunophenotyping, which is significant in diagnosing blood malignancies, especially leukemia. For acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), specific CD markers are expected based on the affected lymphocyte population.
For early precursor B-cell ALL, we expect CD34, CD19, TDT, and HLA-DR. For intermediate pre-B-cell ALL, we expect CD10, CD19, HLA-DR, and TDT. For T-cell ALL, we expect CD2, CD3, CD4, CD5, CD7, and CD8.
For selected mature B-cell neoplasms, we have chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL). Each has specific CD markers that are important for differentiation.
This concludes our last part of the hematology lecture series. Thank you!