Lab Chronicles: My Experience in the Hematology Laboratory

The clinical hematology laboratory contains two different types of testing: hematological and coagulation testing. On the hematological side of testing, the main test performed is the complete blood count (CBC), which directly measures the number of red blood cells, white blood cells, platelets, and hemoglobin, as well as indirectly measuring other values by calculations (such as the mean corpuscular hemoglobin). These tests are performed using a well-mixed whole blood sample that has been collected in a tube containing ethylene diamine triacetic acid, also known as EDTA. Before hematological results are sent out to the healthcare team, they are reviewed by the medical laboratory technologist (MLT). Any abnormal results that are seen by the MLT (either increased or decreased beyond reference ranges) are made into slides and further microscopic analyses are performed. Some results may even be “critical” in the sense that they are so abnormal that the patient may be at risk of harm if the result is not reported immediately. While reviewing parameters, certain “checks” are also performed as an internal quality control system for the sample. One of these kinds of systems include delta checks, which are a comparison of a patient’s previous results to those most current. While changes in values can occur due to the physiological state of the patient, these internal checks are also great for identifying if a sample was taken from the wrong patient. In addition, H&H checks are performed between the patient’s hemoglobin and hematocrit. The hematocrit should match the hemoglobin within certain values, and if not, an issue with the specimen quality is present, and this should be addressed before results are reported for that patient.

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The UniCel DxH 800 by Beckman Coulter is the hematology analyzer used in our laboratory

As previously mentioned, all abnormal specimens have slides that are created, stained, and reviewed microscopically. While reviewing hematological slides, the technologist comments on red blood cell size, abnormal shapes, the different types of white blood cells present, abnormalities in platelets, etc. Blood cells can be abnormal for a variety of reasons, either nutritional (such as iron deficiency anemia or megaloblastic anemia), genetic (such as thalassemia or sickle cell anemia), due to microorganisms (such as mononucleosis or malaria), or even cancerous (such as leukemias or lymphomas). It is incredible to be able to see and determine these different maladies from the changes they cause in the blood.

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Peripheral blood smear of chronic myelogenous leukemia (CML) with myelocytic cells ranging from blasts to segmented neutrophils

Other miscellaneous testing performed by the hematology laboratory include several manual tests, such as the estimated sedimentation rate (ESR), which is a nonspecific test for inflammation and is used many times in the differential diagnosis to rule in or out inflammatory conditions. Other manual tests performed include the MonoSpot test for the detection of infectious mononucleosis caused by the Epstein-Barr virus. Sickledex® is another manual test performed in the hematology laboratory that is a screening test for sickle cell anemia. In this test, test tubes containing buffer have a patient’s blood sample added to them and if a patient has sickle cell anemia, the characteristic sickling of red blood cells will cause the buffer to become cloudy in the test tube.

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Sickledex test: positive control exhibits cloudiness, while negative control is clear. Patient 1 is positive for the presence of sickle cells, while second patient is negative. Further tests will need to be completed in order to determine if patient has sickle cell anemia or sickle cell trait.

Specimens other than blood samples can be tested in the hematology laboratory, such as bone marrow aspirates or body fluids. Whenever a patient has leukemia, a bone marrow aspirate can be collected and the sample is stained using a variety of stains to be reviewed morphologically. One of these stains is a Wright’s stain, a routine hematological stain used to observe morphology of cells present in the bone marrow. In addition, bone marrow aspirates are also stained with Perl’s Prussian Blue stain that determines the presence and amount of iron in the bone marrow. Body fluids are another type of sample that can be analyzed in the hematology laboratory, in performing body fluid cell counts and microscopic reviews in order to detect diseases or malignant processes. Finally, cerebrospinal fluid is also analyzed whenever a patient is suspected of having meningitis. These specimens are analyzed immediately for cell counts, and slides are made in order to be viewed under the microscope.

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Bone marrow aspirate stained smear with the presence of spicules.

Another section of the hematology laboratory is the coagulation laboratory, which tests the components responsible for the clotting of blood. Samples for this part of the laboratory are collected in a blue top tube containing sodium citrate that removes calcium in the blood to prevent it from clotting. Specimen collection is crucial for these types of samples as the amount of anticoagulant present in relation to sample is very important, therefore, the sample should fill the tube. The blood is then centrifuged in order to separate the red blood cells from the plasma, and the plasma is used for testing as it contains the coagulation factors that participate in the clotting of blood. Coagulation factors react in sequence, termed the coagulation cascade, in order to produce the final product of a fibrin clot. The coagulation cascade is separated into three groups: the intrinsic, extrinsic and common pathways. One coagulation pathway that is stimulated by substances present in the blood, such as collagen, is termed the intrinsic pathway. Coagulation factors within this pathway include: factors XII, XI, and IX. The Activated Partial Thromboplasin Time (APTT) tests for deficiencies present within these factors, as well monitoring heparin therapy. A coagulation pathway that is stimulated by the presence of external substances from injured tissue, such as tissue factor, is the extrinsic pathway. Only one coagulation factor is present within this pathway, factor VII. The Prothrombin Time (PT) tests for the presence of any deficiencies within this coagulation factor, as well as for monitoring warfarin therapy. The International Normalized Ratio (INR) is calculated along with the PT result in order to standardize the results across a wide range of reagents. Finally, the common pathway includes coagulation factors that are shared between both intrinsic and extrinsic pathways, and factors present within this pathway are factors X, II, and V. Both the APTT and PT are used in the detection of deficiencies within this pathway. Whenever there are deficiencies observed in any of these coagulation pathways, a mixing study is performed in order to rule out the presence of inhibitors against coagulation factors. This study is performed by mixing the patient’s plasma sample with normal patient plasma that contains normal levels of coagulation factors. If the result is corrected after adding this normal plasma, then the factor is decreased due to a deficiency. If the result is not corrected, then an inhibitor may be present and further testing needs to be performed in order to identify the inhibitor.

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The coagulation cascade including intrinsic, extrinsic and common pathways.

Additional tests commonly performed in the coagulation laboratory are the Thrombin Time (TT), Fibrinogen, and D-Dimer tests. The TT is performed to measure deficiencies or dysfunction in fibrinogen. In addition, the Fibrinogen assay measures fibrinogen activity. Fibrinogen is an important coagulation factor, because once activated, it creates the final product of coagulation, the fibrin clot. Finally, the D-Dimer test measures the breakdown products of fibrin by plasmin. A negative result of this test is commonly used to rule out venous thrombosis, however a positive result may be present in a variety of disorders, such as deep venous thrombosis, as well as pregnancy or liver disease. Therefore, the D-Dimer test is not very specific and additional tests will have to be performed to rule in venous thrombosis.