• Quality Control
• Specimen Handling /Safety
• Automated Hematology Instrumentation
• Differentials and RBC Morphology
• Body Fluids
• RBC Pathophysiology
• Leukocyte Pathophysiology
• Routine and Miscellaneous Hematology Procedures
• Coagulation
• Molecular Diagnostic and Immunologic Assays

1. Explain the rationale for a quality control program in the clinical hematology lab.
2. List specific areas which require surveillance.
3. Demonstrate how the performance of specific reagents are evaluated and how often.
4. Discuss the necessity of keeping written records of all surveillance.
5. Discuss the importance of periodic review of surveillance records and documentation of corrective actions taken.

SPECIMEN HANDLING/SAFETY
The first step in the accurate diagnosis of disease is the proper collection and handling of specimens. Specimen handling involves the following steps: proper specimen collection, appropriate specimen containers, accurate labeling of forms and specimens, timeliness of transport. Thorough knowledge of safety procedures is essential before performing any duties in the clinical laboratory which might be hazardous to personnel. Upon completion of this unit, the student will be able to:

1. Explain the importance of proper collection and transport of specimens.
2. Summarize criteria for evaluating specimen quality and corrective actions to be taken to resolve problems.
3. Organize and label specimens accurately.
4. Report results according to laboratory protocol.
5. Demonstrate safe techniques in handling and disposal of infectious materials according to laboratory protocol.

AUTOMATED HEMATOLOGY INSTRUMENTATION
Automated Cell Counters and Coagulation Instrumentation
Upon completion of this unit, the student will be able to:

1. Operate automated cell counters and coagulation instruments producing accurate patient results.
2. Identify inaccurate instrument results.
3. Correlate patient results with clinical significance.
4. Locate and explain the basic operating components of each analyzer.
5. Demonstrate an understanding of the maintenance routines for each instrument.
6. Perform daily maintenance routines.
7. Describe, prepare, and explain the function of each reagent used on automated cell counters and coagulation instruments.
8. State how reagents are stored when not in use on the analyzer.
9. Discuss calibration of the hematology automated cell counter and coagulation instruments.
10. Explain where to find basic troubleshooting information about the analyzer.

Perform an adjustment of the stain, buffer, and rinse on the laboratory stainer, and be able to produce a quality-stained blood smear.

1. Discuss the basic principle of flow cytometry.
2. List common fluorescent dyes used in flow cytometry.
3. Discuss hematological applications of flow cytometry.
4. Discuss cellular applications of flow cytometry.
5. Discuss flow cytometry markers used in the differentiation of various leukocytic disorders.

DIFFERENTIALS AND RBC MORPHOLOGY
Upon completion of the Hematology practicum, the student will be able to:

1. Identify all the cells in the myelocytic, monocytic, and lymphocytic series including a discussion of the major characteristics pertinent to each.
2. Compare and contrast all of the cells in the erythroid series.
3. Differentiate between the following: eosinophils, basophils, lymphocytes, monocytes.
4. Differentiate between the following: atypical lymphocytes, plasma cells, lymphoblasts.
5. Differentiate platelets from other cells on a Wright's Stained blood smear, and be able to perform a successful estimate from the smear.
6. Prepare good wedge smears and buffy coat preparations.
7. Prepare a good manual stained slide.
8. Judge if the white count matches what is seen on the smear.
9. Distinguish abnormal platelet shapes and large platelets and explain their significance.
10. Calculate a corrected white count, based on the number of NRBC seen on the smear.
11. Differentiate and grade the following on a Wright Stain smear:
•        sickle cells
•        acanthocytes
•        echinocytes
•        ovalocytes
•        elliptocytes
•        schistocytes
•        target cells
•        spherocytes
•        macrocytes
•        microcytes
•        teardrop cells
•        stomatocytes.
12. Evaluate a blood smear for RBC morphology and correctly assess and grade the following:
•        polychromasia
•        basophilic stippling
•        Howell-Jolly bodies
•        Pappenheimer bodies
•        hypochromia
13. Identify malarial parasites.
14. Correctly assess a blood smear for rouleaux.
15. Distinguish between normal and hypersegmented PMNs on a blood smear and explain their significance.
16. Judge from the stained smear if the RDW (Red Cell Distribution Width) is correct.
17. Evaluate a Wright's stained blood smear for toxic granulation and Dohle bodies and explain their significance.
18. Differentiate vacuolation in PMNs from other intra cellular structures and explain its significance.
19. Distinguish between normal and pyknotic PMN, and explain their significance.
20. Identify smudge cells on a Wright's stained blood smear.
21. Interpret RBC and WBC abnormalities in relation to pathological conditions.

BODY FLUIDS
Upon completion of the Hematology praticum, the student will be able to:

1. Determine the body source of each of the following fluids:
•        CSF
•        synovial
•        pleural
•        peritoneal
•        pericardial
•        thoracentesis
2. Determine color and clarity of each body fluid specimen and explain their significance.
3. Determine:
•        the number of tubes normally taken for CSF (3-4)
•        which tubes go to which department and why
4. Perform a cell count on the following CSF:
•        clear & colorless
•        slightly hazy and colorless
•        cloudy and white
•        cloudy and red
•        grossly bloody
•        amber and clear
5. Explain how to determine if a specimen was a bloody tap.
6. Explain the significance of a 500:1 RBC to WBC ratio.
7. Determine when a dilution is necessary, what dilution to make, and how to multiply the number of cells counted by the dilution factor to get an accurate result.
8. Make a cytospin preparation from body fluids and stain smears.
9. Examine a prepared smear of a body fluid and identify the cells present.
10. Examine cytospin preparations and identify the cell types present.
11. State the common characteristics (e.g., cell type) of a) viral meningitis and b) bacterial meningitis and be able to explain why it is important to differentiate between the two.
12. Discuss the reasons why CSF from leukemic patients should always have a cell count and differential (or cytospin if available).
13. Explain the significance of crenated RBC in CSF.
14. Explain why body fluid cell counts should be performed immediately upon arrival in the laboratory.
15. Distinguish a CSF that is contaminated with bone marrow.
16. Explain "xanthochromia" in body fluids.
17. Explain the general characteristics of malignant cells.

RBC PATHOPHYSIOLOGY
Glucose-6-Phosphate Dehydrogenase Screen
Upon completion of the Hematology practicum, the student will be able to:

1. Discuss the principle of the glucose-6-phosphate dehydrogenase (G6PD) screening test.
2. Discuss the problems involved with the test when using blood from a patient that has been transfused.
3. Discuss the problems involved with the test when using blood from a patient that has a high percentage of reticulocytes, e.g., after a hemolytic episode.
4. Discuss the function and importance of G6PD in the Hexose Monophosphate Shunt.

1. Explain the principle involved in solubility tests that analyze for the presence of sickling hemoglobin.
2. Describe the technique and method used to set up a sample for a sickle solubility test.
3. Discuss and describe a quality control procedure that is acceptable for the above procedures.
4. Discuss the importance of the Hemoglobin-S concentration in the solubility tests and the need to adjust for a low hemoglobin.

LEUKOCYTE PATHOPHYSIOLOGY

Cytochemical Stains
Upon completion of the Hematology practicum, the student will be able to:

1. Discuss the purpose for performing cytochemical stains.
2. Discuss the principles of the following cytochemical stains:
•        Periodic Acid Schiff
•        Peroxidase
•        Sudan Black
•        NASDA/NASDA-F
•        Non-specific esterase
•        Chloracetate esterase
•        Acid Phosphatase (TRAP)
3. Differentiate the expected results for each of the stains listed in objective #2 for the following disease states, and be able to interpret the results from each.
•        Acute Lymphocytic Leukemia
•        Acute Myelocytic Leukemia (M0, M1, M2, M3)
•        Acute Myelomonocytic Leukemia
•        Acute Monocytic Leukemia
•        Erythroleukemia
•        Acute Megakaryoblastic Leukemia
4. Compare and contrast the expected staining reactions for the stains listed in objective #2 for the following:
•        Normal myeloid cell series
•        Normal lymphocytes
•        Normal monocytes
•        Normal platelets, megakaryocytes
•        Normal nucleated RBC's

Leukocyte Alkaline Phosphatase Stain
Upon completion of the Hematology practicum, the student will be able to:

1. Explain the principle in the Leukocyte Alkaline Phosphatase (LAP) staining procedure.
2. Explain the procedure involved and be able to perform a Kaplow count.
3. Explain the necessity of staining a normal control slide with each set of patient slides.
4. Explain the necessity for establishing a normal range for each laboratory.
5. Discuss the importance of the LAP test in distinguishing a leukemic myeloid process from a non-leukemic myeloid reaction and be able to evaluate the disease (condition) based on the LAP score.

ROUTINE AND MISC. HEMATOLOGY PROCEDURES

Erythrocyte Sedimentation Rate
Upon completion of the Hematology practicum, the student will be able to:

1. State the normal reference ranges for an adult male, adult female, and children.
2. Discuss the procedure involved in setting up a sedrate.
3. Discuss anemia and its relationship to the sedrate.
4. Explain how coldness of the blood, polycythemia, rouleaux, and agglutination will affect the sedrate.
5. Explain how certain RBC shapes cause a decreased sedrate.
6. Judge whether a specimen is acceptable for a sedrate determination.
7. Evaluate the sedrate as a diagnostic tool.

1. Explain the procedure used in staining reticulocytes.
2. Describe the method for counting the reticulocytes and how to calculate the percent.
3. Discuss the implications of considering only the % reticulocytes rather than the absolute number of reticulocytes.
4. Clarify the type of stain that is used to stain reticulocytes and the significance of this type of stain.
5. Discuss the role of a reticulocyte. This includes shift macrocytosis, response to stress, and normal reference range.
6. Compare polychromasia on a smear to the reticulocyte count.
7. Explain the importance of proper mixing of the specimen immediately before the smears are made.
8. Discuss the clinical implications of the immature reticulocyte fraction (IRF).

1. State the site from which bone marrow is most commonly aspirated.
2. Review the general procedure of bone marrow aspiration.
3. Explain the technique used by the Hematology technologists in preparing bone marrow smears and staining procedures.
4. Define hypoplasia and hyperplasia of the bone marrow.
5. State the normal myeloid:erythroid ratio of the bone marrow.
6. Participate in as many bone marrow aspirations as possible and examine at least one bone marrow sample under the microscope.

1. State the normal reference range for a platelet count.
2. Explain the method for diluting a specimen for a manual platelet count and perform it properly.
3. State the reason for using ammonium oxalate as the unopette diluent.
4. Clarify the method employed in counting the platelet count and include the type of hemocytometer used, type of microscope used, area of hemocytometer counted, and the factor.
5. Explain the rationale for placing the hemocytometer in a moist petri disk for 10-15 minutes before performing the platelet count.
6. Describe the method employed in doing a platelet estimate.
7. Compare the platelet count and estimate, and decide if they correlate.
8. Discuss procedures that are followed when the platelet count and the platelet estimate don't match.
9. Distinguish dirt, bubbles, inclusion bodies, and uneven distribution of platelets on hemocytometer.
10. Explain the effect an uneven distribution of platelets on the hemocytometer can have on the platelet count. Describe corrective action to be taken should this occur.
11. Discuss the principle of the phase microscope.
12. Discuss disease states that may have abnormal platelet counts or morphology.
13. Explain how the patient's hematocrit and the thickness of the smear can affect the platelet estimate.

Total Eosinophil Counts*
Upon completion of this unit, the student will be able to:

1. Explain and perform the technique used in diluting blood for a total eosinophil count.
2. Discuss and perform the technique used in plating and counting a total eosinophil count.
3. Explain the derivation of the factor used in the total eosinophil count for each of the hemocytometers.
4. Compare the total eosinophil count to the absolute number and decide if they correlate. Describe steps to be taken if the two methods fail to show good correlation.

1. Define urine hemosiderin.
2. Discuss the significance of urine hemosiderin, including how it is formed.
3. Clarify the procedure involved in the smear preparation.
4. Explain the principle of the Prussian Blue stain.
5. Examine any specimens received for urine hemosiderin.
6. Explain and perform the procedure involved in performing a Prussian Blue stain.
7. Discuss the importance of filtering reagents for a Prussian Blue stain.
8. State the procedure for specimen collection.

1. Discuss the importance of nasal smears.
2. Explain the procedure used to collect the specimens.
3. Clarify the procedure used to prepare and stain the smears.
4. Summarize the smear examination procedure.
5. Examine any nasal smears that are available.

1. State the normal reference ranges for white blood cell counts and red blood cell counts for adult males and females.
2. Clarify the principle ingredients in the RCB and WBC diluting fluid and the purpose of each.
3. Perform the steps required to dilute whole blood for a manual WBC and RBC count.
4. Perform the method used in plating and counting a WBC and RBC count and discuss the proper counting areas for WBC & RBC counts.
5. Distinguish between properly and improperly filled counting chambers.
6. Perform the calculation of WBC and RBC counts and include correction for dilution and derivation of the appropriate correction factor for volume of a hemocytometer with Neubauer ruling.

COAGULATION
Upon completion of the Hematology practicum, the student will be able to:

1. Explain platelet function following subendothelial exposure and include the steps involved in adhesion, release, and aggregation.
2. State site of production of the proteins involved in coagulation, including factor VIII:C and factor VIII:VWF.
3. Discuss the coagulation factors dependent on vitamin K for their complete synthesis and the role of vitamin K in the hepatic synthesis of these factors.
4. List the coagulation factors that are components of the intrinsic system.
5. Describe the coagulation factors that are components of the extrinsic system and how this system is initiated in vivo.
6. Explain the action and function of antithrombin and it's relationship to heparin.
7. Clarify the function of protein C including site of production and the clinical significance of a protein C deficiency.
8. Name components of hemostatic function that are measured by the bleeding time.
9. Explain the principle of platelet aggregometry including the most common substances that either aggregate platelets directly or induce the release of platelet ADP.
10. Discuss the principle of the prothrombin time (PT) test including reagents used and their major components, methodology, coagulation factors measured, and normal reference range.
11. Compare prolonged prothrombin times with clinical conditions and/or disease states.
12. Discuss the purpose of the International Normalized Ratio (INR) for reporting prothrombin time test results for patients who are undergoing oral anticoagulant therapy.
13. Given the patient's prothrombin time and the International Sensitivity Index (ISI), calculate the INR.
14. Discuss the principle of the activated partial thromboplastin time (APTT) test including reagents used and their major components, methodology, coagulation factors measured, and normal reference range.
15. Compare prolonged activated partial thromboplastin times with clinical conditions and/or disease states.
16. Discuss the principle of "mixing studies" performed on patients with prolonged PT and/or APTT results. Discuss what is indicated when a prolonged patient APTT is "corrected" by the addition of normal plasma and what is indicated when a prolonged patient APTT is not "corrected" by the addition of normal plasma.
17. Compare and contrast the principle of specific coagulation factor assay tests.
18. Discuss the principle of the thrombin time test, including reagents used, and methodology.
19. Explain the principle of quantitative fibrinogen determinations using a modification of the thrombin time test, including reagents used and normal reference range. Be able to correlate abnormal results with clinical conditions and/or disease states.
20. Discuss the affect of the lupus anticoagulants on the PT, APTT, factor assay, and mixing studies.  Also indicate the action of lupus anticoagulants on both in vivo and in vitro coagulation systems.
21. Compare the principles of the laboratory tests used most frequently to diagnose von Willebrand's Disease for bleeding time, PT, APTT, factor VIII:C assay, factor VII, ristocetin cofactor activity, factor VIII:VWF antigen, and platelet aggregation studies.
22. Compare patterns of inheritance for hemophilia A and hemophilia B, and state the specific factor deficiency for each disorder.
23. Explain the effect of circulating factor VIII:C antibodies on hemophilic patients.
24. Explain the effect of vitamin K deficiency on PT and APTT results.
25. Clarify the effect of coumarin compounds on the synthesis and activation of the vitamin K-dependent coagulation factors. Discuss the rationale for coumarin therapy and state optimal therapeutic range of the PT for patients on coumarin therapy. Describe the effect of this therapy on the APTT.
26. Explain the uses of therapeutic heparin and the effect this anticoagulant has on coagulation tests.
27. Discuss the process of disseminated intravascular coagulation. List common clinical problems associated with excessive activation on the extrinsic pathway.
28. Explain the effect of DIC on antithrombin, protein C, coagulation factor and platelet levels. Explain why examination of a peripheral blood smear is important when DIC is suspected.
29. Compare and contrast the type of expected coagulation results (D-dimers, PT, APTT, thrombin time, platelet count, fibrinogen) in patients with DIC and associated fibrinolysis who are bleeding.
30. Discuss different endpoint detection methodologies used to detect clot formation by coagulation analyzers.
31. Explain the significance of improperly filled sodium citrate (blue top) tubes and the proper steps to be taken when this situation arises.
32. Explain the principle of the D-dimers test, the significance of elevated levels of D-dimers, and the importance of a negative D-dimers in terms of deep venous thrombosis.
33. Discuss the laboratory procedures used in the determination of Factor V Lieden, APC resistance, Prothrombin mutation and hyperhomocysteinemia.
34. Interpret coagulation results in relation to pathological conditions.
35. Discuss the clinical and laboratory implications of aspirin resistance.

1. At each affiliate laboratory, identify the molecular diagnostic assays utilized.
   • Explain the principle of each assay listed in #1.  
   • For each assay listed in #1, discuss its clinical significance (e.g., impact on diagnosis or treatment of associated disease).
   • Perform molecular diagnostic assays available at the affiliate site.
2. List the assays and identify the analyte in the affiliate's laboratory that utilize diagnostic immunologic techniques.
• Explain the principle of each assay listed in #2.
• For each assay listed in #2, discuss its clinical significance (e.g. impact on diagnosis)
• Perform immunological assays offered by the affiliate.

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