Hematology Exam 1 Guide and Answers PDF

Hematology 2 Exam 1 Objectives and Answers Chapter 21—Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes Level I 1. Recall neutrophilia from hematologic data and name the common disorders associated with neutrophilia - Neutrophilia refers to an increase in the total...

Hematology 2 Exam 1 Objectives and Answers Chapter 21—Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes Level I 1. Recall neutrophilia from hematologic data and name the common disorders associated with neutrophilia - Neutrophilia refers to an increase in the total circulating absolute neutrophil concentration - Neutrophilia that is not caused by malignancy most often occurs as a response to a physiological or pathologic process and is termed reactive neutrophilia (can be immediate, acute or chronic - About immediate neutrophilia o Can occur w/o pathologic stimulus and is probably a simple redistribution of the marginating pool to the circulating pool o It is transient (so only lasts 20 – 30 min) o The process is Independant of BM output and tissue egress (movement of neutrophils out of the circulation and into tissues) ▪ So no actual change in the number of neutrophils in the vasculature o The neutrophils are typically mature and normal cells o Not pathological - About acute neutrophilia o When foreign microbes are present o Fever, chills o Occurs when neutrophils egress from the BM storage pool into the PB o Withing hours following a pathologic stimulus the circulating pool of neutrophils can increased by 10 times o Increase in immature neutrophils ▪ More bands start to appear - About chronic neutrophilia o Worsening physical symptoms of fever, and nausea o Generally, follows acute neutrophilia and occurs if the stimulus for neutrophils continues beyond a few days o Causes depleted storage pool and increased production of mitotic pool in an attempt to meet the demand for neutrophils o The BM ▪ Increased myeloblasts, promyelocytes and myelocytes o The PB ▪ Increase in bands, metamyelocytes, and myelocytes 2. Describe the quantitative and qualitative neutrophil response to acute bacterial infections. - Bacterial infection is the most common cause of neutrophilia - Quantitative: o Count is usually 10 – 25 x 10^3/mcL - Qualitative o As the demand for neutrophils at the site of infection increases, the early response of the BM is to increase output of storage neutrophils to the PB and causing a left shift 3. Locate immature granulocytes and morphologic changes (toxic granulation, Döhlebodies, intracellular organisms, and vacuoles) often seen in reactive neutrophilia. 4. Describe and locate leukemoid reaction, and pyknotic nuclei on stained blood films and microscopic pictures. - Leukemoid reaction o Reaction to severe infections or necrotizing tissue o There is increased leukocyte count and a left shift o The cells have toxic granulation and Dohle bodies suggesting an infectious or toxic reaction leukocytosis ▪ Toxic granulation and vacuoles are associated with infection o In a neutrophilic-leukemoid reaction, the number of circulating neutrophil precursors is increased including bands, metamyelocytes, myelocytes and promyelocytes, and rarely, blasts. o A leukemoid reaction is transient, disappearing when the inciting stimulus is removed - Pyknotic nuclei: are smooth nuclear fragments seen when the cell is dying and disintegrating 5. Locate the conditions associated with false or pseudo-neutropenia. - Pseudo-neutropenia o It is produced by alterations in the circulating and marginated pools o Results from the transfer of circulating neutrophils to the marginated neutrophil pool with no change in the total PB neutrophil pool o In some infections with endotoxin production and of hypersensitivity reactions - False neutropenia o Could be from laboratory in vitro manipulation of blood o Examples: ▪ EDTA induced neutrophil adherence to RBC ▪ Disintegration of neutrophils over time prior to testing ▪ Disruption of abnormally fragile leukocytes during preparation of the blood for testing ▪ Neutrophil aggregation 6. Locate Alder-Reilly granules, or Chédiak-Higashi inclusions a. Alder-Reilly anomaly - Benign condition characterized by the presence of functionally normal leukocytes with large purplish granules in their cytoplasm when stained - It’s a lysosomal storage disorder - Can also appear as lymphocytes - Occur in clusters in the shape of dots and are surrounded by vacuoles - Found in the BM b. Chediak-Higashi anomaly - Rare autosomal recessive disorder associated with mutations of the CHS1 gene, which codes for a lysosomal trafficking regulatory protein - Death in the first or second decade of life - Has giant gray-green bodies and giant lysosomes in the cytoplasm - Neutropenia and thrombocytopenia are frequent complications as the disease progresses - The patients have skin hypopigmentation, silvery hair and photophobia 7. List the common conditions associated with abnormal eosinophil, basophil, and monocyte counts. - Eosinophil 8. Describe Gaucher and Niemann-Pick diseases. - Gaucher o characterized by a deficiency of B-glucosidase (an enzyme needed to break down the lipid glucocerebroside). o the macrophage is unable to digest the stroma of ingested cells, and the lipid glucocerebroside accumulates. o The clinical findings (splenomegaly and bone pain) of the disease are related to the accumulation of this lipid in macrophages mainly in the spleen, liver, and bone marrow. o The macrophages (Gaucher cells) are large with small eccentric nuclei, and the cytoplasm appears wrinkled or striated o Differential diagnosis of Gaucher disease can be confirmed by demonstrating decreased leukocyte B-glucosidase activity, whereas the enzyme level is normal or increased in myeloproliferative disorders. - Niemann-Pickc disease o Signs of the disease begin in infancy with poor physical development. The spleen and liver are greatly enlarged o the defect is a deficiency of sphingomyelinase (an enzyme needed to break down lipids), resulting in excessive sphingomyelin storage o Macrophages with a foamy appearance are found in lymphoid tissue and the bone marrow ▪ The foam cells are large (20–100 mcM) with an eccentric nucleus and globular cytoplasmic inclusions. o Leukopenia and thrombocytopenia can occur from increased sequestration by the enlarged spleen, and blood lymphocytes can contain several vacuoles that are lipid-filled lysosomes 9. Locate and contrast the abnormal macrophages seen in Gaucher disease, Niemann-pick disease, and sea-blue histiocytosis syndrome. - Gaucher disease and Niemann-pick disease are above in obj.8 - Sea blue histiocytosis syndrome o Characterized by splenomegaly and thrombocytopenia o These sea blue staining macrophages are found in the liver, spleen and BM o The cell is large in diameter with a dense eccentric nucleus and cytoplasm that contains blue or blue green granules 10. Locate neutropenia from hematologic data and name the common disorders associated with neutropenia. - True neutropenia can occur because of o decreased bone marrow production o increased cell loss (from immune destruction or increased neutrophil egress to the tissue) o pseudo-neutropenia (increased neutrophilic margination) 11. Locate and outline the clinical features of the inherited granulocyte functional abnormalities (Chédiak-Higashi, Alder-Reilly, May-Hegglin, and chronic granulomatous diseases) and differentiate their cellular abnormalities. a. May-Hegglin Anomaly - Granulocytes contain inclusions consisting mainly of RNA from rough ER - They are distinguished from Dohle bodies because they are usually larger and rounder in shape - The neutrophils function normally - Variable thrombocytopenia with giant platelets is characteristic b. Chronic granulomatous disease - Characterized by defects in the respiratory burst oxidase (NADPH oxidase) system - Patients suffer from recurrent, often life threatening, infections with opportunistic pathogens that result in the formation of granulomas - The affected cells are morphologically normal but cannot generate antimicrobial oxygen metabolites o Therefore, the neutrophils can phagocytize microorganisms but cannot kill them. - The peripheral blood neutrophil count is normal but increases in the presence of infection. - Immunoglobulin levels are often increased because of chronic infection. 12. Locate, assess, and choose appropriate corrective action for false neutropenia - Could be from laboratory in vitro manipulation of blood o Examples: ▪ EDT induced neutrophil adherence to RBC If observed on the stained smears, blood can be recollected by finger stick to make manual dilutions and blood smears without utilizing EDTA ▪ Disintegration of neutrophils over time prior to testing ▪ Disruption of abnormally fragile leukocytes during preparation of the blood for testing ▪ Neutrophil aggregation Level II 1. Discuss leukemoid reaction and laboratory data. - Reaction to severe infections or necrotizing tissue - There is increased leukocyte count and a left shift - The cells have toxic granulation and Dohle bodies suggesting an infectious or toxic reaction leukocytosis o Toxic granulation and vacuoles are associated with infection - In a neutrophilic-leukemoid reaction, the number of circulating neutrophil precursors is increased including bands, metamyelocytes, myelocytes and promyelocytes, and rarely, blasts. - A leukemoid reaction is transient, disappearing when the inciting stimulus is removed 2. Locate neutrophil nuclear alterations including Pelger-Huët, hypersegmentation, and pyknotic forms on stained blood films and microscopic pictures - All discussed in the level 3 objective 2 3. Correlate the hematologic and clinical features for leukemoid reaction. - Already discussed above Level III 1. Evaluate neutropenia to include etiology and associated conditions as well as blood and bone marrow findings. - Bone marrow o Neutropenia can develop as a result of decreased bone marrow production. o In this case, the bone marrow shows myeloid hypoplasia, and the myeloid-to-erythroid (M:E) ratio is decreased  o Defective neutrophil production depletes the bone marrow storage pool, decreases neutrophil egress to tissues, and reduces both the peripheral blood circulating and marginating pools. - Blood o Neutropenia is a characteristic finding in megaloblastic anemia and myelodysplastic syndromes o In these cases, however, the marrow is usually hyperplastic. 2. Assess the nuclear abnormalities of neutrophils including Pelger-Huët, pseudo-Pelger-Huët, hypersegmentation, and pyknotic nuclei, and reconcile them with the appropriate clinical conditions of the patient. - Pelger-Huet anomaly o The nucleus does not segment beyond the two lobe stage and can appear as a single, round nucleus with no segmentation o The bilobed nucleus is called pince-nez cells o The cell is functionally normal - Pseudo-Pelger Huet o Can be present in myeloproliferative disorders and myelodysplastic states o They are hypo granular because of a lack of secondary granules and the nuceli acquire a round rather than a dumbbell shape o The chromatin appears with intense clumping, aiding in differentiation of these - Hyper segmentation o Six or more segments - Pyknoctic nuclei o Apoptotic nuclei are found in dying neutrophils in blood or body fluid preparations o The nuclear chromatin condenses and the segments disappear, becoming smooth, dark staining spheres o In this image, the nucleated cell at the top is a dying neutrophil (you can see the smooth nucleus 3. Assess the cytoplasmic abnormalities of neutrophils including toxic granulation, Döhle bodies, vacuoles, intracellular organisms, and morulae and reconcile them with the patient’s appropriate clinical condition. - Mostly found in infectious states - Dohle bodies o They frequently come together with toxic granulation - Cytoplasmic vacuoles: o Represent the end stage of phagocytosis o They can also appear as artifact from the smear making 4. Appraise alterations in the relative and/or absolute numbers of eosinophils, basophils, and monocytes and associate them with the clinical condition of the patient. - Eosinophils  - Monocytes 5. Assess the etiology, laboratory findings, and clinical features of lysosomal storage disorders. - Most are autosomal recessive - Disruption of membrane bound sac characterized by defects in various enzymes that are involved in degradative processes leading to the accumulation of either nondegraded substrates or catabolic products that are unable to be transported out of the lysosome - Two categories based on accumulated material o Mucopolysaccharidoses (MPS) ▪ The accumulating macromolecules are found primarily in connective tissue o Lipodoses  6. Evaluate the etiology, associated conditions, and peripheral blood findings for immediate, acute, chronic, and reactive neutrophilia. Chapter 22—Nonmalignant Lymphocyte Disorders Level I 1. Locate the infectious agent and describe the clinical symptoms associated with infectious mononucleosis. - Self-limiting lymphoproliferative disease caused by Epstein Barr virus - Symptoms: o Throat soreness and reddening, coughing o Tonsil Reddening, white patches, and swelling o Swelling of the lymph nodes o Fever, chills, aches o Photophobia, fatigue, loss of appetite, malaise, headache o Enlargement of spleen o Nausea and vomiting 2. Discuss and recognize the reactive morphology of lymphocytes found in infectious mononucleosis. o Causes lymphocytosis, over 20% of lymph's reactive o Classic reactive lymph: stretched nucleus, less densely packed chromatin, basophilia of peripheral cytoplasm, scalloping around RBCs 3. Given a differential and leukocyte count, calculate an absolute lymphocyte count and differentiate it from a relative lymphocyte count. o Absolute count: WBC count x percentage of lymphocytes in decimal form o Or is it: Absolute count = (the lymphocyte %/100) x WBC count 4. Locate reactive cell morphology associated with viral infections and compare it to normal lymphocyte morphology. - Pictures throughout the guide 5. Discuss the pathophysiology of infectious mononucleosis. - EBV attaches to a receptor on the B lymphocyte membrane designated CD21, which is the receptor for the C3d complement component - Binding of the virus to the B lymphocyte activates the cell and induces the expression of the activation marker CD23, which is the receptor for a B lymphocyte growth factor - Once internalized, the virus is incorporated into the B lymphocyte genome, instructing the host cell to begin production of EBV proteins - These viral proteins are then expressed on the cell membrane. - Thus, EBV-infected cells express markers of activated B-lymphocytes as well as viral markers. The viral genome is maintained in the lymphocyte nucleus and passed on to the cell’s progeny. - This results in EBV-immortalized B lymphocytes and possible latent infection. 6. Locate absolute and relative lymphocytopenia and lymphocytosis, and list conditions associated with these abnormal counts. - Lymphocytosis (adults) o > 4.8 x 10^9/L (absolute) o > 25-35% of differential (relative)  - Lymphocytosis (6 months to 4 years) o > 8.5 x 10^9/L (absolute) o > 65% of differential (relative) - Lymphocytopenia (the chart below is only for lymphocytopenia) o Occurs when absolute lymphocyte count < 1.0 × 109/L (adults), < 2.0 × 109/L (children) 7. Discuss clinical symptoms of disorders in which a leukocytosis is caused by lymphocytosis. - Lymphocytosis is usually a self-limiting, reactive process that occurs in response to an infection or inflammatory condition. - Both T and B lymphocytes are commonly affected, but their function remains normal. - Conditions o Infectious Mononucleosis o Bordetella Pertussis 8. Discuss the abnormal hematological findings associated with AIDS. o Leukopenia, lymphocytopenia, reactive lymph's, possible neutropenia, possible thrombocytopenia o Mild-moderate anemia, low iron o MCV > 110 fL after receiving AIDS prevention medication 9. Outline the pathophysiology of HIV infections and describe how it affects lymphocytes. o HIV virus infects helper T lymphocytes via the CD4 receptor. o They deplete these helper T lymphs, as well as effecting the monocytes and macrophages o The normal CD4: CD8 ratio is 2:1 but HIV patients usually have a 1:2 inverted ratio, meaning they have significantly less CD4 cells 10. List the complications associated with cytomegalovirus (CMV) infections. o CMV infection in the immunosuppressed can be life-threatening. o Most common viral infection complicating tissue transplants and significant cause of mortality in immunocompromised patients o Produces flu like symptoms 11. Describe the pathophysiology of CMV infection, and give clinical findings associated with it. o Caused by Herpes-group cytomegalovirus o Lymphocytosis o Congenital or acquired o Usually subclinical or mild flu-like symptoms o Negative heterophile, requires serological testing o Causes decrease in CD4+ cells and increase in CD8+ cytotoxic T lymphocytes o Classic reactive lymphocytes, some may have cleaved nucleus o Laboratory evaluation of the CBC indicates a leukocytosis with an absolute lymphocytosis.  12. Write the pathophysiology of toxoplasmosis infections and explain the resulting lymphocytosis. o Infection of the parasite Toxoplasma gondii o Lymphocytosis o Transmitted through ingestion of oocytes in cat feces or undercooked meat o Results in leukocytosis with increased reactive lymph's and increased relative/absolute lymph counts o Differentiate from mono: toxoplasmosis has a negative heterophile antibody test and mono is positive for the test. Specific serological tests for T. gondii for diagnosis o Reactive lymph's darker/more basophilic than classic reactive lymph 13. Discuss the cytopenia, locate the defect, and recognize the laboratory features in congenital qualitative disorders of lymphocytes. - Congenital disorders are usually characterized by a decrease in lymphocytes and impairment in either cell-mediated immunity (T lymphocytes), humoral immunity (B lymphocytes), or both - NOTE: I don’t know what else to write 14. Discuss hyper IgE syndrome (HIES) and explain its role in the pathophysiology of the disorder. - are rare, primary immunodeficiency diseases that involve both humoral and cellular immunity deficiencies. - They are characterized by elevated serum IgE, eczema, recurrent staphylococcal skin abscesses, recurrent lung infections, and eosinophilia. - Autosomal-dominant HIES, the most common disease in this group, results from STAT3 gene mutations and has a variety of connective tissue and skeletal abnormalities - STAT3 is necessary for normal IL-17 cytokine signaling and regulation. Deficiencies in IL-17 due to STAT3 mutations lead to decreased differentiation of T helper 17 (TH17) lymphocytes and result in a primary immunodeficiency. Both T and NK lymphocytes are decreased. Level II 1. Correlate the heterophile antibody test to infectious mononucleosis.  - They are rapid, cheap and specific tests that can be performed from the onset of symptoms of infectious mononucleosis - Positive: heterophiles are present --> infectious mononucleosis Level III 1. Evaluate and resolve/explain conflicting results from peripheral blood morphology and serologic tests in suspected Epstein-Barr virus (EBV) infection. - In peripheral blood - In bone marrow o Bone marrow aspirations in EBV infection are not indicated, but when performed show hyperplasia of all cellular elements except neutrophils. - In serologic tests o Serologic tests are used to differentiate this disease from similar more serious diseases (e.g., diphtheria, hepatitis).  o The blood of patients with infectious mononucleosis contains greatly increased concentrations of transient heterophile antibodies that agglutinate sheep or horse erythrocytes but are not specific for EBV o Antibodies specific for EBV can be identified by first absorbing the nonspecific heterophile antibodies from patient serum with guinea pig antigen and testing the absorbed serum with horse erythrocytes. o The infectious mononucleosis IgM antibodies react with horse erythrocytes. o Positive agglutination of horse erythrocytes by treated serum indicates EBV infection. o A patient with all clinical manifestations and peripheral blood findings of infectious mononucleosis occasionally does not have a positive heterophile test (heterophile-negative syndrome). o In 10–20% of adult cases and 50% of children younger than 10 years of age, the test is negative in the presence of EBV infection. o In other cases, the heterophile-negative syndrome is caused by a non-EBV viral infection. o The most likely causative agent is cytomegalovirus. o Antibody responses might not be detected in immunocompromised individuals (those in whom the immune response is suppressed either naturally, artificially, or pathologically). o Viral load testing can be important in immune-compromised patients 2. Evaluate and confirm antibody titers found in infectious mononucleosis with respect to the various EBV viral antigens. 3. In Bordetella pertussis infection, research the cause of lymphocytosis and recognize laboratory features associated with it. - Aka whooping cough - A pertussis toxin secreted by the bacteria causes an accumulation of lymphocytes in the blood by recruiting lymphocytes into the peripheral circulation and blocking their migration back into lymphoid tissue.  - Infection with B. pertussis causes a blood picture very similar to that of reactive viral lymphocytosis - Hematologic picture in whooping cough is leukocytosis with lymphocytosis o The rise in leukocytes is caused by an absolute lymphocytosis of T, B, and natural killer (NK) lymphocytes along with increases in neutrophils and monocytes. o The lymphocytes appear as small cells with folded nuclei Chapter 30—Morphologic Analysis of Body Fluids in the Hematology Laboratory Level I 1. List the types of body fluids studied in the clinical laboratory, and describe the body cavities in which they are found. - Pericardial = heart & Pleural = lungs o lined with mesothelial cells; serous fluid predominantly mononuclear - Bronchoalveolar Lavage (BAL) = sputum; bronchial cells - Cerebrospinal = ependymal/arachnoid cells, choroid plexus cells o Predominantly monocytes/lymphocytes - Peritoneal--> torso/stomach cavity = ASCITES ; mesothelial cells - Synovial --> joint fluid; synovial cells; mononuclear cells - Spinal Cord --> CSF o Xanthochromic= evidence of lysis/trauma to cells “in vivo”; past injury 2. List the physical characteristics of various fluids. - CSF: normally clear and colorless with viscosity like water - Serous fluids: pale yellow and transparent. Slightly thicker than water - Synovial fluid: transparent, viscous, colorless or pale straw colored 3. Describe cell-counting and slide preparation techniques. - To ensure accurate cell counts, the preferred sample for hematology analysis is one submitted in an anticoagulant tube containing EDTA or sodium heparin. - Viscous synovial fluids should be treated with hyaluronidase before performing cell counts or preparing cytocentrifuge slides. - A common rule is to perform the cell count within 1 hour of collection. This 1-hour limit is critical for accurate CSF analysis because studies have shown significant WBC loss, particularly neutrophils, at 1 hour post collection. - Serous and synovial fluids have been shown to have improved cellular stability when refrigerated for 24 hours, but these guidelines may not apply to fluids that form because of sepsis or inflammation. - The WBC count performed on body fluids is more correctly reported as a total nucleated cell count (TNCC). - Cell counts performed on CSF traditionally include both the TNCC and the RBC count because the RBCs can be helpful in detecting a subarachnoid hemorrhage - You can also do it manually by a hemocytometer o Clear fluids can be counted undiluted o The pipet used to insert the fluid into the counting chamber can be rinsed with a stain such as new methylene blue before drawing up the fluid to assist in differentiating RBCs from WBCs when performing the cell count. o To prevent any dilutional effect, the stain should be thoroughly expelled from the pipet before drawing the sample. o Dilutions should be made for hazy, cloudy, or bloody fluids based on an estimate of cellularity that can be performed by observing a cover-slipped drop of fluid on a slide - Manual slide preparation o Samples with low nucleated cell counts (less than 1,000 or 2,000/mcL) should be concentrated before making the smear. o An aliquot (1–2 mL) of the fluid can be centrifuged to concentrate the cells in a button on the bottom of the tube. o Centrifuging at a low speed (1000–1500 rpm) for 10 minutes causes less disruption of the cells than centrifuging at a higher speed. o The supernatant is removed, and the button of cells is gently resuspended. o One drop of 10–22% albumin should be added to the resuspended cells for serous and CSF samples. o A drop of the concentrated cells can then be used to prepare a wedge smear similar to a peripheral blood smear; the wedge smear can be made directly from the sample on highly cellular fluids 4. Describe the appearance of bacterial and fungal organisms in Wright-stained preparations. - Intracellular and extracellular bacterial cocci in joint fluid  o Most pathogenic yeasts are found in CSF rather than in pleural, pericardial, or peritoneal fluids and can be found intracellularly - Level II 1. Identify the procedure used to obtain each type of body fluid. 2. Evaluate the significance of microorganisms found in Wright-stained body fluid preparations. (I will put all the pictures of the cells we took here too) - Neutrophils  o Might have vacuoles because they are lacking the nutrients for these cells o The one with an arrow is a dying neutrophil - Monocytes o Monocytes that have undergone a transformation to phagocyte o They are also categorized as histiocytes - Lymphocytes o They are not reactive here; it looks like this because of the making of the smear - Mesothelial cells  - Intracellular and extracellular bacterial cocci in joint fluid o Most pathogenic yeasts are found in CSF rather than in pleural, pericardial, or peritoneal fluids and can be found intracellularly 3. Identify crystals that can be found in joint fluids and associate them with particular disorders. - Answered in the synovial fluid section below Chapter 39—Automation in Hematology and Hemostasis Level I 1. Cite the electrical impedance principle of cell counting and identify the instruments that use this technology. - Based in increased resistance that a blood cell with poor conductivity passes through an electrical field - The number of pulses indicates the blood count, and the amplitude/ height of each pulse is proportional to the size of the cell - Examples: Beckman Coulter, Sysmex, an Abbott diagnostics 2. Describe the use of radio frequency in cell counting, and identify the instruments that use this technology. 3. State the principles of light scatter used in cell counting, and identify the instruments that use this technology.  - Based on light scattering measurements obtained from a single cell when it is passed through a beam of light or laser - Photodetectors detect forward and side scatter - Degree of forward scatter (low angle) is the measure of the cell's size/ volume - Degree of side scatter (high angle) is the measure of cell complexity or granularity - Examples: Siemens Healthcare instruments 4. List the reported parameters for each blood cell–counting instrument. - Coulter® LH Series & Sysmex XE-Series™: o CBC o Five-part leukocyte differential o Nucleated erythrocytes o Reticulocyte count 5. Categorize cell parameters as directly measured and derived from a histogram, scattergram or cytogram, or calculation. - Coulter o Histogram ▪ The shaded area represents those cells used in the red cell distribution width (RDW) calculation ▪ WBC histogram and count o Scatter plot - Sysmex o NRBC Scattergram o Scattergram  6. Describe the principle of reticulocyte count enumeration by automated blood cell– counting instruments. - Sample is mixed with new methylene blue reagent in heated reticulocyte dilution chamber - Residual RNA gets precipitated within reticulocytes - Sample sent to VCS flow cell where contour gating takes place. - This process identifies the fraction of immature reticulocytes and classifies reticulocytes vs. mature RBCs Level II 1. Compare and contrast the methods of analysis for the described histograms, scatterplots, scattergrams, and cytograms, and interpret the results. - Described above 2. Describe and interpret the automated reticulocyte parameters. - Described above Cerebrospinal Fluid 1. Describe the formation of CSF and state at least three functions that the CSF performs. - Produced primarily (70%) from secretions of the four ventricles of the brain by the choroid plexus; small amount from ependymal cells (the cells that line the brain and spinal cord)  - It is described as a selective secretion from plasma NOT as an ultrafiltrate evident by ▪ Higher levels of sodium, chloride, and magnesium than does plasma ▪ Lower concentration of potassium, calcium and protein than does plasma - Functions ▪ Protects and supports the brain and spinal cord from trauma ▪ Provides a medium for the transport and exchange of nutrients and metabolic wastes ▪ Maintain blood brain barrier 2. Describe the procedure for lumbar puncture and the proper collection technique for CSF. - Collected by aseptic lumbar puncture in third or fourth lumbar interspace (for kids) with local anesthesia - Involves significant patient discomfort and can cause complications, so it must be done properly - The spindle needle is advanced into the lumbar interspace, and often a “pop” is heard on penetration of the dura mater - Immediately after the dura mater has been entered and before CSF has been removed, the physician takes initial or “opening” pressure with manometer attached to the spindle needle - If the pressure is within normal range (50 to 180 mmHg), then 20 mL of CSF can be collected - If pressure is less than or greater than normal, only 1 to 2 mL should be removed - It is then collected into three sequentially labeled tubes: ▪ Tube 1: Chemistry ▪ Tube 2: Microbiology (we don’t want to introduce microbial flora, so it should never be the first tube ▪ Tube 3: Cell counts (hematology) 3. Discuss the importance of timely processing and testing of CSF and state at least three adverse effects of time delay on CSF specimens. - Delay in testing could cause: ▪ Falsely low cell counts caused by the lysis of WBC ▪ Falsely high lactate levels because of glycolysis ▪ The recovery of viable microbial organisms is jeopardized 4. State the physical characteristics of normal CSF and discuss how each characteristic can be modified in disease states. - Normal: clear and colorless with viscosity like water - Clarity or turbidity related to number of cells ▪ Pleocytosis; increased number of cells in CSF causes it to appear cloudy ▪ Cloudy CSF associated with WBCs greater than 200 cells/mL or RBCs more than 400 cells/mL  - Xanthochromia: abnormal color of CSF, usually yellow (bilirubin), orange (combination of both), or pink (brain bleed) due to various conditions ▪ Often caused by the presence of breakdown products of red blood cells - Presence of visible blood ▪ Must differentiate between traumatic puncture (colorless when spinned) and subarachnoid (hemorrhage – when spinned there will be blood in the bottom and yellow liquid) intracerebral bleeding ▪ Traumatic tap Greatest amount of blood in tube 1, least in tube 3 ▪ Hemorrhage Consistent amount of blood in all three tubes After centrifugation, xanthochromic supernatant Macrophages with phagocytized RBCs Macrophages stain positive for hemosiderin and may include hematoidin crystals Pink: oxyhemoglobin Yellow: bilirubin Orange: combination of both 5. Discuss the clinical importance of the microscopic examination of CSF + Cell counts and types of WBCs present - In adults, normal cell count is 0 to 5 WBCs per microliter, specifically lymphocytes and monocytes ▪ Increased in diseases of CNS and other conditions - RBCs are not normally present - Cell counts are preformed immediately to prevent lysing of WBCs - Cells counts performed with hemacytometer on undiluted (or diluted depending on clarity) specimen counting all nine squares on both sides ▪ Adults: WBCs 0–5 cells/mcL ▪ Neonates: WBCs 0–30 cells/mcL ▪ Children to 1 yr: WBCs 0–20 cells/mcL ▪ 1 yr to adolescence: WBCs 0–10/mcL ▪ Predominant WBCs are monocytes and lymphocytes. - Pleocytosis: ▪ Neutrophils In bacterial meningitis, up to 90% of WBCs can be neutrophils Early in viral, fungal, TB or parasitic infections Some noninfectious conditions ▪ Lymphocytes Increased in viral, TB, fungal or syphilitic meningitis especially in later stages ▪ Plasma cells Are abnormal when seen in multiple sclerosis and acute viral and chronic inflammatory conditions - Other cells  ▪ Monocytes ▪ May be increased in a mixed cell pattern such as TB or funal meningitis, chronic bacterial meningitis, or rupture of cerebral abscess o Eosinophils ▪ 10% or greater with parasitic, fungal or allergic reactions ▪ Following injection of radiographic contrast media or medications ▪ Can also result from an allergic reaction to malfunctioning intracranial shunts o Macrophages ▪ Often found after hemorrhage because of phagocytic ability 6. Interpret data from a CSF analysis and correlate the data to a probable condition trauma vs brain bleed. Synovial Fluid 1. Describe the formation and function of synovial fluid. - Formed by the ultrafiltration of plasma across the synovial membrane and from secretions by synoviocytes (synovial cells) - Results in viscous fluid to serve as a lubricant for joints and a nutrient source for metabolically active articular cartilage - Composition: o Glucose and uric acid same as plasma o Total protein and immunoglobulins vary from one-fourth to one-half that of plasma - Function: Cushions the ends of bones and reduces friction when you move your joints 2. Summarize the four principal classifications of joint disease. - The following are only used as a guide - Four principles categories ▪ Noninflammatory: joint disorder ▪ Inflammatory: lupus, rheumatoid arthritis ▪ Septic: hazy of cloudy caused by bacteria, fungi, crystals can show ▪ Hemorrhagic - Categories can overlap - Several conditions can occur in the joint at the same time - Variations in test results can occur depending on the stage of disease process in the patient 3. Classify synovial fluid as normal, non-inflammatory, inflammatory, septic, or hemorrhagic using various laboratory results. 4. Discuss appropriate tubes for the collection and distribution of synovial fluid specimens; discuss the importance of timely specimen processing and testing. - It is removed by arthrocentesis (percutaneous aspiration from a joint using aseptic technique and disposable sterile equipment) - If possible, patient should be fasting a minimum of 4 – 6 hours to allow chemicals to balance - Normal fluid volume is 0.1 to 3.5 mL - Sample normally collected in three portions ▪ Tube 1: no anticoagulant tube for chemical and immunologic studies (red top tube) Chemical examinations: lactate, lipids (cholesterol, triglycerides), protein, uric acid, glucose ▪ Tube 2: anticoagulant tube for microscopic studies Microscopic examinations: total cell count, differential cell count crystal identification, cytologic studies ▪ Tube 3: sterile anticoagulant tube for microbiological studies Microbiological studies: cultures - If processing and testing is delayed the following could occur; ▪ Cells in the synovial fluid can alter the chemical composition ▪ Detection of microbial organisms can be jeopardized ▪ Blood cells (RBCs, WBCs) can lyse o Note: refrigeration adversely effects the viability of microorganisms and could induce in vitro crystalline precipitation 5. State the physical characteristics of normal synovial fluid and discuss how each characteristic can be modified in disease states. - Normal: pale yellow or colorless and clear - Color o Abnormal Colors:  ▪ Red or brown: associated with trauma during collection procedure or disorders that disrupt synovial membrane allowing blood to enter joint cavity ▪ Greenish or purulent: in some infectious conditions ▪ Milky (white): tuberculous arthritis, systemic lupus erythematosus) - Clarity o Abnormal clarity Numerous substances that can affect synovial fluid clarity can be identified by microscopic examination o WBCs, RBCs and synoviocytes o Crystals, fat droplets o Fibrin, cellular debris, rice bodies o Rice bodies are white, free-floating substances made up of collagen covered by fibrinous tissue Resemble polished, shiny grains of rice of various sizes Seen in many arthritic conditions o Dark-pepperlike particles called ochronotic shards can be present in synovial fluid from individuals with ochronotic arthropathy, a consequence of alkaptonuria and ochronosis These are pieces of pigmented cartilage that has eroded and broken loose into fluid o Viscosity: all below 6. Identify the substance responsible for the viscosity of synovial fluid. - Synovial fluid has high viscosity due to high concentration of mucoprotein hyaluronate (a polymer that serves as a lubricant) - During inflammatory conditions, hyaluronate can be depolymerized by enzyme hyaluronase present in bacteria and some neutrophils, thus becoming water like - Other diseases can inhibit production and secretion of hyaluronate by syoviocytes - Asses by expelling fluid from collection syringe and observing for a normal string formation at least 4 cm long before breaking 7. Correlate the cells and crystals observed during the microscopic examination of synovial fluid with various joint diseases. Normal o Total nucleated cell count