Body Fluid Examination

Transcription

1 Specimen Handling Body Fluid Examination CSF: Treated as STATS because of cell lysis Studies have shown approximately 40% of CSF WBCs will have lysed after 2 hours at RT. Only 15% had lysed if the fluid was held at 4 degrees C Amniotic Fluid and Synovial Fluid also requires one hour rule because of cell lysis Pleural, Peritoneal and Pericardial Fluids may be examined ASAP. Smears may be made up to 24 hours later on refrigerated specimens. Body Fluid Examination QUALITY CONTROL: To QC Cell Counts Cell-Chex Chex from Streck LI and LII completed each shift To QC Cell Identification CAP Surveys Challenge Slides 2

2 April Challenge This is a cytospin prep of pleural fluid from an 80 year old male with CHF (Congestive Heart Failure). Identify the cells pictured. a. Monocyte/macrophages b. Reactive mesothelial cells c. Malignant cell d. Normal mesothelial cells Correct ANSWER: Normal Mesothelial cells Manual Cell Count Examine the fluid in the tube macroscopically and microscopically~ if there are cellular clumps or clots, MANUAL cell counts are performed. Body Fluid Examination 3

3 Body Fluid Examination Electronic Cell Count Blank is run prior to specimen WBC limit < 0.09 RBC limit < Results considered in the reportable range WBC: > 0.20 = 200 RBC: > = 10,000 Limitations: Cellular interference High WBC counts make a significant impact on the RBC count Never run BAL or Amniotic Fluid Differential Protocol: When to prepare a Cytospin Prep All CSF with over 5 Nucleated Cells/mm3 All body fluids, unless M.D. requests only a cell count. It is important to scan body fluids for the presence of Malignant Cells. All orders for CSF Cytology. 4

4 Optimum Cytospin Slide Quality Agitate fibrinous clots Saline wash technique for fibrinous, mucoid or highly viscous fluids specimens Pour 0.5 to 1 ml of fluid into a tube and add 10 ml of Isoton. Mix. Spin down gently in conventional centrifuge (1800 RPMs for 7 min) and make push smears from sediment. Remove supernatant fluid and prepare a cytospin prep from the sediment. Be sure to add 1 drop of albumin to the final dilution. Optimum Cytospin Slide Quality Tips for bloody: 1 drop fluid + Isoton until faint blood tinge Mucoid fluids: Mucolytic product like Mucolexx Highly viscous fluids (Synovial Fluids): Weigh 5 mg. aliquots (a pinch) of hyaluronidase and store in the freezer. Add a small volume of fluid (~0.5-1 ml) to the hyaluronidase and mix well or vortex for several seconds. Typically, the specimen will liquefy quickly (within 5 minutes). Longer incubation and/or placing the fluid in the 37 o dri-bath warmer may be needed for more resistant specimens. There is no dilution factor when hyaluronidase is used. Cytospin preps can be made from the liquefied specimen. 5

5 Optimum Cytospin Slide Quality Dilution of cellular specimens Develop a dilution scheme to avoid introducing too many cells to the cytospin chamber. Overcrowding the preparation distorts cellular morphology. Both the WBC and RBC count must be considered in developing a scheme. Suggested scheme: Maximum cell count/suspension: WBC 500/uL RBC 5,000/uL Amount cell suspension/chamber: 5 drops Optimum Cytospin Slide Quality Improper Dilution: Individual Cells can not be identified Proper Dilution of cellular specimens Proper Dilution: Same patient- Reactive Neutrophilia 6

6 Optimum Cytospin Slide Quality Cellularity Cytospin prep must show ample cellularity Cells are concentrated 20-fold by cytocentrifugation Even hypocellular samples with chamber counts of zero can yield ~35 cells per smear THE KEY: Use the maximum (0.5 ml) Optimal Cellularity WBC = 7 PREP MUST BE CELLULAR TO INSURE THAT ABNORMAL CELLS/or CONDITIONS ARE DETECTED. The Negative Gram Stain Use established guidelines when making fluid dilutions. These are not absolute criteria and should be modified as necessary to produce a cellular prep. IMPORTANT: Always use the maximum volume (0.5 ml) for preps when the WBC count is less than 50. 7

7 Optimum Cytospin Slide Quality Cytospin chamber technical tips Insure that the clip, funnel, and filter card are in place to deposit a thin layer of cells in a clearly defined area of a microscope slide. The filter card absorbs any excess fluid. For sample volumes of up to 0.5 ml, use the White Filter Card It provides a cell deposition area of 6 mm (28 mm squared). For samples volumes of up to 0.4 ml, such as Spinal Fluids for example, use the Brown Filter Card and Cap. It allows for a slower absorption of fluids. Use both brown and white card for bloody specimens. Add drop of 22% Albumin to the cytospin funnel before the addition of CSF. The albumin enhances the adherence of cells to the glass slide and reduces cell smudging or disintegration, particularly for low protein specimens such as CSF. Optimum Cytospin Slide Quality The use of albumin is not recommended for fluids other than CSF as body fluids typically contain abundant protein. Adding albumin to other body fluids may make it more difficult to produce a good prep as the excess protein clogs the filter card and reduces fluid absorption. 8

8 Optimum Cytospin Slide Quality When the fluid has a high protein content: Better slide preparation will be obtained if albumin is NOT added. All amniotic and many body fluids fall in this category. CSF specimens with a high h protein value also should not have albumin added. MOST CSF preps benefit from the addition of albumin. Optimum Cytospin Slide Quality When using Albumin, watch for INTRAcellular vs EXTRAcellular BACTERIA 9

9 DO NOT let unspun fluid run onto slide Tilt the cytospin funnel forward when removing it from the cytospin head. Artifactual changes will occur if residual fluid remaining in the cell chamber is allowed to flow back onto the slide. When this occurs, the stained cells may appear shrunken or rounded-up. Optimum Cytospin Slide Quality Cytospin Artifact Cells in Interior may be smaller with a denser nucleus than cells at the periphery 10

10 Cytospin Artifact: Nucleus Prominent Nucleolus Distorted Shape to nuclear membrane Fragments or Budding Nuclear Holes Cytospin Artifact: Nucleus Hypersegmentation g Peripheral p nucleus 11

11 Cytospin Artifact: Cytoplasm Fragmentation Cytoplasmic Shredding Localization of granules Cytospin Smear Quality Control Scan the entire cytospin prep under 10x BEFORE performing the differential If 10x scan is not done, it is possible that low volume malignant cells may be missed! Maximum volume (0.5 ml) must be used for low counts. Remake all slides that do not show good cellular distribution. If possible, review the patient diagnosis and previous reports to be alerted to the type of malignant cell that may be present. 12

12 Cerebrospinal Fluid Normal Reference Values Macroscopic Findings Microscopic Findings Cellular Morphology = Clinical Significance Malignant Cells Formed in the choroid plexus by filtration and active transport. The normal rate of CSF production is approximately 20 ml per hour. 20% of the CSF is contained in the ventricles, the rest contained in the subarachnoid space in the cranium and spinal cord. In normal adults, the CSF volume is 125 to 150 ml CSF Production The choroid plexus consists of projections of vessels and pia mater that protrude into the ventricular cavities as frond-like villi. The villi contain capillaries in a loose connective stroma. A specialized layer of ependymal cells called the choroidal epithelium overlies these villi Science Photo - Choroid plexus secretory cells, SEM 13

13 Normal Reference Values For Cerebrospinal Fluid Leukocyte count Leukocyte differential 0-5mononuclearcells cells Adults Lymphocytes 60% + 20 Monocytes 30% + 15 Neutrophils 2% + 4 Neonates Lymphocytes 20% + 15 Monocytes 70% + 20 Neutrophils 4% + 4 Macroscopic Findings: Color Appearance Normal= Clear Abnormal Xanthochromic= = RBC breakdown, increased bilirubin Bloody= = Intracranial hemorrhage, traumatic tap Cloudy= = Meningitis, CNS leukemia Turbidity: Graded 0-4+ based on the ability to read print through the CSF. Semi-quantitated: slightly hazy, hazy, slightly cloudy, cloudy, turbid 14

14 Tramatic Tap vs Subarachnoid Hemorrhage Indications of a traumatic tap: Clearing of the fluid as it is aspirated, with > blood in tube #1, < blood in tube #3 Colorless supernatant Clot in the sample Indications of pathologic bleeding: Xanthrochromia Erythrophagocytosis Positive D-dimer for cross-linked fibrin DEPOCYTE Macroscopic Appearance Cloudy = Bacterial Infection Things are not always as they seem Beware the Depocyte Used for the intrathecal treatment of neoplastic meningitis due to breast cancer Gross appearance- Milky to Cloudy Cytospin prep does not yield WBCs WBC 15

15 Microscopic Findings: Normal CSF Cells Mature Lymphocytes Reactive Lymphocytes: Reported as Few, Mod, or Many PLASMA CELL Eccentric nucleus Perinuclear halo Deep blue cytoplasm Monocytes Normal CSF Cells Intracranial Hemorrhage In body fluids, monocytes will become MACROPHAGES/HISTIOCYTES 16

16 Normal CSF Cells Erythrophage : Macrophage with variable number of phagocytosed erythrocytes Siderophage : Hemosiderin granules are golden-brown when unstained, but a deep Blue-purple stained with Wright stain. Macrophage with Hematin Crystal: Orange, rhomboid-shaped Crystals of hematoidin from the breakdown of hemoglobin Normal CSF Cells Lipophage : Macrophage with multiple small clear vacuoles. 17

17 Normal CSF Cells Immature neutrophils Mature Neutrophils Hypersegmentation maybe present Immature neutrophils are not seen normally Normal CSF Cells NRBCs/Nucleated Red Blood Cells Associated disease states and conditions: Peripheral blood contamination due to traumatic tap or hemorrhage 18

18 NRBCs/ Nucleated Red Blood Cells Normal CSF Cells With a Neutrophilia, beware the pyknotic BONE nucleus MARROW which resembles CONTAMINATION a NRBC Associated disease states and conditions: Peripheral blood contamination due to traumatic tap or hemorrhage Bone marrow contamination Case #1: This CSF Slide from a Neonate male is being evaluated. Identify the cells seen in cluster. 1. Malignant clump 2. Monocytes 3. Chondrocytes 4. CSF Lining cells 19

19 Benign Cells Uncommonly Found: Chondrocyte: Cartilage Cell Capillary Associated disease states and conditions: Cartilage Cells and/or capillaries may be present in postoperative states or after a traumatic tap Benign Cells Uncommonly Found: Ventricular Lining Cells 20

20 Benign Cells Uncommonly Found: Choroid plexus Lining cells Benign Cells Uncommonly Found: Ependymal Cells Lining cells 21

21 Case #2: CSF Slide from one-month old female with hydrocephalus. Identify the cells seen in cluster. 1. Lymphocytes 2. Malignant clump 3. Monocytes 4. CSF Lining Cells Benign Cells Uncommonly Found: Mitotic Cells Mitotic Figure Bone Marrow Contamination Rare in benign CSF Associated with Lymphocytosis, Usually Reactive 22

22 Case #3: CSF Slide from 4-month old female with hydrocephalus. What significant findings are demonstrated and should be included in the comments on this CSF exam? 1. Lymphocytes 2. Basophils 3. Capillaries and Brain tissue RED FLAG Use caution when examining a CSF that has been collected from a intra-ventricular shunt. Low Power BRAIN TISSUE CAPILLARY 23

23 Benign Cells Uncommonly Found: Endothelial cells which line the capillary RBCs captured traveling through the capillary Benign Cells Uncommonly Found: CSF collection from an intra-ventricular shunt Brain tissue Nucleus Nissle bodies Note: Individual neurons may be present Dendrites 24

24 Cellular Morphology= Clinical Significance Pleocytosis= when there are normal cells present in abnormal numbers Cells present which may be diagnostic of a disease process Microorganisms Malignant cells from metastatic tumors Predominance of Neutrophils ACUTE PHASE: Intracellular & Extracellular Bacteria PROLIFERATIVE: Neutrophage Mixed Cellular Reaction REPAIR PHASE: Reactive Lymphocytes 25

25 Predominance of Lymphocytes MULTIPLE SCLEROSIS Note Plasmacytoid Lymphocytes Viral Degenerative Neurological Disorders Inflammatory Conditions Case #4: CSF from 23 year old male admitted to the ER with a headache, neck stiffness and altered mental status. The cell identified by the arrow is? 1. Malignant cell 2. Benign cell 26

26 Mixed Cellular Reaction Monocytes, Macrophages, Lymphocytes, y Neutrophage Variable # of neutrophils Erythrophage Associated disease states and conditions: Lipophage Proliferative phase of bacterial meningitis Generally seen in chronic inflammatory conditions involving the meninges caused by tuberculous, leptospiral or fungal meningitis Eosinophils and Basophils EOSINOPHILS: > 5% Parasitic infection Foreign body reaction BASOPHILS: Inflammatory diseases Parasitic infections Foreign body reactions 27

27 Immediate: >6000 RBC/ul 12 hours-1 Week: Erythrophages Cellular Reaction to CNS Hemorrhage 2 days-20 weeks: Siderophages/Hematin Crystals Lipophage: Tissue necrosis Bone Marrow Contamination Immature granulocytes Nucleated RBCs Mitotic figures 28

28 Hemocytometer Microorganisms India Ink Wright Stain Cryptococcus neoformans Immunodeficient patient Microorganisms AIDS patient Candida species Burn patient Note intra- and extracellular forms 29

29 What should you report? 1. Fiber contaminant 2. Yeast with Pseudohyphae Case History: 12 year old male complaining of neck pain and fever for 24 hours, arrives in the E.R. disoriented and unresponsive. History of recent swimming party at a friend s lake home. Cytospin displayed many segmented neutrophils and necrotic debris um in diameter, with sky blue cytoplasm and a distinct, finely granular, violet nucleus? Is this a degenerating macrophage? 30

30 NOT Normal CSF Cells Primary amebic meningoencephalitis due to Naegleria fowleri Malignant Cells: Carcinoma Breast Most common sites to metastasize BREAST LUNG GASTRIC Lung Lung 31

31 Malignant Cells: Carcinoma Colon Cancer Metastatic Malignant Melanoma Malignant Cells: Clump of malignant cells are individually much larger than the RBC. The cytoplasm contains numerous dark, variable sized pigment granules. Compare the melanoma cell to the large, mono/macrophage which has phagocytized degenerate material. 32

32 Malignant Cells: Carcinoma Metastatic Malignant Melanoma What should you report? 1. Monocyte/Macrophage 2. Ventricular Lining Cell 3. Malignant Cell 33

33 Malignant Cells: Primary Brain Tumors Less than 15%h have positive CSF Cytology Choroid Plexus Carcinoma Malignant Cells: Primary Brain Tumors Glioblastoma Cells Medulloblastoma Cells Difficult to distinguish from leukemia or lymphoma cells 34

34 Case #5: CSF Slide from a 5 year old oncology patient previously diagnosed with Germinoma. Diagnosis? Malignant Cells: Primary Brain Tumors Germinoma Tumor Cells 35

35 Malignant Cells: ALL L1 L2 RED FLAG: Increased Smudges may suggest malignant or fragile cells Lymphoblasts: Note convoluted nucleus Prominent nucleoli Burkitt s Leukemia/ Lymphoma or ALL-L3 L3 Malignant Cells: ALL The malignant cell has prominent punched-out vacuoles. Normal cells do not have vacuoles overlying the chromatin in the nucleus. 36

36 Malignant Cells: Lymphoma HALLMARK: Convoluted nucleus Malignant Cells: Lymphoma Lymphoma: Displays clumping under low power Compare large Lymphoma cell to normal lymphocyte 37

37 Malignant Cells: AML 3 Year Old with AML Pleural, Peritoneal, & Pericardial Fluid cells 1. Cells listed under Cerebrospinal Fluid 2. Mesothelial cells Normal Reactive or atypical Malignant 3. Metastatic tumor cells Transudate vs. Exudate TRANSUDATE: Disease outside cavity Clear, pale, yellow EXUDATE: Disease inside cavity Cloudy, turbid, may clot due to presence of fibrinogen = Exudate 38

38 Macroscopic Appearance Case Study Neonatal patient with turbid pleural fluid Immature Lymphocytes Increased Lymphocytes Increase Lipophages Chylomicrons present Macroscopic Appearance Case Study Adult patient with turbid pleural fluid Increased Triglycerides Increase Chylomicrons History of surgery on the esphagus 39

39 Microscopic Evaluation Diagnosis: Damage or Obstruction to Thoracic Duct Eccentric nucleous Basophilic cytoplasm Perinuclear clear zone Cells Counts Increased WBC Pleural > 1,000 Peritoneal > 300 Effusion is exudate, primary disease in the body cavity 40

40 O v a r i a n Cell Counts A d e n o c a r c i n Increased RBC Traumatic tap Organ hemorrhage Malignancy Hemothorax Pancreatitis TB Serous Fluid Cells Cellular Morphology= Clinical Significance Neutrophils Associated disease states and conditions: Bacterial infection, blood contamination, crystal-induced arthropathies 41

41 Neutrophils at Work Fungal Organisms Peritoneal Fluid Broncho-pneumonia Pleural Fluid Serous Fluid Cells L.E. Cell Lupus Erythematosis Cell in Pleural Fluid Associated disease states and conditions: Lupus erythematosus Rheumatoid arthritis Oh Other autoimmune disordersd 42

42 How would you classify this cell? 1. NRBC 2. Segmented Neutrophil 3. Degenerating Cell Serous Fluid Cells Lymphocytes Associated disease states and conditions: Viral infections, especially acute EBV and CMV infection Chronic inflammatory conditions Chylous effusions Chronic: Congestive heart failure, cirrhosis, Nephrotic syndrome 43

43 How would you classify this cell? 1. Lymphocyte 2. Plasma cell 3. Reactive Lymphocyte How should you classify this cell? 1. Mesothelial Cell 2. Lymphocyte 3. Mitotic figure 44

44 Eosinophils Seen in Pneumothorax Serous Fluid Cells Associated disease states and conditions: Parasitic infections Allergy, hypersensitivity or foreign body reactions or asthma Collagen vascular disease Ventriculo-peritoneal shunts Chest tubes Pneumothorax or other trauma Fluids from patients with myeloproliferative disorders Serous Fluid Cells Basophils Peritoneal Associated disease states and conditions: Peritoneal dialysis Fluids from patients with myeloproliferative disorders MeningitisM i i i Inflammatory conditions Foreign body reactions Parasitic infections 45

45 Macrophages/ Mesothelial Neutrophage Mesothelial Cells/ Macrophages present in variable numbers in chronic effusions. Lipophage : Serous Fluid Cells Macrophage with multiple small clear vacuoles. In pleural and pericardial fluids, derived from cell injury. Lipophages lipids are from the products of cell membrane destruction ti and breakdown, so they are found in fluids with increased necrosis. 46

46 Azurophilic cellular material = Intracellular microorganism? Histoplasma capsulatum Pleural Undetermined cellular debris Pleural- Smoker Mesothelial/ Macrophages Signet Ring Descriptive term seen equally as often in benign and malignant cells 47