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Urine Sediment Photomicrographs/Photographs Case History CM/CMP-17

The urine sample is from a 38-year-old female with three days of nausea, vomiting and diarrhea. Laboratory data include: Specific Gravity = 1.020, pH = 7.0, blood, leukocyte esterase, glucose, ketones and nitrite = negative, Protein = 1+.

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Granular cast 33 100.0 1747 95.7 2767 98.3 Good

CM

/CM

P-17

The arrowed object was correctly identified as a granular cast by 98.3% of participants. Granular casts are semitransparent cylinders containing fine or coarse granules. The size and number of granules is variable. Distinction between finely and coarsely granular casts is of no importance. The granules are the result of breakdown of cells in a cellular cast or are aggregates of plasma proteins. They have little clinical significance, unless present in very large numbers. They may be increased after strenuous exercise, fever, dehydration and stress. Large numbers are associated with renal glomerular or tubular disease.

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Urine Sediment Photomicrographs/Photographs Case History CM/CMP-18

The urine sample is from a 38-year-old female with three days of nausea, vomiting and diarrhea. Laboratory data include: Specific Gravity = 1.020, pH = 7.0, blood, leukocyte esterase, glucose, ketones and nitrite = negative, Protein = 1+.

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Yeast/ fungi, extracelluar 23 100.0 1821 99.6 2812 99.9 Good

CM

/CM

P-18

The arrowed objects were correctly identified as yeast/fungi by 99.9% of participants. Yeast are ovoid and colorless, with a refractile appearance. Yeast may show branching, or hyphae. They may be confused with red blood cells, but are smaller, measuring only about 2 mm in size, and have smooth thick walls. Yeast in urine may be a contaminant from air or skin, or may be due to a true urinary tract infection. Large numbers of white cells will be seen with a true infection. The most common type is Candida. Infections are more common in diabetics and immunocompromised patients.

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Urine Sediment Photomicrographs/Photographs Case History CM/CMP-19

The urine sample is from a 14-year-old female who is asymptomatic. Laboratory data include: Specific Gravity = 1.012, pH = 6.0, blood = 1+ (small), leukocyte esterase, glucose, ketones, nitrite and protein= negative.

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Erythrocyte 33 94.0 1571 85.9 2656 94.3 Good

CM

/CM

P-19

The arrowed object was correctly identified as a red blood cell by 94.3% of participants. Red blood cells are intact biconcave discs in the urine, measuring about 7.6 microns. They swell in hypotonic urine and shrink in hypertonic urine. When the urine is both alkaline and dilute, “shadow” or “ghost” cells may appear. These cells have burst and released most of their hemoglobin. The presence of red cells in the urine is termed erythrocyturia. They may be confused with yeast, but are larger, usually round with thinner walls and no budding.

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Urine Sediment Photomicrographs/Photographs Case History CM/CMP-20

The urine sample is from a 14-year-old female who is asymptomatic. Laboratory data include: Specific Gravity = 1.012, pH = 6.0, blood = 1+ (small), leukocyte esterase, glucose, ketones, nitrite and protein= negative.

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Uric acid 14 100.0 1817 99.5 2800 99.5 Good

CM

/CM

P-20

The arrowed object was correctly identified by 99.5% of participants. Uric acid crystals are considered to be a normal constituent of urine, and are very common. They occur in acidic urine and may be yellow or red-brown. They take a variety of forms including rhombic, rosettes, wedges, 4-sided plates, barrel and lemon shapes. They are soluble in 10% sodium hydroxide and at 60 degrees centigrade. They are insoluble in glacial acetic acid. Uric acid crystals are strongly birefringent with compensated polarized light. Rarely, the needle-shaped crystals of monosodium urate are seen in urine.

Roberta Zimmerman, MD and Rachel Harjes Hematology and Clinical Microscopy Resource Committee

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Body Fluid Photomicrographs/Photographs

Case History CM/CMP-21 The patient is a 58-year-old female with remote history of Hodgkin lymphoma, status post radiation, and chemotherapy 30 years ago. The patient was diagnosed with breast carcinoma two years ago and recently completed chemotherapy for metastases secondary to breast cancer. She was also recently diagnosed with therapy-related myelodysplastic syndrome. Pleural effusion developed, and pleural fluid sample laboratory findings include: WBC = 262/μL (0.26 x 109/L) and RBC = 11,300/μL (11.3 x 109/L).

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Erythrocyte 17 100.0 1209 99.2 1987 99.6 Good

CM

/CM

P-21

Erythrocyte was correctly identified by 100.0% of the referees and 99.6% of the participants. The arrowed cells are mature erythrocytes. These cells have round to oval shaped and are anucleated with homogeneous pink cytoplasm.

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Body Fluid Photomicrographs/Photographs

Case History CM/CMP-22 The patient is a 58-year-old female with remote history of Hodgkin lymphoma, status post radiation, and chemotherapy 30 years ago. The patient was diagnosed with breast carcinoma two years ago and recently completed chemotherapy for metastases secondary to breast cancer. She was also recently diagnosed with therapy-related myelodysplastic syndrome. Pleural effusion developed, and pleural fluid sample laboratory findings include: WBC = 262/μL (0.26 x 109/L) and RBC = 11,300/μL (11.3 x 109/L).

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Macrophage 16 94.0 853 70.4 1444 72.5 Good Macrophage w/ small lipid vacuoles 1 6.0 320 26.4 499 25.1 Acceptable

CM

/CM

P-22

The arrowed cell indicates macrophage. This was correctly identified by 100.0% of the referees and 97.6% of the participants. The nuclear chromatin has a dense reticular pattern. The cytoplasm is light blue and contains multiple small vacuoles and phagocytic debris.

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Body Fluid Photomicrographs/Photographs

Case History CM/CMP-23 The patient is a 58-year-old female with remote history of Hodgkin lymphoma, status post radiation, and chemotherapy 30 years ago. The patient was diagnosed with breast carcinoma two years ago and recently completed chemotherapy for metastases secondary to breast cancer. She was also recently diagnosed with therapy-related myelodysplastic syndrome. Pleural effusion developed, and pleural fluid sample laboratory findings include: WBC = 262/μL (0.26 x 109/L) and RBC = 11,300/μL (11.3 x 109/L).

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Malignant cell (non-hematopoietic) 16 94.0 950 78.5 1589 80.0 Educational

CM

/CM

P-23

This was correctly identified as tumor cells by 94.0% of the referees and 80.0% of the participants. These cells are enlarged and have high nuclear to cytoplasmic ratios. Note the large size of the tumor cells in contrast to the size of the red blood cells that are also present in the field. Malignant tumor cells can form clumps or aggregates.

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Body Fluid Photomicrographs/Photographs

Case History CM/CMP-24 The patient is a 58-year-old female with remote history of Hodgkin lymphoma, status post radiation, and chemotherapy 30 years ago. The patient was diagnosed with breast carcinoma two years ago and recently completed chemotherapy for metastases secondary to breast cancer. She was also recently diagnosed with therapy-related myelodysplastic syndrome. Pleural effusion developed, and pleural fluid sample laboratory findings include: WBC = 262/μL (0.26 x 109/L) and RBC = 11,300/μL (11.3 x 109/L).

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Macrophage containing neutrophil(s)

(Neutrophage) 17 100.0 1116 92.2 1918 96.5 Educational

CM

/CM

P-24

The arrowed cell was correctly identified as macrophage with neutrophil by 100.0% of the referees and 96.5% of the participants. The cytoplasm contains small vacuoles and a relatively intact neutrophil that is recognizable by its lobulated nucleus. The macrophage nucleus is distorted and appears to be ‘pushed’ to one side. These nuclear features can be seen in macrophages that contain large amounts of phagocytic debris.

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Body Fluid Photomicrographs/Photographs

Case History CM/CMP-25 The patient is a 58-year-old female with remote history of Hodgkin lymphoma, status post radiation, and chemotherapy 30 years ago. The patient was diagnosed with breast carcinoma two years ago and recently completed chemotherapy for metastases secondary to breast cancer. She was also recently diagnosed with therapy-related myelodysplastic syndrome. Pleural effusion developed, and pleural fluid sample laboratory findings include: WBC = 262/μL (0.26 x 109/L) and RBC = 11,300/μL (11.3 x 109/L).

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Neutrophil 17 100.0 1194 98.4 1960 98.4 Good

CM

/CM

P-25

The arrowed cell is a neutrophil and was correctly identified by 100.0% of the referees and 98.4% of the participants. The neutrophil nucleus is U-shaped with coarse and clumped nuclear chromatin and small granules are in the cytoplasm.

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Body Fluid Photomicrographs/Photographs

Case History CM/CMP-26 The patient is a 58-year-old female with remote history of Hodgkin lymphoma, status post radiation, and chemotherapy 30 years ago. The patient was diagnosed with breast carcinoma two years ago and recently completed chemotherapy for metastases secondary to breast cancer. She was also recently diagnosed with therapy-related myelodysplastic syndrome. Pleural effusion developed, and pleural fluid sample laboratory findings include: WBC = 262/μL (0.26 x 109/L) and RBC = 11,300/μL (11.3 x 109/L).

Referees CM

Participants CMP

Participants

Performance Identification No. % No. % No. % Evaluation

Cholesterol crystal 27 96.4 1202 99.8 1972 99.6 Good

CM

/CM

P-26

The arrowed cell was correctly identified as a cholesterol crystal by 99.6% of the referres and 99.8% of the participants. In this field, the cholesterol crystals appear as flat plates. The arrowed crystal has a notch in the corner, a finding characteristic of cholesterol crystals. Cholesterol crystals may occur when there is rapid cell breakdown.

Discussion This case depicts a patient, who developed a second malignancy (breast carcinoma) after treatment of Hodgkin lymphoma. The patient was found to have metastatic breast cancer involving the lungs with the development of recurrent pleural effusions.

This patient was initially diagnosed 30 years ago with Hodgkin lymphoma and received radiation and chemotherapy. She has been in remission with no evidence of lymphoma recurrence. Approximately two years ago, mammography studies indicated the presence of a breast mass that was biopsied and showed to be invasive carcinoma. Despite extensive chemotherapy, the patient developed metastatic tumor involving the lungs. Pathology findings confirmed the presence of metastatic breast carcinoma. The patient also developed recurrent large pleural effusions. These large effusions necessitated thoracentesis to remove the fluid, which was subsequently analyzed in the laboratory. Clinical Information From a clinical perspective, this case is challenging. The patient has a history of two distinct malignancies with differences in clinical presentation, pathogenesis, and treatment modalities. Second malignancies can occur in patients who have been successfully treated for Hodgkin lymphoma.

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Myelodysplasia or the development of a second malignancy can occur as late treatment related complications and occur greater than 15 years following successful treatment of the lymphoma. Various factors have been attributed to the development of a second malignancy. For Hodgkin lymphoma, factors include initial age of the patient at diagnosis and treatment and the types of treatment modalities. Breast cancer is a greater risk for young patients, i.e. especially those individuals less than 30 years of age when diagnosed and treated for Hodgkin lymphoma. Another risk factor is mantle radiotherapy, a treatment previously used to treat Hodgkin’s lymphoma. In mantle radiotherapy, radiation was administered to a relatively large area of the body especially to the main lymph node regions in the neck, chest, and under-arm locations. Portions of the lung, heart, and shoulders were shielded from the radiation beam, and the ‘protected’ area was shaped like a cloak or mantle. Hence, the term mantle radiotherapy was applied. With the increased use of chemotherapy in the treatment of Hodgkin’s lymphoma, radiation beams are now applied to a much smaller region of the body.

Breast cancer may develop in a chest field that has been previously irradiated. The doses and number of cycles for radiation treatments and/or chemotherapy can contribute to the development of second malignancies. The contribution of these factors in the evolution of second malignancies continues to be investigated.

Pleural Fluid/ Laboratory Evaluation Both lungs are surrounded by thin membranous structures known as pleura. The pleura are comprised of two layers, consisting of an outer portion (parietal pleura) and an inner portion (visceral pleura). The parietal pleura attach to the chest wall, and the visceral pleura cover the lungs. Between the parietal and visceral pleura is a thin space or cavity, which contains a minute amount of fluid. Pleural fluid can accumulate when there are changes in capillary pressure, permeability, or lymphatic drainage. Fluid can accumulate within the pleural cavity following trauma or can be secondary to an infectious process or to malignancy. Large amounts of fluid may accumulate and removal of fluid from the pleural cavity becomes necessary.

Thoracentesis is an invasive procedure whereby a needle and cannula are placed into the thorax for the removal of fluid. This procedure can be used as a diagnostic or therapeutic tool. The fluid removed may improve a patient’s pulmonary function and comfort (therapeutic outcome) or be analyzed in the laboratory for hematological, chemical microbial, and cellular evaluations (diagnostic outcome).

A fluid may be classified as an exudate or transudate. Exudates occur when there is damage to blood vessels that result in the escape of proteins and cells from the blood across capillary walls. This can occur in infections and malignancies. Transudates form when the fluid outflow exceeds the normal resorption across a membrane. This can be seen in congestive heart failure, liver failure of cirrhosis, and kidney failure. A transudate has a low protein count, (less than 3.0 g/dl) and a specific gravity that less than 1.015. An exudate has a higher protein content and specific gravity than those of a transudate. For an exudate, the protein content is generally more than 3.0 g/dL and the specific gravity is greater than 1.015. In general, the number of cells is lower in a transudate than in an exudate.

Pleural effusions are not uncommon in patients with metastatic breast carcinoma. Fluid can be evaluated for the presence of malignant cells. The detection of malignant cells in fluids is clinically important since the identification of such cells may be the first indication of a serious and potentially life threatening disease. The presence or absence of malignant cells provides useful information to clinicians in planning appropriate oncological treatment modalities.

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The presence of non-malignant cells in pleural fluid can provide additional medical information on a patient. Infections may be suspected in patients with increased white blood cells including neutrophils or lymphocytes. The presence of cholesterol crystals indicates rapid cellular breakdown (cell death). Cholesterol crystals can be seen in patients who have recurrent chronic inflammatory effusions. These crystals can also be seen in patients who have malignant effusions or in those who are receiving chemotherapy.

In summary, this case highlights the importance of continued clinical followup for the development of second malignancies in patients who have been treated for Hodgkin lymphoma. The appropriate identification of cells in a body fluid sample is of clinical importance in the diagnoses and treatment of patients. References 1. Galagan KA, Blomberg D, Cornbleet PJ, Glassy EF (eds). Color Atlas of Body Fluids. Northfield, IL:

College of American Pathologists; 2006. 2. Wolden SL, Hancock SL, Carlson RW, et al. Management of Breast Cancer After Hodgkin’s Disease. J

Clin Oncol 18: 765-772, 2000. 3. Underwood JCE. (ed). General And Systemic Pathology 4th ed. Philadelphia, PA: Churchill Livingstone;

2004. 4. Kumar V, Abbas A, Fausto N. Robbins and Cotran Pathologic Basis Of Disease 7th ed. Philadelphia, PA:

Elsevier Saunders; 2005. 5. 2009 Hematology, Clinical Microscopy, And Body Fluids Glossary published by the College of American

Pathologists

Patricia A. Devine, MD Hematology and Clinical Microscopy Resource Committee

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Clinical Microscopy Miscellaneous Photomicrographs/Photographs

Referees CMM

Participants CMMP

Participants

Performance Identification No. % No. % No. % Evaluation

Ferning is present 37 100.0 749 99.5 1306 99.7 Good

CM

M/C

MM

P-36

This vaginal wet preparation exhibits ferning. The fern test is used to detect ruptured amniotic membranes and the early onset of labor. A vaginal pool sample is collected and the fluid is allowed to air dry on a glass slide. The slide is examined using a microscope to detect ferning, an elaborate arborized crystallization pattern. Ferning, in conjunction with the Nitrazine test and the medical history, is highly sensitive for the detection of ruptured membranes. The “fern test” was initially described in 1955 and its ease of use and clinical utility has been confirmed by multiple published studies.

Referees CMM

Participants CMMP

Participants

Performance Identification No. % No. % No. % Evaluation

No neutrophils are present 37 100.0 985 98.5 1748 98.2 Good

CM

M/C

MM

P-37

This stool specimen is negative for neutrophils. Assessment of stool specimens for neutrophils is a test that can be used in conjunction with a bacterial culture in the evaluation of enteritis/colitis. While the presence of neutrophils is consistent with a bacterial infection, the findings are not specific. Stool cultures are more sensitive and specific for the evaluation of enteric pathogens.

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Clinical Microscopy Miscellaneous Photomicrographs/Photographs

Referees CMM

Participants CMMP

Participants

Performance Identification No. % No. % No. % Evaluation

Pinworm or pinworm eggs are

present 37 100.0 906 99.9 1576 99.7 Good

CM

M/C

MM

P-38

This stool specimen has Enterobius vermicularis (pinworm) present. Enterobius vermicularis is also called human pinworm. (Adult females: 8 to 13 mm, adult male: 2 to 5 mm.) Humans are considered to be the only hosts of E. vermicularis.

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Life Cycle:

Eggs are deposited on perianal folds . Self-infection occurs by transferring infective eggs to the

mouth with hands that have scratched the perianal area . Person-to-person transmission can also occur through handling of contaminated clothes or bed linens. Enterobiasis may also be acquired through surfaces in the environment that are contaminated with pinworm eggs (e.g., curtains, carpeting). Some small number of eggs may become airborne and inhaled. These would be swallowed and follow the same development as ingested eggs. Following ingestion of infective

eggs, the larvae hatch in the small intestine and the adults establish themselves in the colon . The time interval from ingestion of infective eggs to oviposition by the adult females is about one month. The life span of the adults is about two months. Gravid females migrate nocturnally

outside the anus and oviposit while crawling on the skin of the perianal area . The larvae contained inside the eggs develop (the eggs become infective) in 4 to 6 hours under optimal

conditions . Retroinfection, or the migration of newly hatched larvae from the anal skin back into the rectum, may occur but the frequency with which this happens is unknown. References: http://www.cdc.gov

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Clinical Microscopy Miscellaneous Photomicrographs/Photographs

Referees CMM

Participants CMMP

Participants

Performance Identification No. % No. % No. % Evaluation

Eosinophils are present 38 100.0 912 99.9 1606 99.2 Good

CM

M/C

MM

P-39

This photomicrograph demonstrates a Wright-Giemsa nasal smear. Three eosinophils with bright orange-red spherical granules are seen. Nasal smears for eosinophils are useful in distinguishing the nature of a nasal discharge, eosinophils being associated with allergic rhinitis. These cells have nuclei with two lobes separated by a thin filament. Nonallergic causes of nasal discharge will typically be acellular or show a predominance of neutrophils. Several rod-shaped bacteria which stain deeply basophilic with the Wright-Giemsa stain are also present.

Referees CMM

Participants CMMP

Participants

Performance Identification No. % No. % No. % Evaluation

No yeast or other fungal element is

present 39 100.0 1070 97.7 1927 98.1 Good

CM

M/C

MM

P-40

This photomicrograph demonstrates an unstained KOH smear. Use of a 10 percent KOH (potassium hydroxide) solution can assist in the detection of fungi. KOH disrupts proteinaceous material and dissolves cellular more material more rapidly than it does the chitinous cell wall of fungi. The resulting cleared background enables the detection of fungal hyphal elements, yeast cells, and arthrospores. In this photo, no yeast cells are seen.

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Clinical Microscopy Miscellaneous Photomicrographs/Photographs

Referees CMM

Participants CMMP

Participants

Performance Identification No. % No. % No. % Evaluation

Spermatozoa are present 38 100.0 1016 99.8 1859 99.9 Good

CM

M/C

MM

P-41

This photomicrograph demonstrates an unstained vaginal wet preparation. The wet preparation is often examined to diagnose causes of vaginal discharge or a postcoital wet preparation can be used to assess for sperm and the interaction between sperm and cervical mucus. A sample of vaginal secretions is taken from the posterior vaginal pool using a cotton or dacron-tipped swab. It is mixed with nonbacteristatic saline on a slide. Spermatozoa are identified in this photo. The sperm head is about 4 to 6 micrometers long while the slender tails are about 40 to 60 micrometers long.

Deborah A. Perry, MD and Sarah L. Lott, MD

Hematology and Clinical Microscopy Resource Committee