VEENHOVEN ET AL.
1008
charge of the platelets. Recently, Grant and Zucker 3 demonstrated an increase of negative surface charge and loss of aggregability on incubation of citrated platelet-rich plasma with EDTA at 37 C, but not at room temperature. In practice, a low platelet count, especially in the absence of hemorrhagic manifestations in the patient investigated, should always raise suspicion of an invitro effect. Platelet numbers should then be checked by examination of a blood smear, by counting blood obtained from a fingerprick, and by counting in venous blood anticoagulated with heparin or citrate and kept at 37 C.
4. 5. 6. 7. 8. 9. 10.
References 11. 12.
surface charge associated with the loss of aggregability. Assessment by partition in aequous two-phase polymer systems and electrophoretic motility. Blood 52:515-523, 1978 Kjeldsberg CR, Hershgold EJ: Spurious thrombocytopenia. JAMA 227:628-630, 1974 Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-254, 1965 McMillan R: The pathogenesis of immune thrombocytopenia. CR Clin Lab Sci 8:303-331, 1977 Rhee CY: Spurious thrombocytopenia. JAMA 228:1098, 1974 Rowan RM, Fraser C, Gray JH, et al: Evaluation of a semiautomated platelet-counting system. J Clin Pathol 30:361366, 1977 Van der Schans GS, Veenhoven WA, Snijder JAM, et al: The detection of platelet iso-antibodies by membrane immunofluorescence. J Clin Lab Med 90:4-10, 1977 Shreiner DP, Bell WR: Pseudothrombocytopenia: Manifestations of a new type of platelet agglutinin. Blood 42:541-549, 1973 Walsh PN, Mills DCB, White JG: Metabolism and function of human platelets washed by albumin density gradient separation. Br J Haematol 36:281-296, 1977 Watkins SP Jr, Shulman NR: Platelet cold agglutinins. Blood 36:153-158, 1970
Metastatic Neuroblastoma in Bone Marrow Aspirate Smears DAVID R. HEAD, M.D., M.C., PETER S. KENNEDY, M.D., M.C., RICHERT E. GOYETTE, M.D., M.C.
Head, David R., Kennedy, Peter S., and Goyette, Richert E.: Metastatic neuroblastoma in bone marrow aspirate smears. Am J Clin Pathol 72: 1008-1011, 1979. The authors observed rosette formation and partially fibrillar, blue-grey, extracellular material in Wright-Giemsa-stained bone marrow aspirate smears in five preparations from three cases of neuroblastoma metastatic to bone marrow. These features are little publicized in the literature and textbooks. These findings in neuroblastoma metastatic to bone marrow should prove helpful in differentiating that entity from acute leukemia and from other metastatic small-cell neoplasms in bone marrow. (Key words: Metastatic neuroblastoma; Bone marrow aspirate; Rosette formation.) METASTATIC NEUROBLASTOMA is a common tumor in children less than 15 years of age, comprising 8-10% of childhood malignancies. It frequently metastasizes to bone, especially in children more than 1 year of age, and is the most common childhood tumor to metastasize to bone. 1,6,7 The morphology of neuroReceived November 24, 1978; received revised manuscript and accepted for publication March 6, 1979. Address requests for reprints to Dr. Head: Clinical Pathology Service, Department of Pathology and Area Laboratory Services, Brooke Army Medical Center, Fort Sam Houston, Texas 78234.
Clinical Pathology Service, Department of Pathology and Area Laboratory Services, Brooke Army Medical Center, Fort Sam Houston, Texas
blastoma in formalin-fixed paraffin-embedded sections stained with hematoxylin and eosin is common knowledge. It is characterized by small cells with hyperchromatic, round-to-elongate nuclei, scant cytoplasm, formation of Homer-Wright rosettes, and production of an eosinophilic neurofibrillar background material; ganglion-cell differentiation may be seen. 1,5,6 Its ability to mimic Ewing's sarcoma in sections of bone and acute lymphocytic leukemia in aspirate smears has been described. 2 - 5 We have recently observed five bone marrow aspirates from three cases of neuroblastoma metastatic to bone marrow that demonstrated two of these features in Wright-Giemsa-stained aspirate smears: rosette formation and partially fibrillar background matrix. The occurrence of rosettes in metastatic neuroblastoma in bone marrow smears has been little publicized. 2-48 Partially fibrillar blue-grey back-
0002-9173/79/1200/1008 $00.70 © American Society of Clinical Pathologists
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
1. Cimo PL: Pseudothrombocytopenia. JAMA 229:766-767, 1974 2. Feissly R, Ludin H: Microscopie par contrastes de phases. Rev Hematol. 4:481, 1949 3. Grant RA, Zucker MB: EDTA-induced increase in platelet
A.J.C.P. • December 1979
Vol. 72 • No. 6
BRIEF SCIENTIFIC REPORTS
1009
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
FIG. 1. Photomicrograph of bone marrow aspirate from Case 1, showing rosette with associated blue-grey background matrix. Wright-Giemsa stain x500. FIG. 2 .
Photomicrograph of bone marrow aspirate from Case 2, showing rosette with associated blue-grey background matrix. Wright-Giemsa stain x500.
HEAD, KENNEDY, AND GOYETTE
1010
ground matrix in metastatic neuroblastoma in aspirate smears has also been little publicized. 8 These two features may aid in differential diagnosis of this tumor in bone marrow aspirate smears. Report of Three Cases
Case 3. A 2'/i-year-old white girl was admitted in January 1977 with a two-month history of intermittent vomiting, diarrhea, and persistent, progressive anorexia. On physical examination, weight was 26 pounds, 3 ounces, temperature 37 C, pulse rate 130 beats/min, and blood pressure 120/40 mm Hg. Abdominal palpation demonstrated a firm, nonmovable mass, 6 x 5 x 6 cm, in the left flank. Laboratory studies included hematocrit 27%, hemoglobin 8.4 g/dl, platelet count 162,000/mm3, leukocyte count 12,700/mm3, with 66% segmented cells, 28% lymphocytes, 5% monocytes, and 1% eosinophils. Urinalysis disclosed no abnormality. Blood glucose, blood urea nitrogen, serum electrolytes, and serum calcium, phosphorous and uric acid were normal. Alkaline phosphatase was 86IU, serum glutamic oxaloacetic transaminase (SGOT) 87 IU, serum glutamic pyruvic transaminase (SGPT) 10 IU, and lactate dehydrogenase (LDH) 5,000 IU. Intravenous pyelogram demonstrated a left suprarenal mass, 5 x 5 cm. Bone survey revealed lytic lesions in the left femur and right tibia. Urinary vanillylmandelic acid and catecholamines were negative. A bone marrow aspirate from the posterior iliac crest demonstrated the presence of small, round, hyperchromatic cells, 16-35 jxm in diameter, withfineto ropy chromatin, scant to moderate bluegrey cytoplasm, prominent single to multiple large blue nucleoli, and irregular angulated nuclear shape. Some cells formed Homer-Wright rosettes, and an amorphous, blue-grey, extracellular material was seen. On the basis of the results of bone marrow aspiration and the intravenous pyelogram (IVP), a diagnosis of metastic neuroblastoma was made. Because of religious convictions, the family refused further therapy. The child was discharged against medical advice and lost to follow-up. Discussion The combination of small, round, hyperchromatic cells, Homer-Wright rosettes, and fibrillar eosinophilic extracellular background material is the hallmark of primary and metastatic neuroblastoma in hematoxylineosin-stained sections.1,5>6 However, the occurrence of rosettes in aspirate smears of metastatic neuroblastoma, and the occurrence of a blue-grey background matrix peculiar to metastatic neuroblastoma in Wright-Giemsa-stained aspirate smears, have not been widely recognized. The rosettes are quite characteristic on aspirate smears, and are best observed at low scanning power (4x objective). The blue-grey background material, corresponding to eosinophilic fibrillar background material on hematoxylin-eosinstained sections, is also very characteristic. It is located both in the center of Homer-Wright rosettes and in association with sheets of tumor cells. In some areas a fibrillar organization is seen; in others it appears amorphous. The staining of the background material with basic (oxidized methylene blue) rather than acidic (eosin) dye in the air-dried marrow smears was consistent in our three cases; it was presumably related to the difference in pH of the hematoxylin (pH 2.5) and eosin (pH 5.0) stains and the WrightGiemsa stain (buffer p H 7.0). At the higher p H of the Wright-Giemsa buffer, weak acids would be ionized and would stain with basic (blue) dyes, while at the lower p H of hematoxylin and eosin, these
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Case 1. A 9-year-old white boy wasfirstnoticed to have a mass in the left flank in July 1974. At diagnostic laparotomy he was found to have Stage III neuroblastoma characterized by hyperchromatic cells 15-30 fitn in diameter, with scant cytoplasm, irregular round-toelongate nuclear shape, eosinophilic fibrillar background, and Homer-Wright rosette formation. Incomplete surgical resection was followed by whole abdominal radiotherapy and adjuvant chemotherapy with Cytoxan®, Adriamycin®, and Vincristine®. Metastases developed in the clavicle, madible, and left indexfingerover the next 11 months. In January 1977, the patient was hospitalized with an acutely inflamed knee. Admission laboratory data included hematocrit 27%, hemoglobin 8.6 g/dl, and leukocyte count 12,900/ mm3 with 80% segmented cells, 5% bands, 7% lymphocytes, and 8% monocytes. Examination of a peripheral blood smear disclosed both anisocytosis and poikilocytosis and the presence of nucleated erythrocytes. A bone marrow aspirate from the posterior iliac crest showed infiltration by a small-cell tumor with formation of rosettes with partiallyfibrillar,blue-grey, extracellular material. Tumor cells were 15-30 /urn in diameter, with fine to ropy chromatin, irregular angulated nuclei, moderate blue-grey cytoplasm, and occasional nucleoli (Fig. 1). Thefibrillarnature of the background matrix was not apparent in all areas. Case 2. An 18-year-old black girl wasfirstseen in 1973 because of headaches, projectile vomiting, and abdominal pain. Diagnostic studies demonstrated bony destruction of the left parieto-occipital region. An emergency craniotomy disclosed a neuroblastoma involving dura and the inner cranial table. The tumor was characterized by uniform, 15-25-/xm round cells; hyperchromatic, somewhat irregular nuclei; and an eosinophilic fibrillar background. Further study demonstrated a left suprarenal mass, which was treated with 4,000 rads of external radiation to the whole abdomen, followed by chemotherapy with Vincristine and Cytoxan given during alternating weeks for one and a half years. In January 1976, the patient had recurrent symptoms of increased intracranial pressure. Recurrent cranial and dural tumor with some ganglion-cell differentiation was documented, characterized by cells 20-70 /nm in diameter, scant to moderate blue-grey cytoplasm, eosinophilicfibrillarbackground material, Homer-Wright rosette formation, and ganglion-cell differentiation. The patient was treated with 4,000 rads delivered to the whole brain. Postoperatively, chemotherapy that included Cytoxan, Vincristine, Adriamycin, and DTIC was begun. Chemotherapy was interrupted because of sustained leukopenia. In August 1976, hematocrit was 33%, hemoglobin 10.8 g/dl, leukocyte count 4,800/ mm3 with 67% segmented cells, 1% bands, 22% lymphocytes, 5% monocytes, and 5% eosinophils. A bone marrow aspirate from the posterior iliac crest demonstrated a slightly hypoplastic marrow and several clumps of large hyperchromatic cells arranged in rosettes against a background of partiallyfibrillar,blue-grey, extracellular material (Fig. 2). Tumor cells were 20-50 pm in diameter, with ropy chromatin, large blue single to multiple nucleoli, scant to abundant blue-grey cytoplasm, and irregular angulated nuclear shape. The biopsy specimen resembled that obtained from the recurrent cranial tumor in 1975. The patient's condition was clinically stable, and therefore she was not treated. In August 1977 and January 1978, repeat bone marrow aspirate and biopsy were performed and confirmed the continued presence of tumor with the features described above.
A.J.C.P. • December 1979
Vol. 72 • No. 6
BRIEF SCIENTIFIC REPORTS
weak acids would be in the nonionized state, and the material would stain with acidic dyes (eosin). The combination in bone marrow aspirates of a metastatic small-cell tumor and either rosette formation or blue-grey extracellular material—amorphous or faintly fibrillar—is strong presumptive evidence of metastatic neuroblastoma. These findings should be helpful in differentiating metastatic neuroblastoma from acute leukemia, Ewing's sarcoma, and other metastatic small-cell neoplasms, directing the subsequent tumor search in the proper direction to confirm this diagnosis. References 1. Anderson WAD (editor): Pathology. Sixth edition. St. Louis, C. V. Mosby, 1971, pp 1470-1471
1011
2. Dutcher TF: Metastatic neuroblastoma. American Society of Clinical Pathologists, Check Sample Hematology NR H-41, 1971 3. Gaffney PC, Hansman CF, Fetterman GH: Experience with smears of aspirates from bone marrow in the diagnosis of neuroblastoma. Am J Clin Pathol 31:213-221, 1949 4. Kato K, Wachter HE: Adrenal sympathicoblastoma in children. J Pediatr 12:449-462, 1938 5. Parkin JC, Reed RJ: Tumors of the Peripheral Nervous System, Atlas of Tumor Pathology. Second series, Fascicle 3. Armed Forces Institute of Pathology, Washington D. C, 1969, pp 138-140 6. Payling-Wright G, St. Clair Symmers W: Systemic Pathology. London, Green and Co. Ltd., 1966, p 1089 and p 1427 7. Rubin P (editor): Clinical Oncology for Medical Students and Physicians. Sixth edition. The American Cancer Society, University of New York, New York, 1974, pp 488-494 8. Sandoz Atlas of Haematology. Second edition. Sandoz Ltd., Basle, 1973, plate 63
SETH CROSBY
Crosby, Seth: A quick and easy method for the staining of reticulocytes. Am J Clin Pathol 72: 1011-1013,1979. This report presents a simplification of the conventional method for the staining of reticulocytes that is easier, faster and requires no extraneous equipment. Blood is applied directly to a spot of dried stain on a microscope slide. An identical slide is placed over the first and the blood and stain are mixed for about a minute. The slides are held apart by paper labels at each end. The slides are then slid apart to produce a smear on each slide. (Key words: reticulocyte). THE CONVENTIONAL METHOD for staining reticulocytes, essentially that of Brecher, 1 places equal amounts of blood and new methylene blue solution in a capillary pipette and mixes them by tipping the sample from one end of the pipette to the other. The mixture incubates for 10 min, then a drop is touched onto a microscope slide, and a push smear is made. With the new method, finished smears can be produced in about a minute. The technic requires no special skill. Materials and Methods (Fig. 1) Two microscope slides for the procedure are prepared by placing 1 /A1 of buffered, new methylene blue Received September 26, 1978; received revised manuscript and accepted for publication February 5, 1979. Supported in part by funds from NASA Grant #NSG 9061. Address reprint requests to Mr. Crosby: Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037.
Division of Hematology Scripps Clinic and Research Foundation, La Jolla, California
stain directly on the center of each slide and allowing the spot to dry (Fig. L4). The liquid stain contains 1% by weight of new methylene blue powder dissolved in a phosphate buffer adjusted t o p H 5.3. The buffer consists of 97.4% M/15 KH 2 P0 4 solution and 2.6% M/15 Na 2 HP0 4 solution. Two paper labels, one on top of the other, are placed atone end of each slide on the stained side. The patient's finger, after being scrubbed with an alcohol swab and dried, is pricked with a lancet. Enough blood to cover the stain spot is applied from the fingertip. Too much or too little blood results in an undesirably thick or inadequately stained smear. Another slide is placed directly on the first so that all four corners are aligned and the labels are sandwiched between the two slides at opposite ends from each other (Fig. IB). After allowing the slides to sit in this position for about 10 sec, the now partially dissolved stain is thoroughly mixed with the blood. This is accomplished by placing the slides on a fiat surface and applying a rapid series of quick "press-releases" with both index and middle fingers to the center of the upper slide (Fig. 1C). This manipulation is done for about a minute until the mixing is complete and the cells are
0002-9173/79/1200/1011 $00.65 © American Society of Clinical Pathologists
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
A Quick and Easy Method for the Staining of Reticulocytes
1008
charge of the platelets. Recently, Grant and Zucker 3 demonstrated an increase of negative surface charge and loss of aggregability on incubation of citrated platelet-rich plasma with EDTA at 37 C, but not at room temperature. In practice, a low platelet count, especially in the absence of hemorrhagic manifestations in the patient investigated, should always raise suspicion of an invitro effect. Platelet numbers should then be checked by examination of a blood smear, by counting blood obtained from a fingerprick, and by counting in venous blood anticoagulated with heparin or citrate and kept at 37 C.
4. 5. 6. 7. 8. 9. 10.
References 11. 12.
surface charge associated with the loss of aggregability. Assessment by partition in aequous two-phase polymer systems and electrophoretic motility. Blood 52:515-523, 1978 Kjeldsberg CR, Hershgold EJ: Spurious thrombocytopenia. JAMA 227:628-630, 1974 Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-254, 1965 McMillan R: The pathogenesis of immune thrombocytopenia. CR Clin Lab Sci 8:303-331, 1977 Rhee CY: Spurious thrombocytopenia. JAMA 228:1098, 1974 Rowan RM, Fraser C, Gray JH, et al: Evaluation of a semiautomated platelet-counting system. J Clin Pathol 30:361366, 1977 Van der Schans GS, Veenhoven WA, Snijder JAM, et al: The detection of platelet iso-antibodies by membrane immunofluorescence. J Clin Lab Med 90:4-10, 1977 Shreiner DP, Bell WR: Pseudothrombocytopenia: Manifestations of a new type of platelet agglutinin. Blood 42:541-549, 1973 Walsh PN, Mills DCB, White JG: Metabolism and function of human platelets washed by albumin density gradient separation. Br J Haematol 36:281-296, 1977 Watkins SP Jr, Shulman NR: Platelet cold agglutinins. Blood 36:153-158, 1970
Metastatic Neuroblastoma in Bone Marrow Aspirate Smears DAVID R. HEAD, M.D., M.C., PETER S. KENNEDY, M.D., M.C., RICHERT E. GOYETTE, M.D., M.C.
Head, David R., Kennedy, Peter S., and Goyette, Richert E.: Metastatic neuroblastoma in bone marrow aspirate smears. Am J Clin Pathol 72: 1008-1011, 1979. The authors observed rosette formation and partially fibrillar, blue-grey, extracellular material in Wright-Giemsa-stained bone marrow aspirate smears in five preparations from three cases of neuroblastoma metastatic to bone marrow. These features are little publicized in the literature and textbooks. These findings in neuroblastoma metastatic to bone marrow should prove helpful in differentiating that entity from acute leukemia and from other metastatic small-cell neoplasms in bone marrow. (Key words: Metastatic neuroblastoma; Bone marrow aspirate; Rosette formation.) METASTATIC NEUROBLASTOMA is a common tumor in children less than 15 years of age, comprising 8-10% of childhood malignancies. It frequently metastasizes to bone, especially in children more than 1 year of age, and is the most common childhood tumor to metastasize to bone. 1,6,7 The morphology of neuroReceived November 24, 1978; received revised manuscript and accepted for publication March 6, 1979. Address requests for reprints to Dr. Head: Clinical Pathology Service, Department of Pathology and Area Laboratory Services, Brooke Army Medical Center, Fort Sam Houston, Texas 78234.
Clinical Pathology Service, Department of Pathology and Area Laboratory Services, Brooke Army Medical Center, Fort Sam Houston, Texas
blastoma in formalin-fixed paraffin-embedded sections stained with hematoxylin and eosin is common knowledge. It is characterized by small cells with hyperchromatic, round-to-elongate nuclei, scant cytoplasm, formation of Homer-Wright rosettes, and production of an eosinophilic neurofibrillar background material; ganglion-cell differentiation may be seen. 1,5,6 Its ability to mimic Ewing's sarcoma in sections of bone and acute lymphocytic leukemia in aspirate smears has been described. 2 - 5 We have recently observed five bone marrow aspirates from three cases of neuroblastoma metastatic to bone marrow that demonstrated two of these features in Wright-Giemsa-stained aspirate smears: rosette formation and partially fibrillar background matrix. The occurrence of rosettes in metastatic neuroblastoma in bone marrow smears has been little publicized. 2-48 Partially fibrillar blue-grey back-
0002-9173/79/1200/1008 $00.70 © American Society of Clinical Pathologists
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
1. Cimo PL: Pseudothrombocytopenia. JAMA 229:766-767, 1974 2. Feissly R, Ludin H: Microscopie par contrastes de phases. Rev Hematol. 4:481, 1949 3. Grant RA, Zucker MB: EDTA-induced increase in platelet
A.J.C.P. • December 1979
Vol. 72 • No. 6
BRIEF SCIENTIFIC REPORTS
1009
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
FIG. 1. Photomicrograph of bone marrow aspirate from Case 1, showing rosette with associated blue-grey background matrix. Wright-Giemsa stain x500. FIG. 2 .
Photomicrograph of bone marrow aspirate from Case 2, showing rosette with associated blue-grey background matrix. Wright-Giemsa stain x500.
HEAD, KENNEDY, AND GOYETTE
1010
ground matrix in metastatic neuroblastoma in aspirate smears has also been little publicized. 8 These two features may aid in differential diagnosis of this tumor in bone marrow aspirate smears. Report of Three Cases
Case 3. A 2'/i-year-old white girl was admitted in January 1977 with a two-month history of intermittent vomiting, diarrhea, and persistent, progressive anorexia. On physical examination, weight was 26 pounds, 3 ounces, temperature 37 C, pulse rate 130 beats/min, and blood pressure 120/40 mm Hg. Abdominal palpation demonstrated a firm, nonmovable mass, 6 x 5 x 6 cm, in the left flank. Laboratory studies included hematocrit 27%, hemoglobin 8.4 g/dl, platelet count 162,000/mm3, leukocyte count 12,700/mm3, with 66% segmented cells, 28% lymphocytes, 5% monocytes, and 1% eosinophils. Urinalysis disclosed no abnormality. Blood glucose, blood urea nitrogen, serum electrolytes, and serum calcium, phosphorous and uric acid were normal. Alkaline phosphatase was 86IU, serum glutamic oxaloacetic transaminase (SGOT) 87 IU, serum glutamic pyruvic transaminase (SGPT) 10 IU, and lactate dehydrogenase (LDH) 5,000 IU. Intravenous pyelogram demonstrated a left suprarenal mass, 5 x 5 cm. Bone survey revealed lytic lesions in the left femur and right tibia. Urinary vanillylmandelic acid and catecholamines were negative. A bone marrow aspirate from the posterior iliac crest demonstrated the presence of small, round, hyperchromatic cells, 16-35 jxm in diameter, withfineto ropy chromatin, scant to moderate bluegrey cytoplasm, prominent single to multiple large blue nucleoli, and irregular angulated nuclear shape. Some cells formed Homer-Wright rosettes, and an amorphous, blue-grey, extracellular material was seen. On the basis of the results of bone marrow aspiration and the intravenous pyelogram (IVP), a diagnosis of metastic neuroblastoma was made. Because of religious convictions, the family refused further therapy. The child was discharged against medical advice and lost to follow-up. Discussion The combination of small, round, hyperchromatic cells, Homer-Wright rosettes, and fibrillar eosinophilic extracellular background material is the hallmark of primary and metastatic neuroblastoma in hematoxylineosin-stained sections.1,5>6 However, the occurrence of rosettes in aspirate smears of metastatic neuroblastoma, and the occurrence of a blue-grey background matrix peculiar to metastatic neuroblastoma in Wright-Giemsa-stained aspirate smears, have not been widely recognized. The rosettes are quite characteristic on aspirate smears, and are best observed at low scanning power (4x objective). The blue-grey background material, corresponding to eosinophilic fibrillar background material on hematoxylin-eosinstained sections, is also very characteristic. It is located both in the center of Homer-Wright rosettes and in association with sheets of tumor cells. In some areas a fibrillar organization is seen; in others it appears amorphous. The staining of the background material with basic (oxidized methylene blue) rather than acidic (eosin) dye in the air-dried marrow smears was consistent in our three cases; it was presumably related to the difference in pH of the hematoxylin (pH 2.5) and eosin (pH 5.0) stains and the WrightGiemsa stain (buffer p H 7.0). At the higher p H of the Wright-Giemsa buffer, weak acids would be ionized and would stain with basic (blue) dyes, while at the lower p H of hematoxylin and eosin, these
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Case 1. A 9-year-old white boy wasfirstnoticed to have a mass in the left flank in July 1974. At diagnostic laparotomy he was found to have Stage III neuroblastoma characterized by hyperchromatic cells 15-30 fitn in diameter, with scant cytoplasm, irregular round-toelongate nuclear shape, eosinophilic fibrillar background, and Homer-Wright rosette formation. Incomplete surgical resection was followed by whole abdominal radiotherapy and adjuvant chemotherapy with Cytoxan®, Adriamycin®, and Vincristine®. Metastases developed in the clavicle, madible, and left indexfingerover the next 11 months. In January 1977, the patient was hospitalized with an acutely inflamed knee. Admission laboratory data included hematocrit 27%, hemoglobin 8.6 g/dl, and leukocyte count 12,900/ mm3 with 80% segmented cells, 5% bands, 7% lymphocytes, and 8% monocytes. Examination of a peripheral blood smear disclosed both anisocytosis and poikilocytosis and the presence of nucleated erythrocytes. A bone marrow aspirate from the posterior iliac crest showed infiltration by a small-cell tumor with formation of rosettes with partiallyfibrillar,blue-grey, extracellular material. Tumor cells were 15-30 /urn in diameter, with fine to ropy chromatin, irregular angulated nuclei, moderate blue-grey cytoplasm, and occasional nucleoli (Fig. 1). Thefibrillarnature of the background matrix was not apparent in all areas. Case 2. An 18-year-old black girl wasfirstseen in 1973 because of headaches, projectile vomiting, and abdominal pain. Diagnostic studies demonstrated bony destruction of the left parieto-occipital region. An emergency craniotomy disclosed a neuroblastoma involving dura and the inner cranial table. The tumor was characterized by uniform, 15-25-/xm round cells; hyperchromatic, somewhat irregular nuclei; and an eosinophilic fibrillar background. Further study demonstrated a left suprarenal mass, which was treated with 4,000 rads of external radiation to the whole abdomen, followed by chemotherapy with Vincristine and Cytoxan given during alternating weeks for one and a half years. In January 1976, the patient had recurrent symptoms of increased intracranial pressure. Recurrent cranial and dural tumor with some ganglion-cell differentiation was documented, characterized by cells 20-70 /nm in diameter, scant to moderate blue-grey cytoplasm, eosinophilicfibrillarbackground material, Homer-Wright rosette formation, and ganglion-cell differentiation. The patient was treated with 4,000 rads delivered to the whole brain. Postoperatively, chemotherapy that included Cytoxan, Vincristine, Adriamycin, and DTIC was begun. Chemotherapy was interrupted because of sustained leukopenia. In August 1976, hematocrit was 33%, hemoglobin 10.8 g/dl, leukocyte count 4,800/ mm3 with 67% segmented cells, 1% bands, 22% lymphocytes, 5% monocytes, and 5% eosinophils. A bone marrow aspirate from the posterior iliac crest demonstrated a slightly hypoplastic marrow and several clumps of large hyperchromatic cells arranged in rosettes against a background of partiallyfibrillar,blue-grey, extracellular material (Fig. 2). Tumor cells were 20-50 pm in diameter, with ropy chromatin, large blue single to multiple nucleoli, scant to abundant blue-grey cytoplasm, and irregular angulated nuclear shape. The biopsy specimen resembled that obtained from the recurrent cranial tumor in 1975. The patient's condition was clinically stable, and therefore she was not treated. In August 1977 and January 1978, repeat bone marrow aspirate and biopsy were performed and confirmed the continued presence of tumor with the features described above.
A.J.C.P. • December 1979
Vol. 72 • No. 6
BRIEF SCIENTIFIC REPORTS
weak acids would be in the nonionized state, and the material would stain with acidic dyes (eosin). The combination in bone marrow aspirates of a metastatic small-cell tumor and either rosette formation or blue-grey extracellular material—amorphous or faintly fibrillar—is strong presumptive evidence of metastatic neuroblastoma. These findings should be helpful in differentiating metastatic neuroblastoma from acute leukemia, Ewing's sarcoma, and other metastatic small-cell neoplasms, directing the subsequent tumor search in the proper direction to confirm this diagnosis. References 1. Anderson WAD (editor): Pathology. Sixth edition. St. Louis, C. V. Mosby, 1971, pp 1470-1471
1011
2. Dutcher TF: Metastatic neuroblastoma. American Society of Clinical Pathologists, Check Sample Hematology NR H-41, 1971 3. Gaffney PC, Hansman CF, Fetterman GH: Experience with smears of aspirates from bone marrow in the diagnosis of neuroblastoma. Am J Clin Pathol 31:213-221, 1949 4. Kato K, Wachter HE: Adrenal sympathicoblastoma in children. J Pediatr 12:449-462, 1938 5. Parkin JC, Reed RJ: Tumors of the Peripheral Nervous System, Atlas of Tumor Pathology. Second series, Fascicle 3. Armed Forces Institute of Pathology, Washington D. C, 1969, pp 138-140 6. Payling-Wright G, St. Clair Symmers W: Systemic Pathology. London, Green and Co. Ltd., 1966, p 1089 and p 1427 7. Rubin P (editor): Clinical Oncology for Medical Students and Physicians. Sixth edition. The American Cancer Society, University of New York, New York, 1974, pp 488-494 8. Sandoz Atlas of Haematology. Second edition. Sandoz Ltd., Basle, 1973, plate 63
SETH CROSBY
Crosby, Seth: A quick and easy method for the staining of reticulocytes. Am J Clin Pathol 72: 1011-1013,1979. This report presents a simplification of the conventional method for the staining of reticulocytes that is easier, faster and requires no extraneous equipment. Blood is applied directly to a spot of dried stain on a microscope slide. An identical slide is placed over the first and the blood and stain are mixed for about a minute. The slides are held apart by paper labels at each end. The slides are then slid apart to produce a smear on each slide. (Key words: reticulocyte). THE CONVENTIONAL METHOD for staining reticulocytes, essentially that of Brecher, 1 places equal amounts of blood and new methylene blue solution in a capillary pipette and mixes them by tipping the sample from one end of the pipette to the other. The mixture incubates for 10 min, then a drop is touched onto a microscope slide, and a push smear is made. With the new method, finished smears can be produced in about a minute. The technic requires no special skill. Materials and Methods (Fig. 1) Two microscope slides for the procedure are prepared by placing 1 /A1 of buffered, new methylene blue Received September 26, 1978; received revised manuscript and accepted for publication February 5, 1979. Supported in part by funds from NASA Grant #NSG 9061. Address reprint requests to Mr. Crosby: Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037.
Division of Hematology Scripps Clinic and Research Foundation, La Jolla, California
stain directly on the center of each slide and allowing the spot to dry (Fig. L4). The liquid stain contains 1% by weight of new methylene blue powder dissolved in a phosphate buffer adjusted t o p H 5.3. The buffer consists of 97.4% M/15 KH 2 P0 4 solution and 2.6% M/15 Na 2 HP0 4 solution. Two paper labels, one on top of the other, are placed atone end of each slide on the stained side. The patient's finger, after being scrubbed with an alcohol swab and dried, is pricked with a lancet. Enough blood to cover the stain spot is applied from the fingertip. Too much or too little blood results in an undesirably thick or inadequately stained smear. Another slide is placed directly on the first so that all four corners are aligned and the labels are sandwiched between the two slides at opposite ends from each other (Fig. IB). After allowing the slides to sit in this position for about 10 sec, the now partially dissolved stain is thoroughly mixed with the blood. This is accomplished by placing the slides on a fiat surface and applying a rapid series of quick "press-releases" with both index and middle fingers to the center of the upper slide (Fig. 1C). This manipulation is done for about a minute until the mixing is complete and the cells are
0002-9173/79/1200/1011 $00.65 © American Society of Clinical Pathologists
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
A Quick and Easy Method for the Staining of Reticulocytes
