Vol. 93 • No. 4
BRIEF SCIENTIFIC REPORTS
20. Otto BS, duPlessis DJ. Detached ciliary tufts in semen. S Afr Med J 1987;72:441. 21. Papanicolaou GN. Degenerative changes in ciliated cells exfoliating from the bronchial epithelium as a cytologic criterion in the diagnosis of diseases of the lung. NY State J Med 1956;56:26432650. 22. Papanicolaou GN, Bridges EL, Railey C. Degeneration of ciliated cells of the bronchial epithelium (ciliocytophthoria) in its relation to pulmonary disease. Am Rev Respir Dis 1961;83:641-659. 23. Patek E. The epithelium of the human fallopian tube. A surface ultrastructural and cytochemical study. Acta Obstet Gynecol Scand 1974,31:1. 24. Pierce CH, Hirsch JG. Ciliocytophthoria: relationship to viral respiratory infections of humans. Proc Soc Exp Biol Med 1958;98: 489-495. 25. Pierce CH, Knox AW. Ciliocytophthoria in sputum from patients with adenovirus infections. Proc Soc Exp Biol Med 1960; 104: 492-495.
545
26. Poropatich C, Ehya H. Detached ciliary tufts in pouch of Douglas fluid. Acta Cytol 1986;30:440-442. 27. Rilke F, Alasio L. Detached ciliary tufts in cervico-vaginal smears. Acta Cytol 1976;20:189-190. 28. Rosenblatt MB, Trinidad S, Lisa JR, Tcherthoff V. Specific epithelial degeneration (ciliocytophthoria) in inflammation and malignant respiratory disease. Dis Chest 1963;43:605-612. 29. Schueller EF. Ciliated epithelia of the human uterine mucosa. Obstet Gynecol 1968;31:215-223. 30. Sidawy MK, Chandra P, Oertel YC. Detached ciliary tufts in female peritoneal washings. Acta Cytol 1987;31:841-843.
31. Smith JW, McQuay RM, Ash L, Melvin DM, Orihel TC, Thompson JH, eds. Atlas of diagnostic medical parasitology. Chicago: American Society of Clinical Pathologists Press, 1984:14. 32. Verhage HG, Bareither ML, Jaffe RC, Akbar M. Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women. Am J Anat 1979;156:505-522.
GERMAN A. PIHAN, M.D. AND BRUCE A. WODA, M.D.
A method for the immunophenotypic analysis of bone marrow cells in cases of failed bone marrow aspiration is described. Cell suspensions are obtained by mechanical disaggregation of bone marrow core biopsies. The isolated cells are stained with the appropriate antibodies and analyzed by flow cytometry. The usefulness of the method is illustrated by presenting immunophenotypic data obtained in eight consecutive cases accessioned by the authors' laboratory. The method is simple and reproducible. It allows for parallel morphologic examination with Romanovskitype stains and is capable of generating multivariate, quantitative, immunophenotypic data useful in the diagnosis of leukemia and lymphoma. (Key words: Leukemia; Lymphoma; Flow cytometry) Am J Clin Pathol 1990;93:545-548 T H E DIAGNOSIS of hematologic neoplasms that involve the bone marrow relies upon the morphologic examination of smears prepared from aspirated bone marrow particles and stained with Romanovski-type stains. In some clinical situations, however, morphologic examination alone does not provide sufficient diagnostic informat i o n . 5 9 " The use of immunophenotypic analysis of bone marrow cells has increased the diagnostic accuracy and sensitivity of bone marrow examination. It also has provided previously unavailable information regarding prog-
Department of Pathology, University of Massachusetts Medical Center, Worcester, Massachusetts
nosis and selection of therapeutic regimens. 2 " 4 , 7 1 2 Traditionally, immunophenotyping has been performed in cells obtained by bone marrow aspiration. 4 " 7 1 2 Occasionally, however, because of a variety of factors, among which bone marrow fibrosis and hypercellular or "packed" bone marrow appear the most important, aspiration does not yield bone marrow particles, and only a core biopsy can be obtained. 8 Routine histologic study of the core biopsy in these cases of failed bone marrow aspiration may be insufficient to render a definitive diagnosis. The authors describe a method to isolate cells from core biopsies and to use the isolated cells for morphologic and immunophenotypic examination. The authors illustrate the usefulness of this approach in the diagnosis of hematologic malignancies by presenting data on eight consecutive cases of failed bone marrow aspiration accessioned by their laboratory. Materials And Methods
Received May 8, 1989; received revised manuscript and accepted for publication July 31, 1989. Address reprint requests to Dr. Woda: Department of Pathology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655.
Cell
Isolation
Bone marrow core biopsies obtained by conventional needle biopsy are placed in a 35 m m Petri dish containing
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Immunophenotypic Analysis of Cells Isolated from Bone Marrow Biopsies in Patients with Failed Bone Marrow Aspiration ('dry tap')
PIHAN AND WODA
546
5 mL of minimal essential medium (MEM) supplemented with 1% calf serum. Cells are released from the core biopsy by a two-step procedure:firstthe core is repeatedly punctured (5-10 times) and infused with medium using a 23gauge needle attached to a 3-mL plastic syringe. After medium infusion, the specimen is further disaggregated by teasing apart with a set of three-pronged tissue disruption needles. The resultant cell suspension is filtered through 40 ^M nylon mesh and is washed twice. The cell density is adjusted to 1 X 107/mL. Viability is determined by trypan blue exclusion.
lambda (Dako, Santa Barbara, CA). The cells were stained for 15 minutes at 4 °C, washed twice, and fixed with 1% paraformaldehyde. For two-color analysis with CD 11 c (Becton-Dickinson) and CD20, cells were incubated with unconjugated CDl lc antibody, washed, and stained with fluorescein labeled F(ab')2 goat antimouse IgG (Organon Teknika-Cappel, West Chester, PA). After washing, the cells were incubated with 200 jug/mL mouse IgG to block any remaining active sites on the anti-mouse IgG. The cells were then washed and incubated with phycoerythrin CD20. Cells were analyzed with a Coulter 753 flow cytometer. In each sample 10,000 cells, as selected by forward angle and right angle light scatter, were analyzed.
Morphologic Evaluation Morphology was evaluated in Wright-Giemsa stained cytocentrifuge preparations. Each cytospin contained 50,000-200,000 cells. Hematoxylin and eosin stained histologic sections of the core biopsies were available for examination.
Results
The isolation of cells usually was accomplished in less than 5 minutes. The viability of the isolated cells, as determined by trypan blue exclusion, was greater than 90% in all samples. The cell yield varied from 2 X 106 to 2 X 107 cells.
The number of antibodies used was dependent upon both the diagnostic problem encountered and on the total number of cells available for analysis. In general 1 X 105 -1 X 106 cells were processed for one or two color immunofluorescence. The antibodies, directly labeled with fluorescein or phycoerythrin, used in this study included: CD3, CD5, CD10, CD19, CD20, (Leu 4, Leu 1, Calla, Leu 12, Leu 16; Becton-Dickinson, Mountain View, CA); CD4, CD8, CD 13, CD33 (T4, T8, My7, My9; Coulter Immunology, Hialeah, FL). Mouse IgG labeled with fluorescein or phycoerythrin (Beckton-Dickinson) was used as a negative control. Surface immunoglobulin was detected with fluoresceinated F(ab')2 antikappa and anti-
Immunophenotypic and Morphologic Analysis: Chronic B-Cell Leukemia or Lymphoma Case 1 was a B-cell lymphoma that expressed monoclonal kappa light chains (Table 1). The cytospin preparation showed a mixture of normal marrow elements and a population of small lymphocytes. Case 2 was a marrow biopsy in a patient with a known small cleaved cell lymphoma. The cells from this marrow expressed monoclonal kappa light chains, CD 19 and CD20. The cytospin preparation showed a population of lymphoid cells with ir-
Marrow Cells Derived from Core Biopsies*
Case No.
CD3
1 2
33
3
27
4
25
5
32
CD 10
CD4
CD5
CD8
6
16
7
0
13
17
14
1
12
CD 13
CD 19
21
0
6
Kappa
Lambda
Diagnosis
36
82
10
54
99
5
B-cell lymphoma low grade Small cleaved cell lymphoma Hairy cell leukemia Chronic lymphocytic leukemia Chronic lymphocytic leukemia Acute myeloblastic leukemia Acute myeloblastic leukemia Acute lymphoblastic leukemia
77
31 34
54 1
46
0
9
27
8
6
2
2
8
90 92
0
8
4
93
10
3
92
4
7
4
1
8
3
6
4
3
6
CDl let
42 33
25
7
CD33
64
25 44
CD20
88
* The percent of cell expressing the indicated antigen and determined by flow cytometry, t This column shows the percentage of CDl 1c B-cells as determined by two-color flow cytometry
withCDI lcandCD20. These are the percentage of CD20f cells that expressed kappa and lambda light chains.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Cell Isolation
Cell Staining and Immunophenotypic Analysis
Table I. Immunophenotype of Isolated J
AJ.C.P. • April 1990
Vol. 93 • No. 4
BRIEF SCIENTIFIC REPORTS
Acute Leukemias The immunophenotype of three of the cases was compatible with acute leukemia. Two cases had the phenotype of acute myeloblastic leukemia (Table 1). In case 6, 90% of the cells expressed CD33. A subpopulation of these cells was CD10 + suggesting a mixed lineage acute leukemia. The blasts were a uniform population of small- to medium-size cells without cytoplasmic maturation. These cells were peroxidase negative. Case 7 expressed CD 13
FlG. 1. Cytocentrifuge preparation of isolated bone marrow cells from case 2 showing a population of lymphoid cells with irregular nuclei admixed with residual normal marrow cells Wright-Giemsa (XI,250).
and CD33. These cells also stained with CD3. In the authors' experience, the presence of T-cell antigens on myeloblastic leukemias is quite uncommon, yet well documented cases of myeloblastic leukemias expressing CD2 and CD4 have been reported.10 A section of the core biopsy showed a marrow infiltrated by primitive cells. The cytospin preparation showed myeloid differentiation as well as peroxidase positivity of the isolated bone marrow blast cells. The immunophenotype of case 8 was compatible with CD 10 negative acute lymphoblastic leukemia. The blast cells were CD19 + , CD20~, CD 10" and surface immunoglobulin negative. A population of residual Tcells was present. A section of the core biopsy showed a marrow packed by small mononuclear cells. The cytospin preparation showed a population of uniform mediumsize cells compatible with lymphoblasts. Discussion This study describes a method to isolate cells from bone marrow biopsies in cases of failed bone marrow aspiration and to analyze them immunophenotypically and morphologically to aid in the diagnosis of hematologic malignancies. Failed bone marrow aspiration ("dry tap") is not an exceedingly rare event in an active hematologic service. Dry tap recently has been reported to have occurred in 6.8% of biopsies in a large series.8 The method described here is simple, rapid, and reliable and can be easily implemented in laboratories that routinely process specimens for flow cytometry. Morphologic examination of sections of the core biopsy was insufficient in all but one of the cases in this study to make a definitive diagnosis. Immunophenotypic analysis, on the other hand, made it evident that, among the cases in which the morphology suggested lymphoma or chronic leukemia, monotypic immunoglobulin was detected on all cases, allowing a definitive diagnosis of B-cell neoplasia. Among the cases of acute leukemia, two had myeloid and one had lymphoid markers. Comparable results to these can be obtained using immunocytochemistry in semithin plastic1 or frozen13 sections of core biopsies. An advantage of immunocytochemical methods in tissue sections is the preservation of the architecture of the sample. The chief disadvantages of these methods are that the interpretation of results may be subjective, quantitation often is difficult, and the procedures are technically demanding. 512 The advantage of immunofluorescence/flow cytometry is the high sensitivity and the generation of more quantitative and objective immunophenotypic data. 512 It also allows a more rapid diagnosis than the morphologic immunophenotyping methods that yield equivalent information. 113 The chief disadvantages are the lack of preservation of architectural features and the potential for artifactual alteration of the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
regular nuclei admixed with residual normal marrow elements (Fig. 1). Case 3 was from a bone marrow biopsy of a patient with suspected hairy cell leukemia. Analysis of cells from this biopsy showed a B-cell neoplasm that expressed dim lambda light chains, which caused appreciable asymmetry in the kappa and lambda flow histograms. A CD1 lc positive B-cell population was present. The cytospin preparation showed both normal marrow elements and a population of mononuclear cells with oval nuclei and abundant light staining cytoplasm. Some cells had "hairy projections." Cases 4 and 5 were marrow biopsies from patients with a history of chronic lymphocytic leukemia. Case 4 showed a B-cell neoplasm that expressed monoclonal kappa light chains, CD 19 and CD20. Histologic sections of the core biopsy showed an hypercellular bone marrow with several large, nodular lymphoid infiltrates. A cytospin preparation from the isolated cells showed a mixture of myeloid elements and small lymphocytes. Case 5 was a B-cell neoplasm that expressed monoclonal lambda light chains, CD20 and CD5. Sections of the core biopsy showed infiltration of the marrow by small round lymphocytes. The cytospin confirmed the small lymphocytic nature of the neoplasm. The phenotype and morphology of this case were consistent with chronic lymphocytic leukemia.
547
548
PI HAN AND WODA
References 1. Beckstead JH. The bone marrow biopsy. A diagnostic strategy. Arch Pathol Lab Med 1986;110:175-179. 2. Chan IX, Pegram SM, Greaves MF. Contribution of immunophenotype to the classification and differential diagnosis of acute leukemia. Lancet 1985;1:475-479.
3. Chessels JM. Acute lymphoblastic leukemia. Semin Hematol 1982;19:155-168. 4. Foon KA, Todd RF. Immunologic classification of leukemia and lymphoma. Blood 1986;68:1-31. 5. Foucar K. Bone marrow examination in the diagnosis of acute and chronic leukemias. Hematol Oncol Clin North Am 1988,2:567584. 6. Griffin JD. The use of monoclonal antibodies in the characterization of myeloid leukemias. Hematol Pathol 1987;1:81-91. 7. Griffin JD, Davies R, Nelson DA. Use of surface marker analysis to predict outcome of adult acute myeloblasts leukemia. Blood 1986;68:1232-1241. 8. Hak Hyun B, Gulati GL, Ashton JK. Bone marrow examination: Techniques and interpretations. Hematol Oncol Clin North Am 1988;2:513-523. 9. Head DR, Cerezo L, Savage RA. Institutional performance in application of the FAB classification of acute leukemias: The Southwest Oncology Group experience. Cancer 1985;55:979-986. 10. Kaplan SS, Penchansky L, Stole V, Contis L, Krause JR. Immunophenotyping in the classification of acute leukemia. Interpretation of multiple lineage reactivity. Cancer 1989;63:1520-1527. 11. Kass L, Elias JE. Cytochemistry and immunocytochemistry in bone marrow examination: Contemporary techniques for the diagnosis of acute leukemia and myelodysplastic syndromes. Hematol Oncol Clin North Am 1988;2:537-555. 12. Little JV, Foucar K, Horvath A, Crago S. Flow cytometric analysis of lymphoma and lymphoma-like disorders. Semin Diagn Pathol 1989;6:37-54. 13. Wood GS, Warnke RA. The immunologic phenotyping of bone marrow biopsies and aspirate: Frozen section techniques. Blood 1982;59:913-922.
Ristocetin-lnduced Platelet Aggregate Formation and Adherence to the Probe of an Impedance Aggregometer JOSEPH D. SWEENEY, M.D., JAMES W. LABUZETTA, B.S., ZALE P. BERNSTEIN, M.D. KENNETH L. BIELAT, PH.D., AND JOHN E. FITZPATRICK, M.D.
The examination of the interaction between ristocetin and platelets generally is performed in an optical aggregometer where aggregate formation is detected by a change in turbidity. Ristocetin also will induce conductivity changes in an impedance aggregometer with either fresh whole blood or fresh plateletrich plasma due to aggregate attachment to the electrical probe. Formaldehyde-fixed platelets, however, do not attach, and although fresh platelets agglutinate at extremes of pH, probe adhesion does not occur unless the pH is within a certain range. Incubation of platelet preparations with neuraminidase, colchicine, or cytochalasin-b affects neither aggregate formation nor probe adherence. Incubation of platelet preparations with a fibronectin tetrapeptide inhibitor eliminates aggregate formation in response to collagen but is without effect on ristocetin-induced
Received December 30, 1988; received revised manuscript and accepted for publication October 2, 1989. Address reprint requests to Dr. Sweeney: Department of Laboratory Medicine, Roswell Park Memorial Institute, Elm and Carlton Streets, Buffalo, New York 14263.
Hemophilia Center of Western New York and Department of Laboratory Medicine and Pathology, Roswell Park Memorial Institute, Buffalo, New York
attachment of aggregates to the impedance probe. It is suggested that the ability of platelet aggregates to attach to an impedance probe in response to ristocetin is dependent on additional aspects of the functional integrity of platelet preparations and that these changes required for probe adherence may have in vivo significance. (Key words: Platelet aggregates; Impedance aggregometer) Am J Clin Pathol 1990;93:548-551 VARIOUS METHODS ARE AVAILABLE for the in vitro evaluation of platelet function.9 However, systems for assessing aggregate formation remain the most popular. Platelet aggregate formation in response to the addition of an agonist may be measured using an optical method based on a change in turbidity, 3 an impedance method
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
relative frequency of different cell populations. Limited direct comparisons between the two methods using the same sample has revealed good concordance. 13 Another potential shortcoming of this technique is the failure to obtain sufficient cells for immunophenotyping. When the cell yield is low, the selection of antibodies to be used should be judicious, and the number of cells stained may have to be adjusted downward so that a sufficient panel of antibodies can be used. The authors have processed samples for flow cytometry containing as few as 1 X 104 cells. In summary, it is possible to circumvent the difficulties posed by failed marrow aspiration in the accurate diagnosis of leukemias and lymphomas involving the bone marrow by performing immunophenotypic analysis of cells isolated from bone marrow biopsies. The methodology is especially well suited for the hematologic laboratory that routinely employs diagnostic flow cytometry.
A.J.C.P. • April 1990
BRIEF SCIENTIFIC REPORTS
20. Otto BS, duPlessis DJ. Detached ciliary tufts in semen. S Afr Med J 1987;72:441. 21. Papanicolaou GN. Degenerative changes in ciliated cells exfoliating from the bronchial epithelium as a cytologic criterion in the diagnosis of diseases of the lung. NY State J Med 1956;56:26432650. 22. Papanicolaou GN, Bridges EL, Railey C. Degeneration of ciliated cells of the bronchial epithelium (ciliocytophthoria) in its relation to pulmonary disease. Am Rev Respir Dis 1961;83:641-659. 23. Patek E. The epithelium of the human fallopian tube. A surface ultrastructural and cytochemical study. Acta Obstet Gynecol Scand 1974,31:1. 24. Pierce CH, Hirsch JG. Ciliocytophthoria: relationship to viral respiratory infections of humans. Proc Soc Exp Biol Med 1958;98: 489-495. 25. Pierce CH, Knox AW. Ciliocytophthoria in sputum from patients with adenovirus infections. Proc Soc Exp Biol Med 1960; 104: 492-495.
545
26. Poropatich C, Ehya H. Detached ciliary tufts in pouch of Douglas fluid. Acta Cytol 1986;30:440-442. 27. Rilke F, Alasio L. Detached ciliary tufts in cervico-vaginal smears. Acta Cytol 1976;20:189-190. 28. Rosenblatt MB, Trinidad S, Lisa JR, Tcherthoff V. Specific epithelial degeneration (ciliocytophthoria) in inflammation and malignant respiratory disease. Dis Chest 1963;43:605-612. 29. Schueller EF. Ciliated epithelia of the human uterine mucosa. Obstet Gynecol 1968;31:215-223. 30. Sidawy MK, Chandra P, Oertel YC. Detached ciliary tufts in female peritoneal washings. Acta Cytol 1987;31:841-843.
31. Smith JW, McQuay RM, Ash L, Melvin DM, Orihel TC, Thompson JH, eds. Atlas of diagnostic medical parasitology. Chicago: American Society of Clinical Pathologists Press, 1984:14. 32. Verhage HG, Bareither ML, Jaffe RC, Akbar M. Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women. Am J Anat 1979;156:505-522.
GERMAN A. PIHAN, M.D. AND BRUCE A. WODA, M.D.
A method for the immunophenotypic analysis of bone marrow cells in cases of failed bone marrow aspiration is described. Cell suspensions are obtained by mechanical disaggregation of bone marrow core biopsies. The isolated cells are stained with the appropriate antibodies and analyzed by flow cytometry. The usefulness of the method is illustrated by presenting immunophenotypic data obtained in eight consecutive cases accessioned by the authors' laboratory. The method is simple and reproducible. It allows for parallel morphologic examination with Romanovskitype stains and is capable of generating multivariate, quantitative, immunophenotypic data useful in the diagnosis of leukemia and lymphoma. (Key words: Leukemia; Lymphoma; Flow cytometry) Am J Clin Pathol 1990;93:545-548 T H E DIAGNOSIS of hematologic neoplasms that involve the bone marrow relies upon the morphologic examination of smears prepared from aspirated bone marrow particles and stained with Romanovski-type stains. In some clinical situations, however, morphologic examination alone does not provide sufficient diagnostic informat i o n . 5 9 " The use of immunophenotypic analysis of bone marrow cells has increased the diagnostic accuracy and sensitivity of bone marrow examination. It also has provided previously unavailable information regarding prog-
Department of Pathology, University of Massachusetts Medical Center, Worcester, Massachusetts
nosis and selection of therapeutic regimens. 2 " 4 , 7 1 2 Traditionally, immunophenotyping has been performed in cells obtained by bone marrow aspiration. 4 " 7 1 2 Occasionally, however, because of a variety of factors, among which bone marrow fibrosis and hypercellular or "packed" bone marrow appear the most important, aspiration does not yield bone marrow particles, and only a core biopsy can be obtained. 8 Routine histologic study of the core biopsy in these cases of failed bone marrow aspiration may be insufficient to render a definitive diagnosis. The authors describe a method to isolate cells from core biopsies and to use the isolated cells for morphologic and immunophenotypic examination. The authors illustrate the usefulness of this approach in the diagnosis of hematologic malignancies by presenting data on eight consecutive cases of failed bone marrow aspiration accessioned by their laboratory. Materials And Methods
Received May 8, 1989; received revised manuscript and accepted for publication July 31, 1989. Address reprint requests to Dr. Woda: Department of Pathology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655.
Cell
Isolation
Bone marrow core biopsies obtained by conventional needle biopsy are placed in a 35 m m Petri dish containing
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Immunophenotypic Analysis of Cells Isolated from Bone Marrow Biopsies in Patients with Failed Bone Marrow Aspiration ('dry tap')
PIHAN AND WODA
546
5 mL of minimal essential medium (MEM) supplemented with 1% calf serum. Cells are released from the core biopsy by a two-step procedure:firstthe core is repeatedly punctured (5-10 times) and infused with medium using a 23gauge needle attached to a 3-mL plastic syringe. After medium infusion, the specimen is further disaggregated by teasing apart with a set of three-pronged tissue disruption needles. The resultant cell suspension is filtered through 40 ^M nylon mesh and is washed twice. The cell density is adjusted to 1 X 107/mL. Viability is determined by trypan blue exclusion.
lambda (Dako, Santa Barbara, CA). The cells were stained for 15 minutes at 4 °C, washed twice, and fixed with 1% paraformaldehyde. For two-color analysis with CD 11 c (Becton-Dickinson) and CD20, cells were incubated with unconjugated CDl lc antibody, washed, and stained with fluorescein labeled F(ab')2 goat antimouse IgG (Organon Teknika-Cappel, West Chester, PA). After washing, the cells were incubated with 200 jug/mL mouse IgG to block any remaining active sites on the anti-mouse IgG. The cells were then washed and incubated with phycoerythrin CD20. Cells were analyzed with a Coulter 753 flow cytometer. In each sample 10,000 cells, as selected by forward angle and right angle light scatter, were analyzed.
Morphologic Evaluation Morphology was evaluated in Wright-Giemsa stained cytocentrifuge preparations. Each cytospin contained 50,000-200,000 cells. Hematoxylin and eosin stained histologic sections of the core biopsies were available for examination.
Results
The isolation of cells usually was accomplished in less than 5 minutes. The viability of the isolated cells, as determined by trypan blue exclusion, was greater than 90% in all samples. The cell yield varied from 2 X 106 to 2 X 107 cells.
The number of antibodies used was dependent upon both the diagnostic problem encountered and on the total number of cells available for analysis. In general 1 X 105 -1 X 106 cells were processed for one or two color immunofluorescence. The antibodies, directly labeled with fluorescein or phycoerythrin, used in this study included: CD3, CD5, CD10, CD19, CD20, (Leu 4, Leu 1, Calla, Leu 12, Leu 16; Becton-Dickinson, Mountain View, CA); CD4, CD8, CD 13, CD33 (T4, T8, My7, My9; Coulter Immunology, Hialeah, FL). Mouse IgG labeled with fluorescein or phycoerythrin (Beckton-Dickinson) was used as a negative control. Surface immunoglobulin was detected with fluoresceinated F(ab')2 antikappa and anti-
Immunophenotypic and Morphologic Analysis: Chronic B-Cell Leukemia or Lymphoma Case 1 was a B-cell lymphoma that expressed monoclonal kappa light chains (Table 1). The cytospin preparation showed a mixture of normal marrow elements and a population of small lymphocytes. Case 2 was a marrow biopsy in a patient with a known small cleaved cell lymphoma. The cells from this marrow expressed monoclonal kappa light chains, CD 19 and CD20. The cytospin preparation showed a population of lymphoid cells with ir-
Marrow Cells Derived from Core Biopsies*
Case No.
CD3
1 2
33
3
27
4
25
5
32
CD 10
CD4
CD5
CD8
6
16
7
0
13
17
14
1
12
CD 13
CD 19
21
0
6
Kappa
Lambda
Diagnosis
36
82
10
54
99
5
B-cell lymphoma low grade Small cleaved cell lymphoma Hairy cell leukemia Chronic lymphocytic leukemia Chronic lymphocytic leukemia Acute myeloblastic leukemia Acute myeloblastic leukemia Acute lymphoblastic leukemia
77
31 34
54 1
46
0
9
27
8
6
2
2
8
90 92
0
8
4
93
10
3
92
4
7
4
1
8
3
6
4
3
6
CDl let
42 33
25
7
CD33
64
25 44
CD20
88
* The percent of cell expressing the indicated antigen and determined by flow cytometry, t This column shows the percentage of CDl 1c B-cells as determined by two-color flow cytometry
withCDI lcandCD20. These are the percentage of CD20f cells that expressed kappa and lambda light chains.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Cell Isolation
Cell Staining and Immunophenotypic Analysis
Table I. Immunophenotype of Isolated J
AJ.C.P. • April 1990
Vol. 93 • No. 4
BRIEF SCIENTIFIC REPORTS
Acute Leukemias The immunophenotype of three of the cases was compatible with acute leukemia. Two cases had the phenotype of acute myeloblastic leukemia (Table 1). In case 6, 90% of the cells expressed CD33. A subpopulation of these cells was CD10 + suggesting a mixed lineage acute leukemia. The blasts were a uniform population of small- to medium-size cells without cytoplasmic maturation. These cells were peroxidase negative. Case 7 expressed CD 13
FlG. 1. Cytocentrifuge preparation of isolated bone marrow cells from case 2 showing a population of lymphoid cells with irregular nuclei admixed with residual normal marrow cells Wright-Giemsa (XI,250).
and CD33. These cells also stained with CD3. In the authors' experience, the presence of T-cell antigens on myeloblastic leukemias is quite uncommon, yet well documented cases of myeloblastic leukemias expressing CD2 and CD4 have been reported.10 A section of the core biopsy showed a marrow infiltrated by primitive cells. The cytospin preparation showed myeloid differentiation as well as peroxidase positivity of the isolated bone marrow blast cells. The immunophenotype of case 8 was compatible with CD 10 negative acute lymphoblastic leukemia. The blast cells were CD19 + , CD20~, CD 10" and surface immunoglobulin negative. A population of residual Tcells was present. A section of the core biopsy showed a marrow packed by small mononuclear cells. The cytospin preparation showed a population of uniform mediumsize cells compatible with lymphoblasts. Discussion This study describes a method to isolate cells from bone marrow biopsies in cases of failed bone marrow aspiration and to analyze them immunophenotypically and morphologically to aid in the diagnosis of hematologic malignancies. Failed bone marrow aspiration ("dry tap") is not an exceedingly rare event in an active hematologic service. Dry tap recently has been reported to have occurred in 6.8% of biopsies in a large series.8 The method described here is simple, rapid, and reliable and can be easily implemented in laboratories that routinely process specimens for flow cytometry. Morphologic examination of sections of the core biopsy was insufficient in all but one of the cases in this study to make a definitive diagnosis. Immunophenotypic analysis, on the other hand, made it evident that, among the cases in which the morphology suggested lymphoma or chronic leukemia, monotypic immunoglobulin was detected on all cases, allowing a definitive diagnosis of B-cell neoplasia. Among the cases of acute leukemia, two had myeloid and one had lymphoid markers. Comparable results to these can be obtained using immunocytochemistry in semithin plastic1 or frozen13 sections of core biopsies. An advantage of immunocytochemical methods in tissue sections is the preservation of the architecture of the sample. The chief disadvantages of these methods are that the interpretation of results may be subjective, quantitation often is difficult, and the procedures are technically demanding. 512 The advantage of immunofluorescence/flow cytometry is the high sensitivity and the generation of more quantitative and objective immunophenotypic data. 512 It also allows a more rapid diagnosis than the morphologic immunophenotyping methods that yield equivalent information. 113 The chief disadvantages are the lack of preservation of architectural features and the potential for artifactual alteration of the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
regular nuclei admixed with residual normal marrow elements (Fig. 1). Case 3 was from a bone marrow biopsy of a patient with suspected hairy cell leukemia. Analysis of cells from this biopsy showed a B-cell neoplasm that expressed dim lambda light chains, which caused appreciable asymmetry in the kappa and lambda flow histograms. A CD1 lc positive B-cell population was present. The cytospin preparation showed both normal marrow elements and a population of mononuclear cells with oval nuclei and abundant light staining cytoplasm. Some cells had "hairy projections." Cases 4 and 5 were marrow biopsies from patients with a history of chronic lymphocytic leukemia. Case 4 showed a B-cell neoplasm that expressed monoclonal kappa light chains, CD 19 and CD20. Histologic sections of the core biopsy showed an hypercellular bone marrow with several large, nodular lymphoid infiltrates. A cytospin preparation from the isolated cells showed a mixture of myeloid elements and small lymphocytes. Case 5 was a B-cell neoplasm that expressed monoclonal lambda light chains, CD20 and CD5. Sections of the core biopsy showed infiltration of the marrow by small round lymphocytes. The cytospin confirmed the small lymphocytic nature of the neoplasm. The phenotype and morphology of this case were consistent with chronic lymphocytic leukemia.
547
548
PI HAN AND WODA
References 1. Beckstead JH. The bone marrow biopsy. A diagnostic strategy. Arch Pathol Lab Med 1986;110:175-179. 2. Chan IX, Pegram SM, Greaves MF. Contribution of immunophenotype to the classification and differential diagnosis of acute leukemia. Lancet 1985;1:475-479.
3. Chessels JM. Acute lymphoblastic leukemia. Semin Hematol 1982;19:155-168. 4. Foon KA, Todd RF. Immunologic classification of leukemia and lymphoma. Blood 1986;68:1-31. 5. Foucar K. Bone marrow examination in the diagnosis of acute and chronic leukemias. Hematol Oncol Clin North Am 1988,2:567584. 6. Griffin JD. The use of monoclonal antibodies in the characterization of myeloid leukemias. Hematol Pathol 1987;1:81-91. 7. Griffin JD, Davies R, Nelson DA. Use of surface marker analysis to predict outcome of adult acute myeloblasts leukemia. Blood 1986;68:1232-1241. 8. Hak Hyun B, Gulati GL, Ashton JK. Bone marrow examination: Techniques and interpretations. Hematol Oncol Clin North Am 1988;2:513-523. 9. Head DR, Cerezo L, Savage RA. Institutional performance in application of the FAB classification of acute leukemias: The Southwest Oncology Group experience. Cancer 1985;55:979-986. 10. Kaplan SS, Penchansky L, Stole V, Contis L, Krause JR. Immunophenotyping in the classification of acute leukemia. Interpretation of multiple lineage reactivity. Cancer 1989;63:1520-1527. 11. Kass L, Elias JE. Cytochemistry and immunocytochemistry in bone marrow examination: Contemporary techniques for the diagnosis of acute leukemia and myelodysplastic syndromes. Hematol Oncol Clin North Am 1988;2:537-555. 12. Little JV, Foucar K, Horvath A, Crago S. Flow cytometric analysis of lymphoma and lymphoma-like disorders. Semin Diagn Pathol 1989;6:37-54. 13. Wood GS, Warnke RA. The immunologic phenotyping of bone marrow biopsies and aspirate: Frozen section techniques. Blood 1982;59:913-922.
Ristocetin-lnduced Platelet Aggregate Formation and Adherence to the Probe of an Impedance Aggregometer JOSEPH D. SWEENEY, M.D., JAMES W. LABUZETTA, B.S., ZALE P. BERNSTEIN, M.D. KENNETH L. BIELAT, PH.D., AND JOHN E. FITZPATRICK, M.D.
The examination of the interaction between ristocetin and platelets generally is performed in an optical aggregometer where aggregate formation is detected by a change in turbidity. Ristocetin also will induce conductivity changes in an impedance aggregometer with either fresh whole blood or fresh plateletrich plasma due to aggregate attachment to the electrical probe. Formaldehyde-fixed platelets, however, do not attach, and although fresh platelets agglutinate at extremes of pH, probe adhesion does not occur unless the pH is within a certain range. Incubation of platelet preparations with neuraminidase, colchicine, or cytochalasin-b affects neither aggregate formation nor probe adherence. Incubation of platelet preparations with a fibronectin tetrapeptide inhibitor eliminates aggregate formation in response to collagen but is without effect on ristocetin-induced
Received December 30, 1988; received revised manuscript and accepted for publication October 2, 1989. Address reprint requests to Dr. Sweeney: Department of Laboratory Medicine, Roswell Park Memorial Institute, Elm and Carlton Streets, Buffalo, New York 14263.
Hemophilia Center of Western New York and Department of Laboratory Medicine and Pathology, Roswell Park Memorial Institute, Buffalo, New York
attachment of aggregates to the impedance probe. It is suggested that the ability of platelet aggregates to attach to an impedance probe in response to ristocetin is dependent on additional aspects of the functional integrity of platelet preparations and that these changes required for probe adherence may have in vivo significance. (Key words: Platelet aggregates; Impedance aggregometer) Am J Clin Pathol 1990;93:548-551 VARIOUS METHODS ARE AVAILABLE for the in vitro evaluation of platelet function.9 However, systems for assessing aggregate formation remain the most popular. Platelet aggregate formation in response to the addition of an agonist may be measured using an optical method based on a change in turbidity, 3 an impedance method
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relative frequency of different cell populations. Limited direct comparisons between the two methods using the same sample has revealed good concordance. 13 Another potential shortcoming of this technique is the failure to obtain sufficient cells for immunophenotyping. When the cell yield is low, the selection of antibodies to be used should be judicious, and the number of cells stained may have to be adjusted downward so that a sufficient panel of antibodies can be used. The authors have processed samples for flow cytometry containing as few as 1 X 104 cells. In summary, it is possible to circumvent the difficulties posed by failed marrow aspiration in the accurate diagnosis of leukemias and lymphomas involving the bone marrow by performing immunophenotypic analysis of cells isolated from bone marrow biopsies. The methodology is especially well suited for the hematologic laboratory that routinely employs diagnostic flow cytometry.
A.J.C.P. • April 1990
