Alkaline Phosphatase-antialkaline Phosphatase Technique Research Articles

A Decrease in the Density of HLA-DR-Positive Cells Occurs Faster in Corneas Stored in Organ Culture than under Hypothermic Conditions

Aims: To compare the number of antigen-presenting cells (APC) at various locations in fresh human corneoscleral disks and in those that are stored for grafting under hypothermic conditions or in organ culture (OC) with the aim of determining the conditions under which the decline in APC numbers is most substantial. Methods: Cryosections obtained from fresh corneoscleral disks and disks stored under hypothermic (Optisol-GS) or OC conditions were used. The density of HLA-DR-positive cells was determined on cross sections using enzyme immunohistochemistry (alkaline phosphatase-antialkaline phosphatase technique). Longitudinal sections were used for detecting ATPase activity. Results: The densities of HLA-DR-positive cells in both the epithelium and stroma increased from the central (3.79 and 0.61/mm²) to the peripheral cornea (5.56 and 1.35/mm²) as well as to the limbus and conjunctiva. A marked decrease in the number of HLA-DR-positive cells occurred after 7 days of storage in all corneal areas, the limbus and conjunctiva, compared to fresh tissue. No positive cells were found in the epithelium of corneas after 14 days in OC and after 21 days in hypothermic storage. Twenty-eight days of storage in OC led to the complete absence of HLA-DR-positive cells in the epithelium of the limbus and conjunctiva, and to a significant reduction in the stroma. Conclusion: Corneas stored in OC longer than 14 days are likely to be less immunogenic than corneas stored under hypothermic conditions, thus resulting in a possible positive effect on prolonging graft survival.

Subclinical activation of latent cytomegalovirus (CMV) infection and anti-CMV immune response in patients with atopic dermatitis.

Microbiological infections are considered to be of pathophysiological importance in atopic dermatitis (AD). As yet, no information is available regarding cytomegalovirus (CMV) infection in this disease. This, however, is of interest because of the high prevalence of latent infections in the general population, the frequent reactivation in inflammatory diseases, and the immunomodulating capacity of CMV. To investigate the prevalence of latent CMV infection, the frequency of active CMV infection, and the immune response to CMV in patients with moderate to severe AD. Methods To detect active infection we analysed CMV antigen expression by peripheral blood mononuclear cells (PBMC) from 27 patients with moderate to severe AD in comparison with 53 healthy volunteers. We used three monoclonal antibodies recognizing different CMV-encoded antigens and immunocytological staining (alkaline phosphatase-antialkaline phosphatase technique). Patients with AD had a higher mean frequency of CMV-positive PBMC: 2.25 per 10 000 vs. 0.74 per 10 000 in controls (P = 0.001) as well as a higher incidence of CMV antigenaemia: 29.6% vs. 7.5% (P < 0.01). Seropositivity for anti-CMV IgG antibodies indicated subclinical activation of latent infection. Remarkably, a clearance of CMV antigenaemia was observed during anti-eczematous treatment. Significantly higher plasma levels of tumour necrosis factor-alpha, which is involved in CMV reactivation, and interleukin-12, which is crucial for an antiviral cellular immune response, were observed in AD patients in comparison with healthy volunteers. Furthermore, a significantly enhanced frequency of circulating activated HLA-DR+ T cells especially in CMV-seropositive AD patients (19.3% vs. 13.5% in seronegative AD patients vs. 10.2% in controls) suggested that the active CMV infection triggers a cellular immune response. This was also supported by a high frequency of CMV-specific interferon-gamma-producing T cells in CMV-seropositive patients with AD. Our data suggest that active, subclinical CMV infection is more frequent in patients with moderate to severe AD and may have immunopathophysiological relevance.

Inflammatory cells, cytokine and chemokine expression in asthma immunocytochemistry and in situ hybridization

The bronchial wall in asthma is infiltrated by a number of activated inflammatory cells—in particular, eosinophils, which can be detected in the submucosa and within the epithelial cells. This has been demonstrated in Panel 1 through use of immunocytochemistry with an antibody against major basic protein and the alkaline phosphatase-antialkaline phosphatase technique.

Inflammatory cells and myocardial fibrosis: spatial and temporal distribution in renovascular hypertensive rats.

The fibroblasts producing collagen are co-localized with inflammatory cells in myocardial fibrosis areas of spontaneously hypertensive rats, suggesting that collagen overproduction in this model may be modulated by inflammatory cells. The present study extends these observations to the Goldblatt model of hypertension in which the renin-angiotensin system is activated. Inflammatory cells were identified with monoclonal antibodies directed against macrophages (ED1+), T helper (CD4+) and cytotoxic lymphocytes (CD8+), and MHC class II-expressing cells (Ia+). The alkaline phosphatase-anti-alkaline phosphatase (APAAP) immuno-staining technique was used. A new computer-assisted morphometric method was utilized to quantify the inflammatory infiltrate in each cardiac compartment with polarized-light microscopy. Cells responsible for the collagen synthesis were identified by in situ hybridization. The collagen content was estimated by morphometry on left ventricle sections stained with Sirius red, and by biochemical quantification of the hydroxyproline concentration. Computer-assisted morphometry under polarized light was well suited to quantify inflammatory cells labeled by the APAAP technique. Inflammatory cells were co-localized with collagen-synthesizing fibroblasts. The main inflammatory cells were CD4+ lymphocytes > Ia+ > ED1+ > CD8+ cells. These cell densities were increased in hypertensive rats in all cardiac areas compared to control rats except for IA+ cells which were concentrated in microscars. Macrophage density was correlated with plasma renin activity. The inflammatory cell density which best correlated with fibrosis was macrophage density, and which best correlated with systolic blood pressure was macrophage and T helper lymphocyte densities. One can speculate that the correlation between macrophage density and blood pressure as well as with plasma renin activity may indicate that angiotensins and/or elevation of blood pressure could participate in the initial signalling which may mobilize inflammatory cells. These inflammatory cells could promote fibrosis by releasing mediators such as growth factors or cytokines which act upon fibroblasts.

Interleukin-7 receptor expression in cutaneous T-cell lymphomas

Keratinocyte-derived interleukin-7 (IL-) is a potent growth factor for some cutaneous T-cell lymphomas (CTCL). We investigated the expression of IL-7 receptor (IL-7R) in several types of cutaneous and nodal lymphomas. We studied 44 CTCL (13 mycosis fungoides, six Sézary syndromes, eight pleomorphic small cell, and 17 pleomorphic medium and large cell), 10 lymphomatoid papulosis (LP), five cutaneous B-cell lymphomas, and five reactive lymphocytic infiltrates. Twenty nodal T-cell lymphomas, and three reactive lymph nodes were also analysed. Frozen sections were stained with monoclonal antibodies directed against IL-7R, CD25, CD30 and T antigens (CD3, CD2, CD5, CD7, CD4, CD8), using the alkaline phosphatase-antialkaline phosphatase technique. No expression of IL-7R was observed in cutaneous B-cell lymphomas, benign cutaneous lymphoid infiltrates, and reactive lymph nodes. IL-7R was expressed by more than 20% of lymphoid cells in 50-75% of all histological subtypes of CTCL, and by more than 50% of cells in 15-50%. IL-7R was expressed by more than 20% and 50% of cells in 40% and 10% of nodal large T-cell lymphomas, respectively. Eighty-nine per cent of CTCL and LP expressing IL7-R also expressed CD25+, compared with 58% of IL-7R--CTCL and LP (P < 0.05). No association of IL7-R and CD30 expression was found. In conclusion, CTCL frequently express IL-7R. This expression is not related to the epidermotropic characteristic of the infiltrate. In CTCL and LP, IL-7R expression is associated to CD25 expression, but not to CD30 expression.

Increased expression of heat shock protein 70 on airway cells in asthma and chronic bronchitis.

Heat shock proteins (HSPs) appear to be involved in inflammation. Because asthma is an inflammatory disease, HSPs may be expressed. We studied the expression of HSP-70 by immunohistochemistry using the alkaline-phosphatase anti-alkaline phosphatase technique on epithelial cells and alveolar macrophages (AMs) from 19 patients with asthma, eight patients with chronic bronchitis (CB), and 13 control subjects and in bronchial biopsies from 15 asthmatics, 15 CB patients, and nine control subjects. The specificity of the antibody was confirmed by Western blotting on heated AM. The specificity of the immunostaining was confirmed by inhibition experiments. The expression of HSPs in the asthmatics in comparison with the CB patients and control subjects was significantly increased on epithelial cells (P < 0.002 and P < 0.007, respectively) and AMs (P < 0.0004 and P < 0.0002, respectively) and was significantly correlated with the severity of asthma (P < 0.005 for AMs) and the percentage of eosinophils in bronchoalveolar lavage fluid (P < 0.004). In biopsies from the asthmatics but not from the CB patients or control subjects, a positive staining for HSP-70 was observed on epithelium, mononuclear cells, and basement membrane and was correlated with the severity of asthma (P < 0.0005, P < 0.002, and P < 0.007, respectively). This study demonstrates that bronchial inflammation in asthma but not in CB may be linked to the production of HSPs.

The use of the APAAP technique as a rapid indicator of peripheral blood progenitor cell levels

Rapid and sustained engraftment following autotransplantation with peripheral blood stem cells (PBSC) depends on adequate numbers of stem cells and progenitor cells. In this study we have compared the number of myeloid progenitor cells quantitated using the colony forming units-granulocyte macrophage (CFU-GM) clonogenic assay with the number of CD34+ cells estimated both by flow cytometry and by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. We have analysed 15 peripheral blood mononuclear cells (PBMNC) samples from 13 normal subjects and 179 PBMNC from 32 patients undergoing PBSC harvests during the recovery phase of high dose cyclophosphamide chemotheraphy. The number of CD34+ cells measured by the APAAP technique correlated well with the number of CD34+ cells measured by flow cytometry (r = 0.727, p = 0.0001), and also with the number of CFU-GM measured in the clonogenic assay (r = 0.721, p = 0.0001). The APAAP method provides a rapid, reliable measure of progenitor cell levels that can be used to monitor the optimal time to harvest peripheral blood stem cells (PBSC), and to estimate the marrow repopulating ability (MRA) of stem cell preparations used for transplantation.

Clinical significance of P‐glycoprotein expression in acute leukaemia as analysed by immunocytochemistry

Multidrug resistance, mediated by the overexpression of an energy-dependent transport protein, P-glycoprotein, has been one of the major targets of interest in solving the mechanisms of clinical drug resistance of malignant cells. To evaluate the correlation between P-glycoprotein overexpression and the response to chemotherapy, we analysed cytospin preparations of gradient-separated blood or bone marrow mononuclear cells from 79 patients with acute leukaemia by means of the P-glycoprotein-directed monoclonal antibody JSB-1 and immunocytochemistry using the alkaline phosphatase-antialkaline phosphatase technique. P-glycoprotein expression was detected in all disease phases of acute leukaemia. Thirteen out of 51 patients at diagnosis, 10/29 patients in relapse or during residual disease and 8/27 patients in remission overexpressed P-glycoprotein. Seven out of the 8 positive remission samples were collected between the cycles of consolidation treatment. Our results suggest that increased P-glycoprotein expression in samples collected between the cycles of consolidation treatment during remission may be induced in normal leukocytes by cytotoxic drug treatment, infections, or by some physiological mechanisms related to the disease. Patients older than 45 years of age were significantly more often P-glycoprotein-positive (11/25) at diagnosis than younger patients (2/26). P-glycoprotein expression at diagnosis was significantly correlated with a low remission rate after the first cycle of induction therapy. Of 34 P-glycoprotein-negative patients, 25 achieved remission after the first cycle as compared to 4/12 of the P-glycoprotein-positive patients. Our results indicate that the method used is specific and sensitive enough for the analysis of P-glycoprotein expression and that the expression at initial presentation is inversely correlated with the outcome of induction therapy.

IgE and its related phenomena in bullous pemphigoid.

This study was designed to analyse IgE and its related phenomena in bullous pemphigoid (BP). We analysed 17 BP sera by indirect immunofluorescence (IIF) and immunoblotting (IB) using a monoclonal antibody to IgE. In addition, inflammatory cells in lesional skin from 11 patients with BP were analysed by the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique using monoclonal antibodies to IgE and Fc epsilon RII/CD23. IgE class anti-basement membrane zone (BMZ) autoantibody was detected in nine of 17 sera (52.9%) by IIF. IgG class anti-BMZ antibody could block the BMZ-binding reactivity of IgE class antibody. Titres of IgE class autoantibody in the sera ranged from 1:40 to 1:320, and statistically correlated with serum IgE levels. Two of 11 sera contained an IgE class autoantibody which recognized a 230-kDa BP antigen by IB. By radio-allergosorbent test (RAST), IgE-specific antibodies to an extended series of common inhalant and food allergens were detectable in six sera with high concentrations of total IgE (over 3,300 IU/ml). IgE-bearing and Fc epsilon RII-expressing cells were demonstrated in the upper dermis and along the BMZ in seven of 11 biopsy specimens by the APAAP technique. The distribution and number of IgE-bearing cells in the lesions were similar to those of the Fc epsilon RII-expressing cells. These results suggest that both IgE-mediated immune responses and autoimmunity characterize BP as distinctive features.

Intraepidermal localization of the clone in cutaneous T-cell lymphoma

Background : No immunohistologic techniques are currently available to demonstrate clon-ality of T-cell lymphomas. Monoclonal antibodies to the variable region of the T-cell receptor (TCR) have been produced that identify minor populations of normal peripheral blood T lymphocytes. Objective : We investigated the expression of TCR V-region genes in cutaneous lymphomas to determine whether immunostaining with these antibodies may be a simple method to detect clonal T-cell proliferations and help to distinguish benign lymphoid infiltrates from malignant lymphoma. Methods : Cutaneous samples were obtained from 18 cutaneous T-cell lymphomas (14 mycosis fungoides, 1 Sézary syndrome, 2 pleomorphic T-cell lymphoma, and 1 large cell anaplastic lymphoma) and 8 benign lymphoid infiltrates. Frozen sections were incubated with monoclonal antibodies and stained by the alkaline phosphatase-antialkaline phosphatase technique. Staining was performed with a panel of 7 anti-TCR V-region antibodies, 6 T-cell markers, 1 anti- β chain antibody, and 1 anti- δ chain antibody. Results : Clonality could be demonstrated in 2 of 18 cutaneous lymphomas. We observed the strictly intraepidermal localization of clonal proliferation in one case of early-stage mycosis fungoides. Conclusion : Anti-TCR V-region antibodies may identify a strictly epidermotropic clone in early mycosis fungoides. However, the panel of antibodies currently available stains only a minority of cutaneous T-cell lymphomas. The usefulness of these antibodies as a clonotypic marker needs to be reevaluated when a larger panel of antibodies becomes available.

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.

The Use of the Alkaline Phosphatase-Antialkaline Phosphatase Technique for Immunophenotyping Acute Myeloid Leukemia

The leukemic cells from 31 cases of acute myeloid leukemia were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) technique, using 7 monoclonal antibodies reactive with cells of myeloid origin. We found a good correlation between the results obtained using the APAAP method and indirect immunofluorescence. In most cases, we observed a slight degree of variation in the percentages of reacting cells when comparing the two methods. Nevertheless, taking 20% of cells being immunolabeled as a threshold for defining a case as positive, we found no discrepancies in the final classification of each case. The main advantages of the APAAP method are: (1) its use with routinely prepared peripheral or blood marrow samples, and (2) the possibility of correlating immunological characterization with morphology. Since the results with the APAAP method were comparable with those obtained using indirect immunofluorescence, we suggest that this former technique can complement, and sometimes substitute, other methods of immunological evaluation.

Comparative study of immunocytochemical staining versus Giemsa stain for detecting dysmegakaryopoiesis in myelodysplastic syndromes (MDS)

23 patients with myelodysplastic syndromes (MDS) and 8 normal controls were analyzed for dysmegakaryopoiesis (DMP) in the bone marrow by alkaline phosphatase anti-alkaline phosphatase (APAAP) technique and by conventional May-Giemsa staining. In the immunocytochemical study, monoclonal antibody (MoAb) against glycoprotein (GP) IIb/IIIa was utilized to demonstrate megakaryocytic cells. 91% (21/23) of MDS cases were detected as having DMP by APAAP method, while only 52% (12/23) were detectable by Giemsa stain. There were difficulties in recognizing small micromegakaryocytes (micro MKs), designated as type 1 atypical MKs, by Giemsa staining. Furthermore, megakaryoblasts (MKBs) were detectable only by APAAP technique. In 8 normal controls, no type 1 and type 3 atypical MKs (round shaped multinuclear MKs) were observed either by Giemsa staining or by the APAAP method, suggesting that they are a distinctive feature of MDS. These results indicate the necessity of immunocytochemical technique for accurate recognition of DMP in MDS.

Immunoglobulin light chains in common acute lymphoblastic leukaemia

A single immunoglobulin light chain lambda was identified in the blast cells of two out of 12 patients with common acute lymphoblastic leukaemia (C-ALL) using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. Inhibition at the cell surface proved that the reaction was a genuine anti-lambda reaction. Immunoglobulin mu chain was not identified in these patients. Results of immunoglobulin typing in 103 patients with B chronic lymphocytic leukaemia (B-CLL) are cited to show the increased sensitivity of the APAAP technique as compared to the indirect immunoperoxidase technique for cellular immunoglobulin identification.

Detection of estrogen receptor proteins in breast tumors using an improved APAAP immunohistochemical technique

A monoclonal antibody (anti-estrogen receptor [ER] from the ER-immunocytochemical assay [ICA] [Abbott Laboratories, Berkshire, England]), which binds to epitopes of the estrogen receptor in surgical biopsy specimens of breast carcinomas, was used in an improved alkaline phosphatase anti-alkaline phosphatase (APAAP) technique for in-situ receptor detection. This method was compared with a standard radioligand binding biochemical assay in assessing the ER status of 48 human breast carcinomas. The monoclonal antibody allowed detection of ER in the presence of estrogens or anti-estrogens and avoided problems of nonspecific intrinsic reactions, due to endogenous steroid hormones. The immunohistologic results (positive and negative) correlated well with the radioligand binding results (rs = 0.760; P less than 0.001) and allowed the additional assessment of cell morphology and tumor heterogeneity in the cryostat sections. The APAAP method provides diagnostic rapidity (approximately 4 hours) combined with sensitivity as compared with the radioligand assay (an approximately 2-day assay). The vivid red color associated with APAAP staining also was a distinct advantage compared with the brown reaction product of ER-ICA in interpreting stained sections. The results of this study indicate that the APAAP technique is an improved method for detection of ER and is applicable in routine hospital practice, thereby contributing to the immediate clinical management of patients with breast tumors.

Chapter 10 Limited expression of myeloid antigens by neuroendocrine tumours of lung

Abstract Frozen sections of small cell lung carcinoma (SCLC) and bronchial carcinoid (BC) and cytospins of the SCLC lines, NCI-H69, NCI-H128 and NCI-H146, were stained with antibodies of the myeloid-monocyte panel assembled by the Third International Workshop on Human Leucocyte Differentiation Antigens by a sensitive alkaline phosphatase-antialkaline phosphatase technique. Two SCLC, one BC and two cell lines were screened with full panel of 145 antibodies. To assess the heterogeneity of expression of leucocyte antigens, antibodies of defined clusters, provisional groups and single unclustered antibodies which were immunoreactive on the initial screen were further tested against a total of 8 SCLC, 3 BC and 3 cell lines. SCLC expressed CD epitopes frequently, with 5 of 8 tumours reacting with one or more of the 14 CD15 antibodies in the panel. No other established CD cluster reacted consistently with SCLC but at least one antibody of provisional group (G) 10 reacted focally with a few of the SCLC tested. Each of the BC reacted with at least one of the CD15 antibodies but labeling reactions of BC differed from those of SCLC in that CD13 epitopes were found on all of the tumours tested. In addition, BC were labelled with broader range of provisional group antibodies including G10, G11, G12, G14 and G15. Many antibodies which could not be clustered at the Workshop labelled both SCLC and BC. These Studies suggest that X-hapten (CD15) is frequently expressed in neuroendocrine tumours of lung and that CD13 antigen and several provisional group antigens are more commonly expressed in BC than in SCLC. Other well defined myelomonocytic epitopes are rarely found on neuroendocrine lung tumour cells.

Immunophenotyping of Acute Myeloid Leukemia Using Monoclonal Antibodies and the Alkaline Phosphatase-Antialkaline Phosphatase Technique

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French-American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR-positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.

Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French- American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR- positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.