Alkaline Phosphatase-antialkaline Phosphatase Method Research Articles

Detection of light chain restriction in chronic B-lymphoid leukaemia and B-non-Hodgkin's lymphoma

The determination of immunoglobulin light chain restriction using monoclonal and polyclonal antibodies is a rapid method for the detection of a neoplastic B-cell-population. Cytocentrifuge preparates of mononuclear blood cells from 42 patients with chronic B-lymphoid leukaemia and of lymph node aspirates from 24 patients with B-non-Hodgkin's lymphoma were examined using the alkaline phosphatase-antialkaline phosphatase (APAAP) method. Monoclonal antibodies from different commercial sources and rabbit polyclonal antibodies were used in this study. Staining with polyclonal antibodies demonstrated light chain restriction in 65 cases. The leukaemic cells of a patient with hairy cell leukaemia did not express light chain immunoglobulins. Monoclonal antibodies from two manufacturers demonstrated monotypic staining for light chains in all cases with light chain immunoglobulins. Monoclonal antibodies from four manufactures failed to show monotypic light chains in 5, 21, 25 and 28 of the 65 cases. All investigated antibodies detected a similar percentage of light chain-positive lymphocytes in 10 healthy persons. We conclude that not all investigated monoclonal antibodies are suitable for detection of light chain restriction in B-non-Hodgkin's lymphomas and chronic B-lymphoid leukaemias. However, using selected monoclonal antibodies or rabbit polyclonal antibodies the APAAP method is very sensitive for detection of light chain restriction in these disorders.

Expression of cytokines and chemokine receptors in the cutaneous lesions of erythema multiforme and Stevens-Johnson syndrome/toxic epidermal necrolysis

Erythema multiforme (EM) and Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are caused by a dysregulation of cellular immunity. To evaluate further the potential role of certain cytokines and chemokine receptors in cutaneous lesions of patients affected by EM and SJS/TEN and to establish whether such diseases are polarized preferentially towards a T-helper (Th) 1 or Th2 pattern. Biopsy specimens from eight patients with EM, six with SJS/TEN and three healthy controls were stained for immunohistochemical examination using the alkaline phosphatase-antialkaline phosphatase method. The monoclonal antibodies used included those to tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-2, IL-5, IL-13, receptor 3 for C-C chemokines (CCR3), receptor 3 for C-x-C chemokines (CXCR3) and CXCR4. The SJS/TEN specimens showed significantly higher expression of all the cytokines and chemokine receptors (except CXCR3) tested than the EM specimens. Both lesional series showed significantly higher expression of all the receptors tested than the healthy control specimens, with the sole exception of a lower expression of CCR3 in EM specimens. Comparison between molecules associated with a Th1 or Th2 response revealed a predominance of Th1 response in EM and no significant imbalance between Th1 and Th2 in SJS/TEN. We have provided further evidence that TNF-alpha is strongly expressed in SJS/TEN lesions and therefore it may be involved in the epidermal necrosis featured in such diseases. IFN-gamma may play an important role both in EM and SJS/TEN. IL-2, IL-5 and IL-13 may contribute to the cutaneous immunoinflammation in these diseases. Chemokine receptors may be involved strongly in the recruitment of inflammatory cells in lesional skin. In our cases we found a sharp polarization towards a Th1 pattern in EM, while the SJS/TEN lesions showed a mixed Th1/Th2 pattern.

CD34+ cell enrichment depletes atypical CD30+ cells from PBPC grafts in patients with HD

European Group for Blood and Marrow Transplantation (EBMT) registry data indicate that patients with relapsed HD given high-dose therapy (HDT), supported with PBPC might have a poorer outcome compared with those given BM. Since this can be due to the infusion of contaminating tumor cells in the PBPC products, we studied the presence of minimal residual disease and tested whether CD34(+) cell enrichment was able to remove atypical CD30(+) cells from PBPC grafts. Eighteen HD patients eligible for HDT were included in the study. By the use of immunocytochemistry (ICC), mononuclear cells from BM and peripheral blood (PB) before mobilization, PBPC products and selected CD34(+) fractions were stained using anti-CD30 MAb (Ber-H2) and the APAAP (alkaline phosphatase-anti-alkaline phosphatase) method. Cells scored as atypical CD30(+) cells were large- to medium-sized, with membranous, cytoplasmatic and/or Golgi positivity for CD30. Nine out of 11 BM tested were positive, while 14 of 14 PB and 18 of 18 PBPC contained atypical CD30(+) cells. The total number of atypical CD30(+) cells was significantly higher in PBPC than in the corresponding BM. CD34(+) cell enrichment employing ISOLEX 300I gave a purity and yield of 99.2% (range 97.8-99.7) and 49.6% (range 30.0-78.4), respectively. After HDT a median of 5.8 x 10(6) (range 2.7-20) CD34(+) cells/kg was infused. Neutrophil counts of > 0.5 x 10(9)/L and platelet counts of > 20 x 10(9)/L were achieved at Day 12 (range 10-17) and at Day 10 (range 7-15), respectively. Sixteen of 18 CD34(+) selected products had no detectable atypical CD30(+) cells, while two had a low number. After HDT, the overall survival was 80% and the event-free survival was 69%, with a median follow-up of 24 months (range 1-36). We show that contaminating atypical CD30(+) cells in PBPC can efficiently be removed by CD34(+) cell enrichment, and the use of such grafts following HDT gives fast and sustained engraftment.

Basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin

Background: Previous studies used indirect methods to identify basophils in the bronchi in asthma, and the numbers were not compared with eosinophils and mast cells. Furthermore, differences in basophil numbers between atopic and nonatopic asthma at baseline and between late-phase skin and asthmatic reactions have not been previously documented. Objective: The basophil granule–specific mAb BB1 was used to identify basophils in (1) bronchial biopsy specimens from atopic asthmatic subjects and nonatopic asthmatic subjects and control subjects, (2) biopsy specimens from atopic asthmatic subjects before and after inhalational allergen challenge, and (3) late-phase skin reactions. Basophil numbers were compared with EG2+ eosinophils and tryptase+ mast cells. Methods: Cells were enumerated in bronchial and skin biopsy specimens by means of immunohistochemistry with the alkaline phosphatase-antialkaline phosphatase method. Results: There were elevated numbers of basophils in baseline biopsy specimens in atopic asthmatic subjects compared with atopic control subjects or normal control subjects, although eosinophils and mast cells were 10-fold higher. There was an intermediate number of basophils in nonatopic asthmatic subjects. Basophils increased after allergen inhalation, but again basophils were less than 10% of eosinophils. In contrast, basophils in cutaneous late-phase reactions were approximately 40% of infiltrating eosinophils. The peak of basophil accumulation was at 24 hours, whereas maximal eosinophil infiltration occurred at 6 hours. One third of cutaneous basophils had morphologic appearances suggestive of degranulation. Conclusion: Numerous basophils infiltrated cutaneous late-phase reactions in atopic subjects. However, this cell was not prominent in bronchial biopsy specimens of asthmatic subjects, either at baseline or after allergen challenge. (J Allergy Clin Immunol 2000;105:99-107.)

Antigenic Phenotyping of Lymphoid Cells and B Cell Gene Rearrangement in Type B Gastritis and in Gastritis Not Associated with Helicobacter pylori Colonization

Marginal-zone B cells of the mucosa-associated lymphoid tissue (MALT) are the normal counterpart of the neoplastic cells in MALT lymphoma. In both cases these lymphocytes express surface immunoglobulins, but are negative when stained for B cell associated antigens like CD10 and CD23. Furthermore, the B cell gene rearrangement has been found in Helicobacter pylori associated chronic gastritis and in extranodal type of marginal-zone lymphoma. The aim of this study was to quantify the number of IgM-, CD10-, and CD23-positive lymphocytes in patients with type B gastritis and to compare the results with the antigen profile of mononuclear cells in patients with gastritis not associated with H. pylori. Additionally, the immunoglobulin heavy-chain (IgH) gene rearrangement in H. pylori positive and H. pylori negative gastritis was studied. From 23 patients with a positive urease test and/or histologically proven H. pylori infection and chronic gastritis and from 22 patients with H. pylori negative chronic gastritis mucosa biopsy specimens were taken. Single-cell suspensions were obtained following enzymatic digestion. For immunocytochemistry, an alkaline phosphatase-antialkaline phosphatase method was applied. IgH gene rearrangement in formalin-fixed, paraffin-embedded specimens was determined by polymerase chain reaction in 11 patients with chronic gastritis. An increase in mu-positive plasma cells and B lymphocytes was detected in patients with H. pylori positive gastritis as compared with patients with H. pylori negative gastritis (10.0 vs. 3.9%, p < 0.001, and 4.3 vs. 1.6%, p < 0.01, respectively). In both groups, the proportion of CD10- and CD23-positive lymphocytes was <1%. IgH gene rearrangement was not restricted to type B gastritis; single bands were also present in 3 of 7 patients with H. pylori negative chronic gastritis. Our finding of IgH gene rearrangement in some of the patients with H. pylori negative chronic gastritis indicates that additional factors may be critical for these genotypical changes and for the pathogenesis of gastric MALT lymphoma.

The expression of beta 1 integrins in human coronary artery.

The beta 1 integrin adhesion receptors mediate the binding of cells to extracellular matrices, facilitating their growth, migration, and capacity to deposit matrix proteins: important factors in arterial restenosis and atherosclerosis. The expression of integrins in human coronary artery is, however, unexplored. The aim of the current study was, therefore, to define the expression of beta 1 integrins by cultured human coronary artery vascular smooth muscle cells (hCAVSMC) and in normal human coronary artery; confirming whether or not this differs from the repertoire found in other species and human vessels. The expression of beta 1 integrins by hCAVSMC was assessed by immuno-precipitation and the alkaline phosphatase anti-alkaline phosphatase (APAAP) immunochemical technique. In addition, mRNA expression was defined by reverse transcription polymerase chain reaction (RT-PCR). Normal adult human coronary arteries (n = 4) were also stained by the APAAP method. In vitro hCAVSMC express alpha 2 beta 1 (a collagen and occasional laminin receptor) and alpha 5 beta 1 (a fibronectin receptor) with lesser expression of alpha 3 beta 1 (a multifunctional receptor). They do, however, possess mRNA for several other integrins. Cells within the media of human coronary artery wall express alpha 3 beta 1 and alpha 5 beta 1 but not alpha 2 beta 1: instead the alternative collagen/laminin receptor, alpha 1 beta 1, is expressed in vivo. This pattern of expression differs subtly from that described in rats through it closely parallels that found in other human arteries.

Positive immunostaining with MLuC1 of bone marrow aspirate predicts poor outcome in patients with small-cell lung cancer

Immunocytochemistry has been proven able to identify tumor cells in bone marrow aspirate (BMA) of patients with SCLC. However, few data exist about the clinical significance of the procedure. 108 BMA taken from 60 patients were incubated with the MoAb MLuC1 (cluster 6) and stained by the APAAP (alkaline phosphatase-antialkaline phosphatase) method. The serum levels of LDH, TPA, NSE and CEA were also studied in relation to bone marrow involvement by means of discriminant analysis. Immunocytochemistry of the aspirate with MLuC1 detected positive cells in 23 patients (38%) (38 of 108 samples) vs. 13% of the conventional biopsies studied without MLuC1 (P < 0.001). With respect to bone marrow positivity, three groups of patients were identified: those with no positive cells in the aspirate and negative biopsy (group A); those with less than 10 positive cells in the aspirate and negative biopsy (group B); and those with more than 10 positive cells or clumps in the aspirate or positive biopsy (group C). Group C patients had poorer median survivals than those in the other two groups (5.5 vs. 11 months, respectively, P = 0.01). Discriminant analysis showed that the four serum markers were poor discriminators of the degree of bone marrow involvement, with only 55% of grouped cases being correctly classified. These results show that detection of bone marrow involvement i) can be improved by the use of MLuC1 ii) is not predictable by conventional tumor markers, and iii) is related to poor outcome.

A panel of antibodies for the immunostaining of Bouin's fixed bone marrow trephine biopsies.

AIMS: To assess a panel of antibodies on Bouin's fixed bone marrow trephine (BMT) biopsies. These biopsies are widely used in routine diagnosis of various haematological malignancies and may be...

Secretory meningioma: clinical, histologic, and immunohistochemical findings in 31 cases.

Secretory meningioma is a rare histologic variant characterized by a unique epithelial differentiation of meningothelial cells resulting in the production of hyaline inclusions. Most previous reports have presented single case observations. The authors selected 31 cases for a clinicopathologic study to characterize this type of tumor further. Clinical data were compiled and the extent of peritumoral edema was assessed from preoperative computed tomography or magnetic resonance imaging scans. Preparations of surgical specimens of all tumors were studied after both conventional histologic and immunohistochemical preparations were made. Immunostaining was performed by either the avidin-biotin complex method or the alkaline phosphatase-antialkaline phosphatase method using 22 primary antibodies. In the tumor collection used in this study, secretory meningiomas represented 3% of meningiomas. The female-to-male ratio was 9:1. Most tumors were located at the sphenoid ridge or at the frontal convexity, and recurrences were not observed. Eighty-four percent of tumors presented with slight to marked peritumoral edema. The MIB-1 staining index showed a mean of 3.8%. Inclusions and surrounding cells consistently expressed epithelial membrane antigen, cytokeratins, carcinoembryonic antigen, and carbohydrate antigen 19-9. In decreasing frequency, they also contained alpha1-antitrypsin, immunoglobulin (Ig)A, alpha1-antichymotrypsin, IgM, and IgG. Cells positive for vimentin and S-100 did not contain inclusions. All tumors were positive for progesterone receptors. Macrophages were stained with antibodies to factor XIIIa, human leukocyte antigen-DR, and alpha1-antitrypsin. In 64% of cases, tumor vessels lacked expression of glucose transporter protein 1. The classification of secretory meningioma as a distinct variant has been justified on clinical, histologic, and immunohistochemical grounds. The unique epithelial features call attention to the broad spectrum of differentiation properties found in meningiomas.

Immunocytochemical phenotyping of disseminated tumor cells in bone marrow by uPA receptor and CK18: investigation of sensitivity and specificity of an immunogold/alkaline phosphatase double staining protocol.

Phenotyping of cytokeratin (CK)18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15-80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (10(6) bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1-280 in 10(6); double staining 808, range 0-253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant (p < 0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients.

Immunobiological peculiarities of cholesteatoma in children: quantification of epithelial proliferation by MIB1.

Cholesteatoma in children is characterized by a more extensive and rapid growth in the middle ear and mastoid cavities. The growth characteristics of the cholesteatoma in 20 children were studied using the monoclonal antibody MIB 1, which recognizes a nuclear antigen expressed by cells in the G1, S, and G2/M phases. Specimens of normal adult auditory meatal skin (n = 15) and adult cholesteatoma (n = 15) served as controls. The tissue specimens were prepared for immunohistochemical examination using the alkaline phosphatase-antialkaline phosphatase method and an automatic image analyzer. Specimens of normal skin revealed an average MIB 1 score of 9.2 +/- 3.10%. Child and adult cholesteatomas showed higher values. The average MIB 1 score was higher in child cholesteatoma (42 +/- 9.4%) than in adult cholesteatoma (28.2 +/- 6%). This difference was statistically significant (P<.01). Our results confirm a significant increase of the proliferative rate of cholesteatoma keratinocytes in children, giving an explanation for the more aggressive clinical behavior observed in these patients.

Immunohistochemical demonstration of c-myc oncogene product in middle ear cholesteatoma.

Cholesteatoma epithelium is characterized by a dysregulation with a hyperproliferative growth and altered differentiation. In a variety of cells c-myc oncogene was found to be highly linked to the control of growth and differentiation. Expression of c-myc was studied in cholesteatoma epithelium using a monoclonal antibody directed against the 67 kDa c-myc protein product and the alkaline phosphatase-antialkaline phosphatase method. For quantitative analysis a computer-linked analyzing system was used. In contrast to normal skin, keratinocytes of basal and suprabasal layers showed nuclear staining in cholesteatoma epithelium. The extent of nuclear staining of epithelial cells in the cholesteatomas studied was significantly increased. Concurrent cytoplasmic staining was observed in both skin and cholesteatoma, but with a stronger reactivity in the latter. These findings suggest participation of the c-myc oncogene in cholesteatoma epithelium.

Enhanced production of TGF-beta by blood monocytes from patients with active tuberculosis and presence of TGF-beta in tuberculous granulomatous lung lesions.

The expression of TGF-beta, a molecule that affects both immune responsiveness and wound healing, was examined in blood monocytes and granulomatous lesions from patients with active pulmonary tuberculosis. The spontaneous release of TGF-beta was higher in culture supernatants of monocytes from patients as compared with those of healthy subjects by an ELISA (p < 0.0005). TGF-beta activity was also confirmed in a bioassay in supernatants from patients. Next, freshly isolated monocytes from patients with tuberculosis and matched subjects were examined for TGF-beta activity. Cytosmears of monocytes were stained with an Ab against TGF-beta 1 (anti-LC) or isotype-specific Ab by using an alkaline-phosphatase anti-alkaline phosphatase method. In contrast to monocytes from healthy individuals, 60 to 70% of monocytes from patients demonstrated cytoplasmic staining for TGF-beta (n = 3). Upon hypotonic lysis, monocytes from patients with tuberculosis contained immunoreactive TGF-beta (n = 3). By Northern blot analysis, monocytes from three of seven patients with tuberculosis had increased expression of TGF-beta mRNA as compared with concurrently examined monocytes from healthy subjects. Within the granulomas of lung sections from two patients with untreated tuberculosis, TGF-beta immunoreactivity was identified in the Langhan's giant cells mainly and to a lesser extent the epithelioid cells using anti-LC Ab and the peroxidase-anti-peroxidase technique. Thus, both blood monocytes and lung granuloma macrophages from patients with active tuberculosis express TGF-beta. Excess activity of this cytokine in blood monocytes may underlie the depressed T cell responses of patients with tuberculosis. Moreover, within the infected tissues excess TGF-beta activity may interfere with anti-mycobacterial mechanisms and effective granuloma formation.

Detection at diagnosis of tumor cells in bone marrow aspirates of patients with small-cell lung cancer (SCLC) and clinical correlations

Immunocytochemistry has often been used to identify tumor cells in bone marrow aspirate (BMA) of SCLC patients in order to improve the results of conventional histomorphology. However, whether the detection of bone marrow microlocalisation at diagnosis had implications for prognosis has not been clear. Eighty-four slides (44 patients) and 66 bone marrow biopsies (from 42/44 patients) were evaluated. Cytospins of BMA were incubated with the monoclonal antibody (MAb) NCC-LU-243, recognising the cluster 1 antigen (NCAM) and then stained by the APAAP (alkaline phosphatase-anti-alkaline phosphatase) method. The relationship among BMA and PS (performance status), NSE (neuron-specific enolase), stage, survival was also studied. 33/84 (39%) BMA were positive for NCAM, compared with 8/66 (12%) bone marrow biopsies (p = 0.009), (17/44 and 6/42 patients, respectively). Moreover, BMA was positive for NCAM in 6/19 patients with limited disease. The presence of positive BMA did not correlate with PS, NSE or stage, but patients with positive BMA had shorter survivals than those with negative BMA (median survival: 7 and 12 months, respectively, p = 0.007). Bone marrow involvement detected by immunocytochemistry appears to be related to survival but not to parameters of tumor burden (NSE, stage), suggesting that this technique might help to select patients with better prognoses for new therapeutic strategies.

Neural cell adhesion molecule expression in human pituitary adenomas

Neural cell adhesion molecule (NCAM) is a cell surface glycoprotein whose expression may be important in metastatic or local growth of neoplasms. Expression of NCAM in human pituitary adenomas was assessed on snap-frozen and Bouin's and/or formalin-fixed, paraffin-embedded archival tissue using a sensitive alkaline phosphatase-antialkaline phosphatase method of immunostaining with the monoclonal antibody, NKH-1. The 20 pituitary adenoma patients consisted of 13 males and 7 females, ages 19-78 years, with null cell macroadenomas (3), plurihormonal macroadenomas (4), weak gonadotropic macroadenomas (5), growth hormone-prolactin adenomas (2), and prolactinomas (6). Eight of the adenomas were clinically invasive. The snap-frozen material from 14 pituitary tumors showed immunostaining for NCAM in either a cytoplasmic, peripheral membranous, or mixed pattern; 2 of 3 prolactinomas showed patchy immunostaining. The Bouin's and/or formalin-fixed pituitary adenoma specimens from 6 clinically invasive cases showed less distinct NCAM localization but 4 out of 6 showed diffusely positive NCAM immunostaining, with greatest intensity appreciated on the Bouin's-fixed tissue. There was no correlation between NCAM staining pattern and invasive characteristics of the tumors. These results suggest that NCAM expressivity is not a useful marker for assessing pituitary adenoma tumor invasiveness.

Azure B-eosin APAAP staining: a method for simultaneous hematological and immunological cell analysis.

Azure B-eosin APAAP staining allows simultaneous analysis of peripheral blood and bone marrow cells for hematological characteristics and immunological cell marker profiles. A defined sequence of staining procedures maintains characteristic components of the Romanowsky-Giemsa stain whereas cell antigens can be detected immunologically using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) detection system. Antigens are visualized by the staining product of the substrate-naphthol AS GR phosphate and variamine blue salt. The usefulness of the azure B-eosin APAAP method was demonstrated on blood and bone marrow smears of patients with various hematological disorders.

Specificity and sensitivity of immunocytochemistry for detecting P-glycoprotein in haematological malignancies.

To determine the optimal working conditions of the alkaline phosphatase-antialkaline phosphatase (APAAP) method to establish a specific and sensitive assay for the detection of low numbers of MDR positive cells in patients with hematological malignancies. Three monoclonal antibodies (C-219, JSB-1, MRK-16) were used for the detection of P-glycoprotein (P-gp) in cell lines and in samples from 43 patients with haematological malignancies. The results of the APAAP method were compared with western blotting for specificity and sensitivity. Excellent correlation was obtained between optimised APAAP and western blotting, except in the case of multiple myeloma. JSB-1 seemed to be the more useful monoclonal antibody for the APAAP which was more sensitive than western blotting in its ability to detect single P-gp positive cells. Methods for P-gp detection, as defined by multidrug resistant (MDR) cell lines, are not necessarily optimal and specific for clinical samples and may lead to higher false positive and negative results, according to the conditions and the monoclonal antibodies used.

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.

Immunophenotyping of Leukemia and Lymphoma Cells with IGSS: Comparison with APAAP and Flow Cytometry

Leukemia and lymphoma cells were immunophenotyped with immunogold-silver staining, the alkaline phosphatase-antialkaline phosphatase method, and flow cytometry and the results were compared. Peripheral blood, bone marrow aspirates, and lymph node cell suspensions of 26 patients with acute or chronic leukemia, non-Hodgkin's lymphoma or Hodgkin's disease were examined. Thirty-five monoclonal antibodies directed against cell surface antigens of myeloid and lymphoid cells were used. In addition, the efficiency of the 3 methods was determined by establishing monoclonal antibody dilution curves on normal peripheral blood cells. In each patient, the 3 immunocytochemical techniques gave an identical immunodiagnosis. Good correlation coefficients (0.91 to 0.93) were found among the percents of positive cells obtained with the3 techniques. Flow cytometry had the highest labeling efficiency. Some monoclonal antibodies of IgM class gave a less reliable immunostainine with APAAP than with IGSS or flow cytometry. Flow cytometry is recommended for immunophenotyping homogeneous cell suspensions. With poor samples or mixed cell suspensions, labeling on cytocentrifuge preparations with IGSS is preferred for its good morphology and stable immunostaining. (The J Histotechnol 16:263, 1993)

Immunohistology of the nasal mucosa in seasonal allergic rhinitis: Increases in activated eosinophils and epithelial mast cells

The immunohistology of the nasal mucosa was examined in 13 grass pollen—sensitive patients and in seven normal nonatopic control subjects before and during the pollen season. Cryostat sections (6 μm) of biopsy specimens from the inferior turbinate were immunostained with the alkaline-phosphatase antialkaline-phosphatase method and a panel of monoclonal antibodies. Mast cell subtypes were measured with a double sequential immunostaining method. Within the submucosa, seasonal increases in total (MBP +, p < 0.01) and “activated” (EG2 +, p < 0.01) eosinophils were observed for the patients, which were significant when these counts were compared with counts for those of control subjects (MBP + p < 0.01; EG2 + p < 0.001). Within the nasal epithelium, seasonal increases in total ( p < 0.05) and “activated” ( p < 0.02) eosinophils were also observed. Mast cell counts revealed seasonal increases in tryptase-only positive mast cell (MC T) ( p < 0.02) but not chymase plus tryptase—positive mast cells (MC TC) within the epithelium that were significant when counts were compared with those of control subjects ( p < 0.03). No significant changes were observed within the submucosa or epithelium for total leukocytes (CD45 + cells) or T-lymphocytes (CD3 +, CD4 +, CD8 +, and CD 25 + cells) for either group. Similarly, no significant changes were observed for neutrophils (antielastase), macrophages (CD68 +), nor HLA-DR + cells. In the subjects with rhinitis, seasonal submucosal CD3 + counts correlated with MBP + eosinophils ( r = 0.56; p < 0.05) and MC TS ( r = 0.65; p < 0.02). Similarly, seasonal epithelial EG2 + eosinophil counts correlated with MC Ts ( r = 0.56; p < 0.05). The results indicate that seasonal allergic rhinitis is associated with local accumulation of “activated” eosinophils and the epithelial migration of MC Ts but not MC Ts during natural grass-pollen exposure.