Leukocyte Alkaline Phosphatase Activity Research Articles

Early Diagnosis of Neonatal Sepsis Using the Leukocyte Alkaline Phosphatase Score: A Cohort Study

Introduction: The clinical features of sepsis in neonates are subtle and non specific, requiring a high index of suspicion for early diagnosis. Blood culture is the gold standard for diagnosis, but it is time consuming. Therefore, there is a need for a costeffective and reliable screening tool. The Leukocyte Alkaline Phosphatase (LAP) activity of neutrophils is known to increase during bacterial infections in adults. Aim: To determine the activity of LAP in Neonatal Sepsis (NS) and compare the results with blood culture. Materials and Methods: This is a prospective cohort study conducted from January 2018 to June 2019 at Mandya Institute of Medical Sciences, Karnataka, India. Total of 200 neonates by their haematological profile were clinically suspected of having sepsis. A peripheral smear was prepared from a drop of blood and stained to assess the LAP activity. In each smear, a total of 100 consecutive segmented neutrophils were examined and were rated from 0 to 4 according to the red granular precipitate intensity within their cytoplasm. The possible range of 0-400. The blood culture report was obtained from the case sheet. The neonates were divided into two groups: Group 1 consisted of culture-proven sepsis cases, and Group 2 consisted of neonates with clinical suspicion of sepsis but negative blood culture results. Descriptive analysis was performed using mean and standard deviation for quantitative variables, and frequency and proportion for categorical variables. Chi-square test was conducted, and a p-value <0.05 was considered statistically significant. Results: Out of the 200 cases studied, 64 neonates showed a positive blood culture. The most common causative organism observed was Klebsiella pneumoniae, which was seen in 56 neonates. The age of the neonates ranged from newborn to 28 days old, with 115 being male. Furthermore, 116 neonates were term neonates, and 146 presented with early onset sepsis. The study revealed a wide range of LAP activity in both groups. In the culture-positive sepsis group, the LAP activity ranged from 36 to 350, while in the clinical sepsis group, it ranged from 10 to 354. Due to the broad range of LAP scores observed, it is concluded that LAP activity is not useful as a screening test. Conclusion: Findings of the present study indicate that LAP activity exhibited a wide range in both the culture-proven and clinical sepsis groups. However, due to this variability, LAP activity does not appear to be a reliable screening test for NS. While LAP activity assessment may still hold diagnostic value, further research is needed to refine its utility and explore its clinical relevance.

妊婦血中好中球アルカリフォスファターゼ(L.Alp)活性の制御機構に関する研究

妊婦血中好中球アルカリフォスファターゼ(L.Alp)活性の制御機構に関する研究

Comparative studies on biochemical and cytological constituents of synovial fluids in some farm animals

The goal of the present study was to evaluate the difference in the synovial fluid constituents in cattle, buffaloes, camels, and donkeys. A total number of 20 clinically healthy adult male animals (cattle (N = 5), buffaloes (N = 5), camels (N = 5), and donkeys (N = 5) were subjected to study. Synovial fluid samples were collected from the metacarpophalangeal joint under complete aseptic conditions. The samples were examined physically, cytologically, and biochemically. Synovial fluid analysis revealed significant variations in specific gravity, total leukocyte counts, total proteins, albumin, globulins, glucose levels, and alkaline phosphatase activity among investigated animal species.

JAK2 V617F uses distinct signalling pathways to induce cell proliferation and neutrophil activation

The acquired JAK2 V617F mutation is observed in the majority of patients with BCR-ABL1 negative chronic myeloproliferative neoplasms (MPN). BCR-ABL1 negative MPN displays myeloproliferation with an elevated leucocyte alkaline phosphatase (LAP) activity, a neutrophil activation marker. We tried to separate the downstream signalling of JAK2 V617F to stimulate myeloproliferation and LAP activity. NB4, a myeloid lineage cell line, was transduced with Jak2 V617F mutation or wild-type Jak2. We found that Jak2 V617F mutation, but not wild-type Jak2 enhanced LAP expression in NB4-derived neutrophils and proliferation of NB4 cells. JAK2 V617F induces constitutive phosphorylation of STAT3 and STAT5, and uses signalling targets such as Ras/MEK/ERK and PI3K/Akt pathways. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, and STAT3 or STAT5 siRNAs, JAK2 V617F was found to specifically use the STAT3 pathway to enhance LAP expression, while STAT5, Ras/MEK/ERK and PI3K/Akt, but not STAT3 pathways, were able to stimulate cell proliferation. These data strongly suggest that JAK2 V617F uses distinct signalling pathways to induce typical pathological features of MPN, such as high LAP activity and enhanced cell proliferation.

Identification of Health Risks in Workers Staying and Working on the Terrains Contaminated with Depleted Uranium

This study investigated health risks in workers residing and working in terrains contaminated by low ionizing radiation doses which originated from ammunition containing depleted uranium (DU). The studied population was composed of two test groups (T-I, T-II) who were occasionally exposed to DU, and two referent (R-I, R-II) groups not exposed at any time to DU. All of them were evaluated for the following: complete clinical examination and blood count, presence of immature forms and blasts, leukocyte alkaline phosphatase activity and cytogenetic tests. The probability of onset of the characteristic complete biomarkers--chromosomal aberrations, was analyzed using logarithmic function of the Poisson regression. The estimated function of the density of probabilities of Poisson distribution of the chromosomal aberrations in the test group T-II was drastically different from the corresponding distribution of the referent group R-I and to a somewhat lesser extent from the group R-II; Wilcoxon test exactly confirms the presence of a significant difference between the reference group R-II and test group T-II, p < 0.05. The damages to chromosomes and cells were highest in the test group T-II of workers additionally occupationally exposed to DU. The group of workers T-I, who had been exposed to DU working on contaminated terrain, have had certain risks of cell and chromosome damages, and that risk was not greater than the risk to the referent group R-II of workers occupationally exposed to ionizing radiation.

CHRONIC NEUTROPHILIC LEUKAEMIA: A DISTINCT CLINICAL ENTITY?

CHRONIC NEUTROPHILIC LEUKAEMIA: A DISTINCT CLINICAL ENTITY?

Flow-Cytometric Analysis of Leukocyte Alkaline Phosphatase in Myelodysplastic Syndromes

The expression of leukocyte alkaline phosphatase (LAP) in neutrophils is reduced in some patients with myelodysplastic syndrome (MDS). We quantitatively assayed for LAP in MDS leukocytes by a flow cytometry based method using a monoclonal antibody raised against human bone alkaline phosphatase. The LAP expression was assayed in blood samples from a group of 46 MDS patients, consisting of 39 patients with refractory anemia (RA), 3 with RA with excess blasts (RAEB), and 4 patients with RAEB in transformation. The percentage of LAP-positive cells was significantly higher in the MDS patients than in the normal subjects and also higher in RA than in RAEB and RAEB in transformation. To investigate the cause of the elevated LAP expression, we measured the serum concentrations of several cytokines. The granulocyte colony-stimulating factor (G-CSF) level was significantly elevated in MDS patients, and the serum G-CSF concentration clearly correlated with the percentage of LAP-positive cells. Thus, the LAP activity in RA is higher than in normal subjects, and G-CSF is thought to be one of the causes stimulating LAP expression in MDS neutrophils.

Sweet’s syndrome associated with chronic myelogenous leukemia: Demonstration of leukemic cells within a skin lesion

We report a case of acute febrile neutrophilic dermatosis, Sweet’s syndrome, associated with chronic myelogenous leukemia (CML) in which we found rearrangement of the bcr gene in DNA obtained from a skin lesion as well as in blood DNA by Southern blot analysis. This indicated the presence of CML cells within the skin lesion. To our knowledge, this is the first report in which the presence of CML cells is shown within skin lesions of Sweet’s syndrome. In our patient, leukocyte alkaline phosphatase activities returned to normal levels when he was suffering from Sweet’s syndrome and decreased again to below normal levels after it subsided. Whether the normalization of leukocyte alkaline phosphatase activity is common among CML patients with Sweet’s syndrome remains to be determined. (J Am Acad Dermatol 1999;40:275-9.)

Benzene exposure in car mechanics and road tanker drivers.

The purpose of this study was to identify professional factors related to benzene exposure and to deduce suitable safety measures. Atmospheric benzene, urinary muconic acid (tt-MA) and leukocyte alkaline phosphatase activity (LAPA) were evaluated among 66 car mechanics, 34 road tanker drivers, and 28 nonexposed workers. Professional and medical questionnaires were filled in at the same time. Atmospheric benzene was significantly higher among road tanker drivers than among car mechanics. The arithmetic mean +/- SD, median, and geometric mean values were, respectively, 0.48 +/- 1.49, 0.14, and 0.06 mg/m3 among car mechanics and 1.88 +/- 4.18, 0.68, and 0.65 mg/m3 among road tanker drivers. In the latter case the increase was caused by transport of unleaded petrol and correlated with the volume of the tank. Among car mechanics, tobacco smoking, windy conditions, dismantling of petrol filters, and handling of petrol increased atmospheric benzene levels. Urinary muconic acid was increased significantly among car mechanics (148 +/- 137, 127, and 111 micrograms/g) and among road tanker drivers (309 +/- 420, 137, and 151 micrograms/g) as compared with the controls (49 +/- 46, 33, and 33 micrograms/g). Among road tanker drivers, alcohol intake and transportation of unleaded petrol increased the excretion of muconic acid, which was also directly related to the volume of the tank. Among car mechanics, professional factors (dismantling of petrol filters, handling of and washing of hands with petrol) and nonprofessional factors (tobacco smoking and damaged skin on the hands and forearms) increased muconic acid excretion. In the control group, tobacco smoking increased its excretion. LAPA was not significantly modified among exposed workers. There was a weak but significant linear correlation between LAPA and muconic acid. These results suggest that to reduce exposure to benzene in unleaded petrol, individual and collective safety measures should be imposed in both occupations.

MPXI and early neutrophilia: New potential therapeutic biomarkers for recombinant human granulocyte colony-stimulating factor

We evaluated the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) given after myelosuppressive chemotherapy in 15 cancer patients. No severe neutropenia (absolute neutrophil count, ANC < 0.5 x 10(3)/microL) was noticed in 10 rhG-CSF primary prophylactic patients, but was noticed in two of five rhG-CSF secondary prophylactic patients. Neutrophilia characterized by shift to the left occurred within 24 hours after starting rhG-CSF prophylaxis. Thereafter, conversion to normal level occurred within 24 hours. The peak of neutrophilia occurred earlier in the primary group than in the secondary prophylactic group. The detection of myeloperoxidase (MPO) using flow cytochemistry blood autoanalyzer (TechniconR H * 1) was evaluated as mean peroxidase index (MPXI). Leukocyte alkaline phosphatase (LAP) using the method of Kaplow (Am J Clin Pathol 39:439-449, 1963) was recorded as LAP score. There was a statistically significant elevation of MPXI in the primary group over the secondary prophylactic patients. The LAP activity was in normal range. There was a slightly decreased red blood cell (RBC) count, hemoglobin (Hb), and platelet count. In conclusion, rhG-CSF induced neutrophilia with efficient enzymatic activity. These findings demonstrate the value of rhG-CSF in patients receiving chemotherapy. MPXI and early neutrophilia may serve as a potential biomarker of therapeutic efficacy of rhG-CSF.

CD16 antigen density on neutrophils in chronic myeloproliferative disorders.

Using flow cytometry, we quantitatively examined the density of the CD16 (IgG Fc receptor III) antigen on neutrophils in healthy control subjects, in patients with neutrophilia due to bacterial infection, and in patients with chronic myeloproliferative disorders (chronic myeloid leukemia [CML], polycythemia vera, or essential thrombocythemia). The density was expressed as the mean fluorescence intensity of neutrophils stained with fluorescein isothiocyanate-labeled anti-CD16 monoclonal antibody. We also determined leukocyte alkaline phosphatase activity semiquantitatively in the same population. The mean (+/- SD) density of the CD16 antigen on neutrophils in patients with CML (n = 13; 240.4 +/- 134.8) was lower (P<.001 ) than in healthy control subjects (n = 25; 656.6 +/- 238.0), and the density was also lower than in patients with bacterial infection (n = 15; 671.5 +/- 288.1), polycythemia vera (n = 7; 552.6 +/- 99.9), or essential thrombocythemia (n = 11; 671.5 +/- 411.5). The density of the CD16 antigen was 300 or more in all healthy control subjects and in all patients examined, except for those with CML. The CD16 antigen density was less than 300 in 10 of the 13 patients with CML. Leukocyte alkaline phosphatase activity was also low in 10 of the 13 patients with CML. These findings indicate that flow cytometric analysis of the density of neutrophil CD16 antigen is useful for the differential diagnosis of CML from other chronic myeloproliferative disorders.

Leukocyte alkaline phosphatase score in plasma cell dyscrasias: correlation with disease severity and circulating levels of granulocyte-colony stimulating factor.

In this study the leukocyte alkaline phosphatase (LAP) score in 106 patients with multiple myeloma (MM) in various phases of the disease (66 at diagnosis, 18 in plateau phase, 22 in relapse) was examined and compared with the score of 68 patients with monoclonal gammopathy of undetermined significance (MGUS) and 53 normal volunteers. In addition, the circulating levels of granulocyte-colony stimulating factor (G-CSF) were measured to explore the possible involvement of this cytokine in the pathogenetic mechanisms that lead to increased LAP activity. The results showed that the mean LAP score in patients with MGUS was comparable to normals and significantly lower than in MM (p < 0.001). Also, it increased with increasing tumor mass, and was lower in myelomas with stable disease than in those with active disease. G-CSF concentrations closely mirrored the behaviour of LAP score (r = 0.850, p < 0.001), significantly differing between each group of individuals. Its mean levels in MGUS were comparable to those of controls, whereas they were significantly increased in MM (p < 0.001), again with escalating values from cases with low tumor mass to advanced stages, and with lower concentrations in patients in plateau phase than in those in relapse. A significant correlation was found between G-CSF and neopterin levels (r = 0.578, p < 0.001), thus indicating an origin of the cytokine from monocytes and macrophages. These findings suggest that LAP scoring may assist in distinguishing benign from malignant paraproteinemias and may be used to follow the progression of plasma cell neoplasias.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative method to measure alkaline phosphatase activities in individual leukocytes by image analysis.

Leucocyte alkaline phosphatase (L-ALP) is well known as leukemia marker, but recent results suggest its usefulness for the diagnosis of several diseases. The aim of this study was to develop a quantitative method to measure alkaline phosphatase activities in individual leukocytes by image analysis. We studied the reaction rate of L-ALP in human polymorphonuclear leucocytes by a microscope attached to a TV camera and a computerized image analyzer. The optical density (OD) measured was standardized by grey filters with known absorbance. We measured IOD for individual cells after a set incubation time by end-point measurements. Studies of kinetic parameters of L-ALP were performed by single-point measurements in the linear phase of the reaction and at increasing substrate concentrations. Cellular IOD increased proportionally with incubation time up to 10 min. The mean KM(mM) and Vmax(delta IOD/min) values were 0.70 +/- 0.11 and 1.76 +/- 0.2 (mean +/- SE, n = 5) respectively. Our findings are comparable to previous results using a polyvynil alcohol method in microphotometry analysis. The image analysis of cellular L-ALP activity appears a valuable tool for quantitative studies.

Retinoic acid and granulocyte colony-stimulating factor synergistically induce leukocyte alkaline phosphatase in acute promyelocytic leukemia cells

In this report we show a strong synergistic interaction between granulocyte colony-stimulating factor (G-CSF) and all-trans retinoic acid (ATRA) on the expression of leukocyte alkaline phosphatase (LAP) in freshly isolated acute promyelocytic leukemia (APL) blasts as well as in NB40 and HL-60 cell lines. The strong synergism observed in these cell types was not evident in two acute leukemia cell lines (K562 and GF-D8), in normal granulocytes, and in monocytes. In freshly isolated leukocytes derived from chronic myelogenous leukemia (CML), in the stable phase of the disease, a weaker interaction between ATRA and G-CSF was documented. The cross-talk between the cytokine and the retinoid was studied in detail in NB4, an immortalized APL leukemia cell line, retaining the 15-17 chromosomal translocation involving the retinoic acid receptor type alpha. The treatment of NB4 cells with G-CSF alone or ATRA alone leads to no increase and to minor induction in LAP activity, respectively. If the cells are treated with the two compounds simultaneously, a dramatic elevation of LAP is observed after 4 days. The synergism between G-CSF and ATRA is evident at concentrations of the retinoid between 10(-7) and 10(-5) mol/L and at concentrations of the cytokine between 1 and 10 ng/mL. The simultaneous presence of the two compounds is necessary to obtain maximal increase of LAP activity and the effect is cell density-dependent. Synergism is specific for G-CSF, and it is not observed with other cytokines and functional inducers of the granulocyte. The augmentation of LAP activity is the consequence of an increased transcriptional rate of the liver/bone/kidney-type (L/B/K-type) alkaline phosphatase gene, as determined by Northern blotting and nuclear run-on analysis using specific cDNA probes. Only one of the two possible alternatively spliced forms of L/B/K-type alkaline phosphatase transcript is detected in NB4 cells after stimulation with G-CSF and ATRA. This mRNA form, which is the one observed in normal polymorphonuclear leukocytes, contains the most upstream leader exon. In NB4 cells, ATRA induces G-CSF, alpha, and beta retinoic acid receptor transcripts, whereas G-CSF has minor effects on the expression of these mRNAs.

Retinoic Acid and Granulocyte Colony-Stimulating Factor Synergistically Induce Leukocyte Alkaline Phosphatase in Acute Promyelocyte Leukemia Cells

AbstractIn this report we show a strong synergistic interaction between granulocyte colony-stimulating factor (G-CSF) and all-trans retinoic acid (ATRA) on the expression of leukocyte alkaline phosphatase (LAP) in freshly isolated acute promy-elocytic leukemia (APL) blasts as well as in NB40 and HL-60 cell lines. The strong synergism observed in these cell types was not evident in two acute leukemia cell lines (K562 and GF-D8), in normal granulocytes, and in monocytes. In freshly isolated leukocytes derived from chronic myelogenous leukemia (CML), in the stable phase of the disease, a weaker interaction between ATRA and G-CSF was documented. The cross-talk between the cytokine and the retinoid was studied in detail in NB4, an immortalized APL leukemia cell line, retaining the 15-17 chromosomal translocation involving the retinoic acid receptor type α. The treatment of NB4 cells with G-CSF alone or ATRA alone leads to no increase and to minor induction in LAP activity, respectively. If the cells are treated with the two compounds simultaneously, a dramatic elevation of LAP is observed after 4 days. The synergism between G-CSF and ATRA is evident at concentrations of the retinoid between 10-7 and 10-5 mol/L and at concentrations of the cytokine between 1 and 10 ng/mL. The simultaneous presence of the two compounds is necessary to obtain maximal increase of LAP activity and the effect is cell density-dependent. Synergism is specific for G-CSF, and it is not observed with other cytokines and functional inducers of the granulocyte. The augmentation of LAP activity is the consequence of an increased transcriptional rate of the liver/bone/kidney-type (L/B/K-type) alkaline phosphatase gene, as determined by Northern blotting and nuclear run-on analysis using specific cDNA probes. Only one of the two possible alternatively spliced forms of L/B/K-type alkaline phosphatase transcript is detected in NB4 cells after stimulation with G-CSF and ATRA. This mRNA form, which is the one observed in normal polymorphonuclear leukocytes, contains the most upstream leader exon. In NB4 cells, ATRA induces G-CSF, α, and β retinoic acid receptor transcripts, whereas G-CSF has minor effects on the expression of these mRNAs.

犬における好中球アルカリフォスファターゼ活性

犬における好中球アルカリフォスファターゼ活性

Leukocyte alkaline phosphatase score in patients with cerebrovascular disease and in patients with primary and metastatic brain tumors

Leukocyte alkaline phosphatase (LAP) activity in peripheral blood was determined in 54 patients with cerebrovascular disease (CVD), 11 patients with primary brain tumor, and 23 patients with metastatic brain tumor. The LAP score of the primary brain tumor patients (155 +/- 40) was significantly higher (P less than 0.03) than the LAP score for cerebrovascular disease patients (96 +/- 87). The LAP score levels of the metastatic brain tumor patients (251 +/- 55) was significantly higher as compared with both the cerebrovascular disease (P less than 0.001) and primary brain tumor groups (P less than 0.001). The LAP score could serve as an additional, although non-specific, marker to assist in the differentiation between cerebrovascular disease and brain tumors.

Induction of alkaline phosphatase activity in chronic myeloid leukemia cells: In vitro studies and speculative hypotheses

Three patients with Ph chromosome + chronic myeloid leukemia (CML) in chronic phase suffered from intercurrent pleuritis of undefined origin. At that time, leucocyte alkaline phosphatase activity (LAPA) score was low for circulating neutrophils, but high for those from pleural effusion. LAP negative circulating CML granulocytes were incubated with the pleural liquid: after 40-70 hr, almost all were intensely LAP positive. This finding suggests that the low LAPA score in resting CML neutrophils is attributable to the absence of appropriate stimuli rather than to an incapacity to synthesize the enzyme.

In vivo leucocyte interactions on Pellethane® surfaces

In vivo leucocyte interactions of three Pellethane® materials of varying hardness were qualitatively and quantitatively characterized using a cage implant system over a 21 d implantation period. Scanning electron microscopy (SEM) and cytochemical staining were utilized to observe the cellular events occurring at the leucocyte-biomaterial interface. Many of the quantitative assays performed, the intracellular alkaline phosphatase activity of exudate leucocytes, the intracellular acid phosphatase activity of adherent leucocytes, the density of adherent leucocytes and the foreign body giant cell network formation tendencies of adherent leucocytes, suggest increased cellular activation with increased Pellethane® hardness. Qualitative SEM evaluation of Pellethane® surfaces revealed a variety of cellular activities. These included macrophage adherence, cytoplasmic spreading and macrophage-macrophage membrane fusions to form foreign body giant cells. The foreign body giant cells exhibited nuclear reorganization and, when compared with adherent macrophages, they displayed an enhanced ability to fuse to neighbouring leucocytes, increased spreading of membrane processes over the polymer surface, the presence of large cytoplasmic vacuoles, and a lengthened duration of enzymatic activity. Contact angle analysis showed the Pellethane® surfaces to be hydrophobic and of low hysteresis. The critical surface tension and the dispersive component of the total surface tension were found to increase with Pellethane® hardness.

Leukocyte alkaline phosphatase in hypothyroidism and hyperthyroidism. Response to initiation of thyroxine replacement therapy

Leukocyte alkaline phosphatase (LAP) activity was determined in normal subjects, and in untreated, symptomatic patients with primary hypothyroidism or thyrotoxicosis. The means ± 1 SD of (n) subjects were, respectively: 61.7 ± 27.5 (16), 149.9 ± 56.3 (9) and 96.9 ± 27.7 (9). The mean LAP values of the hypothyroid and thyrotoxic groups were significantly different from that of the normal group ( P < .01). Values were above the normal range (20 to 120) in seven of the nine hypothyroid patients. LAP values were in the upper half of the normal range in eight of the nine thyrotoxic patients. In the two hypothyroid patients studied at 24-hour intervals, LAP activity was altered markedly within 48 hours of initiation of thyroxine therapy, 25 μg daily. In five hypothyroid patients followed for several months after initiating thyroxine replacement, LAP levels were essentially normal within 1 to 2 months. In the thyrotoxic patients, LAP values declined within the first month of medical management, but tended to remain within the normal range.