INTRODUCTION Infections by pathogenic organisms are the major cause of sufferings to the human beings.

Bacterial infections contribute to majority of microbial infections and a variety of diseases. Respiratory tract diseases like pneumonia caused by Streptococcus pneumoniae or pneumococcus is a significant human pathogen, primarily for young children and elderly adults, which leads to a large number of human deaths in various parts of the world, especially in developing countries like India. Pneumonia is a disease of lungs that is caused by S. pneumoniae which is gram-positive, lancet-shaped cocci, alpha-hemolytic, encapsulated, diplococcus and aerobic. Also variety of other bacterias including Streptococcus, Staphylococcus, Pseudomonas, Chlamydia, Mycoplasma infect the upper respiratory tract and can cause pneumonia. Pneumonia is an inflammatory illness of the lungs. Frequently, it is described as lung parenchyma/alveolar inflammation. During this infection pus and fluid fill the alveoli in one or both lungs, and this interferes with oxygen absorption, making breathing difficult. The symptoms of infectious pneumonia are caused by the invasion of the lungs by microorganisms and by the immune system's response to the infection. Pneumonia is the leading global killer of children under five, responsible for almost 1.6 million deaths per year. It occurs most commonly when a childs still-developing defense system is weakened by malnutrition, air pollution, co-infections with HIV/AIDS and measles, and low birth weight. In wealthier nations, adults over 65 years old and people with chronic health problems bear the greater burden of pneumonia. An estimated 156 million new cases of pneumonia occur each year, 97 percent of them in the developing world, and 7 to 13 percent of them severe enough to require hospitalization. Seventy-four percent of those cases occur in just 15 countries, mostly in Asia and sub-Saharan Africa, with 43 million cases in India alone. The death rate from pneumonia is 215 times higher in low-income countries than in high-income countries. Streptococcus pneumonia , is the most common cause of bacterial pneumonia worldwide, accounting for about 30 percent of the total pneumonia case load and at least half the cases in the developing world. The burden of pneumonia in the developing world is nearly ten times that of the developed world. Treating pneumonia depends on a number of factors, including patients age, general health and the causative organism. Patients with pneumonia need an antibiotic that is effective against the organism causing the disease. Antibiotics often used in the treatment of this type of pneumonia include penicillin, amoxicillin and clavulanic acid (Augmentin, Augmentin XR), and macrolide antibiotics including erythromycin (E-Mycin,Eryc, Ery-Tab, PCE, Pediazole, Ilosone), azithromycin (Zithromax, Z-Max), and clarithromycin(Biaxin). Penicillin was formerly the antibiotic of choice in treating this infection. With the advent and widespread use of broader-spectrum antibiotics, significant drug resistance has developed. Penicillin may still be effective in treatment of pneumococcal pneumonia, but it should only be used after cultures of the bacteria confirm their sensitivity to this antibiotic (3). Vaccination is more important in preventing pneumonia. Pneumococcal vaccine is the prototype of a capsular polysaccharide vaccine. Pneumococcal vaccines are classified into

two major categories which are Pneumococcal Polysaccharide Vaccine (PPV) and Pneumococcal Conjugate Polysaccharide Vaccine (PCV). Pneumovax23 is an example of PPV, which is a 23-valent polysaccharide vaccine. Prevnar is a 7-valent conjugated polysaccharide vaccine (4). The 23-valent pneumococcal polysaccharide vaccine has been shown to be safe and, in the majority of studies, effective in reducing the incidence of invasive disease . These proteinpolysaccharide combinations, known as conjugate vaccines, contain 713 selected polysaccharides bound to a protein carrier and induce a T-cell dependent immune response. These vaccines are likely to be protective even in children under two years of age and may reduce pneumococcal transmission through herd effect. The immunization with 7-valent PCV can be extended to the persons of age group 20-39 and greater than 65 years . Immunization with 23-valent PPV to the older adults over 65 years age against pneumococcal bacteremia with efficiency of 44-47% during 3-5 years (5). There are different types of bio-assays used for testing the immunogenicity of the pneumonia Vaccines. The different bioassays such as ELISA (Enzyme Linked Immuno Sorbent Assay), Opsono Phagocytic Killing Assay (OPA), Multiplexed Opsonophagocytic assay(MOPA) are available. The ELISAs give information about the presence and quantity of serotype specific antibodies especially Immunoglobulin G (IgGs). All the IgGs may not play a functional role in tackling the bacteria, hence the estimation of functional antibody titers are also required to assess the actual efficacy of the vaccine in generating immunogenicity data. The Opsonophagocytic assay (OPA) is used to estimate the phagocytic activity of the serum containing the antibody against S. pneumoniae. This assay measures the complement dependent opsonic activity of serum using culturable phagocytic cells. In this assay HL-60 promyelocytic leukemia cell is used as an effector cells (phagocytic cells). This assay provides an exact in vivo condition in in vitro, that's why this method is widely accepted for measuring the protective capacity of pneumococcal antibodies.

LITERATURE REVIEW Streptococcus pneumoniae Streptococcus pneumoniae, pneumococcus, is an important pathogen that causes both serious invasive infections, such as septicaemia, meningitis and pneumonia, and mild upper respiratory infections. It belongs to the normal nasopharyngeal microbial flora that consists of bacteria with physiologic and genetic properties suitable for colonization and multiplication under certain conditions. These microbes are usually harmless and they even benefit human health by preventing the growth of more pathogenic bacteria.(1) Pneumococcus is a gram-positive, hemolytic, bilesoluble and commonly capsulated streptococcus that is usually identified without problems. Identification is based on the bacterial colony morphology on a blood agar plate, optochin sensitivity, bile solubility and the presence of a capsule. So far, 90 different capsular serotypes have been identified. (1) Infections caused by S. pneumoniae can be divided into two categories. In invasive infections (e.g. meningitis, pneumonia and bacteraemia),where pneumococcus can be isolated from blood or other normally sterile body fluids. In mucosal infections such as sinusitis, otitis media and conjunctivitis, pneumococcus can be isolated from mucosal excretions only.(1) Pneumococci may occur in two forms, unencapsulated and capsulated. The capsule and the amount of produced capsules are significant virulence factors. The capsule protects bacterial cells very effectively from phagocytosis and also inhibits complement activation. The capsule also protects against environmental effects, and the produced components help pneumococci compete with other bacteria, such as H. influenzae, Moraxella catarrhalis and N. meningitidis, for existence in the nasopharynx.(5) Streptococcus pneumoniae is known to cause bacteremia, otitis media, and meningitis in humans, though it is best known for causing pneumonia, a disease of the upper respiratory tract that causes illness and death all over the world. (5) Symptoms of pneumonia include a cough accompanied by greenish or yellow mucous, fever, chills, shortness of breath, and chest pain. The bacteria enter the body most commonly via inhalation of small water droplets. Very young children and the elderly are the most prone to catching pneumonia. The virulence factors of S. pneumoniae include a polysaccharide capsule that prevents phagocytosis by the host's immune cells (5), surface proteins that prevent the activation of complement (part of the immune system that helps clear pathogens from the body), and pili that enable S. pneumoniae to attach to epithelial cells in the upper respiratory tract. (11) The polysaccharide capsule interferes with phagocytosis through its chemical composition, resisting by interfering with binding of complement C3b to the cell's surface. Encapsulated strains of S. pneumoniae are found to be 100,000 times more virulent than unencapsulated strains during invasion of mucosal surfaces. (11) Pili are long, thin extracellular organelles that are able to extend outside of the polysaccharide capsule. They are encoded by the rlrA islet (an area of a genome in which rapid mutation

takes place) which is present in only some isolated strains of S. pneumoniae. These pili contribute to adherence and virulence, as well as increase the inflammatory response of the host. (4) Among the 90 different polysaccharide serotypes, some are more virulent than others. A small number of them, approximately 10 serotypes/groups, are common in pneumococcal infections. According to a recent review, serotypes/groups 1, 3, 5, 6, 14, 19 and 23 are comprehensive types in invasive pneumococcal infections on several Continents. The serotypes/groups 6A, 6B, 14, 15, 18C, 19F and 23F are commonly isolated from the nasopharynx of healthy children on every continent.The serotypes 10, 11, 13, 15, 33 and 35 are also fairly common in the nasopharynx of healthy children, whereas types 1, 5 and 46 are rare even in populations where they are isolated from invasive infections. Pneumonia Pneumonia is a form of acute respiratory infection that affects the lungs. The lungs are made up of small sacs called alveoli, which fill with air when a healthy person breathes. When an individual has pneumonia, the alveoli are filled with pus and fluid, which makes breathing painful and limits oxygen intake.(2) Pneumonia is the single largest cause of death in children worldwide. Every year, it kills an estimated 1.4 million children under the age of five years, accounting for 18% of all deaths of children under five years old worldwide. Pneumonia affects children and families everywhere, but is most prevalent in South Asia and sub-Saharan Africa. Children can be protected from pneumonia, it can be prevented with simple interventions, and treated with low-cost, low-tech medication and care. (2)

Streptococcus pneumoniae is the most common cause of bacterial pneumonia. People who suffer from chronic obstructive pulmonary disease (COPD) or alcoholism most often get pneumonia from Klebsiella pneumoniae and Hemophilus influenzae. Atypical pneumonia, a type of pneumonia that typically occurs during the summer and fall months, is caused by the bacteria Mycoplasma pneumoniae. People who have Legionnaire's disease caused by the bacterium Legionella pneumoniae (often found in contaminated water supplies and air conditioners) may also develop pneumonia as part of the overall infection. Another type of bacteria responsible for pneumonia is called Chlamydia pneumoniae. Pneumocystis carinii pneumonia is a form of pneumonia that usually affects both lungs and is found in patients with weakened or compromised immune systems from such conditions as cancer and HIV/AIDS and those treated with TNF (tumor necrosis factor) for rheumatoid arthritis. (3) Pneumonia is caused by a number of infectious agents, including viruses, bacteria and fungi. The most common are: Streptococcus pneumoniae the most common cause of bacterial pneumonia in children; Haemophilus influenzae type b (Hib) the second most common cause of bacterial pneumonia; respiratory syncytial virus is the most common viral cause of pneumonia;

in infants infected with HIV, Pneumocystis jiroveci is one of the commonest causes of pneumonia, responsible for at least one quarter of all pneumonia deaths in HIVinfected infants.

(2)

Pneumococcal Vaccines At least 1 million children die of pneumococcal disease every year, most of these being young children in developing countries. In the developed world, elderly persons carry the major disease burden. Conditions associated with increased risk of serious pneumococcal disease include HIV infection, sickle-cell anaemia and a variety of chronic organ failures. Vaccination is the only available tool to prevent pneumococcal disease. The recent development of widespread microbial resistance to essential antibiotics underlines the urgent need for more efficient pneumococcal vaccines.(4) Immunity following pneumococcal disease is directed primarily against the capsular serotype involved. The currently licensed pneumococcal vaccine is based on the 23 most common serotypes, against which the vaccine has an overall protective efficacy of about 60%70%. Children under two years of age, and persons suffering from various states of immunodeficiency, for example HIV infection, do not consistently develop immunity following vaccination, thus reducing the protective value of the vaccine in some major target groups for pneumococcal disease. However, in a healthy elderly population the polysaccharide vaccine provides relatively efficient protection against invasive pneumococcal disease. (4) To obtain a more efficient vaccine, there are active efforts to improve currently available vaccines, which are designed to elicit antibodies to pneumococcal capsular polysaccharide (PS). A 23-valent pneumococcal PS vaccine (PPV23) contains capsular PS of 23 different serotypes and is widely used for the elderly . Because PPV23 is not useful for young children, a 7-valent pneumococcal conjugate vaccine (PCV7) was developed, and its use has significantly reduced the incidence of invasive pneumococcal infections. To extend the serotypic coverage of PCV7, 10- and 13-valent conjugate vaccines are now in late stages of development. (6) At present, one pneumococcal PS vaccine is still licensed and marketed, i.e. Pneumovax 23 produced by Merck Research Laboratories, USA. These vaccines contain 23 purified capsular polysaccharide antigens (serotypes 1, 2, 3, 4, 5,6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,20, 22F, 23F, and 33F) accounting for 8590% of pneumococcaldiseases . These vaccines are effective in adults and children above 2 years of age(5) Vaccine Potency A major concern regarding vaccine quality control is the requirement that each individual batch of vaccine is tested before use for safety and effectiveness. For this reason, the potency test are designed to measure the ability of a vaccine to protect against subsequent challenge from the active component responsible for pathogenicity, and represents a valuable tool for

testing the actual relative strength of manufactured batches of vaccine, as well as being essential to the pre-clinical and clinical development of a vaccine. Biological-based manufacturing methods are inherently variable, and potency testing provides a tool to ensure lot-to-lot consistency of commercial vaccines. Once established, the potency test provides the batch release data required by the relevant regulatory authorities to enable the subsequent licensing of a novel vaccine.(6,7) To evaluate novel bacterial vaccines, such as pneumococcal vaccines, an enzyme-linked immunosorbent assay (ELISA) is commonly used alongside a modified in vitro opsonophagocytic-killing assay (OPKA). Immune responses to pneumococcal vaccines have been evaluated with assays that measure total binding antibody, such as radioantigen binding assays and enzyme-linked immunosorbent assays (ELISAs). In addition, since host protection against pneumococcal disease is mainly mediated by phagocytosis, animal models and opsonophagocytic assays which measure functional antibodies are being developed.(9) The development or improvement of pneumococcal vaccines requires measuring their immunogenicity, determined primarily by measuring anticapsular PS antibody levels with the use of enzyme-linked immunosorbent assay (ELISA). Use of the pneumococcal antibody ELISA has established that an antibody level of 0.35 g/ml is associated with protection from invasive pneumococcal infections in children (7). However, old adults generally have more than 0.35g/ml of pneumococcal antibodies and yet are susceptible to pneumococcal infections . Thus, old adults may have nonfunctional pneumococcal antibodies , and ELISA for pneumococcal antibodies may be inadequate to accurately measure immunogenicity of pneumococcal vaccines in the elderly.(6) Opsonophagocytic killing is the best in vitro surrogate for a protective immune response against bacterial pathogens. Not all opsonic antibodies are protective, but all antibodies that provide antibacterial immunity are opsonic. For Gram-positive organisms, there is no direct bactericidal activity from antibodies in concert with complement because of the absence of an outer membrane, and opsonic killing requires the interplay of antibodies, complement, and phagocytes(6,8) Opsonophagocytic assays for S. Pneumonia are traditionally performed with peripheral blood leukocytes (PBLs) as effector cells and a variety of techniques (radioisotopic, flow cytometric, microscopic, and viability assays) measure opsonophagocytic activity (9) Pneumococcal Stock Preparation Characterization of S. pneumoniae Pneumococcus is routinely identified and characterized by four phenotypic characteristics: colony morphology on a blood agar plate, bile solubility, optochin sensitivity and the presence of a capsule.(10) Developed diagnostic molecular methods have made it possible to identify bacterial isolates through the presence of toxins, antimicrobial resistance genes and exact components of genes, e.g. lyt A. However, today most clinical laboratories still use the optochinsensitivity test or the bile solubility test as a screening test to differentiate pneumococcus from other -

hemolytic streptococcal isolates. The molecular methods have provided important tools for epidemiological surveillance (10) Optochin sensitivity Optochin sensitivity is commonly used and often the only characterization test for pneumococcus.Optochin (ethylhydrocupreine hydrochloride), a quinine derivative, has a detergent-like action and causes selective lysis of pneumococci. A sterile disk impregnated with optochin is placed on the first sector of an isolation plate before incubation. A zone of inhibition (area with no growth) of 14 mm. or more in diameter will be seen around the disk after incubation if the organism is Streptococcus pneumoniae. Other alpha-hemolytic streptococci are resistant to (not killed by) optochin. Their colonies will thus grow right up to the disk of optochin or have zones of inhibition less than 14 mm. in diameter. Incubation in the presence of carbon dioxide has been shown to influence the size of the inhibition zone. A trypticase soy medium with sheep blood and incubation in a carbon dioxide atmosphere proved to be the most suitable conditions for testing optochin sensitivity(10). Capsule staining Streptococcus pneumoniae is characterized by a polysaccharide capsule that completely encloses the cell, and plays a key role in its virulence. The cell wall of S. pneumoniae is composed of peptidoglycan, with teichoic acid attached to every third N-acetylmuramic acid, and is about 6 layers thick. Lipoteichoic acid is attached to the membrane via a lipid moiety, and both teichoic and lipoteichoic acid contain phosphorylcholine. Two choline residues may exist on each carbohydrate repeat, which is important to S. pneumniae because the choline adheres to choline-binding receptors located on human cells. (11) S. pneumoniae contains more than 500 different surface proteins. A notable group is the family of choline-binding proteins (CBPs). Twelve of these are bound to the choline moiety of the cell wall and assist in attaching various functional elements onto the surface of the cell. Among the CBPs are found important determinants of virulance, including PspA (protective antigen), LytA, B, and C (autolysins), and CbpA (adhesin). (11) The capsule staining is a negative staining in which the background, not the cells, are stained. In some protocols, the bacteria cells can be counterstained, leaving a colored background, stained bacteria cells, and an uncolored area around the cells, which represents the capsule. (17)

Quellung Reaction The quellung reaction (swelling reaction) forms the basis of serotyping and relies on the swelling of the capsule upon binding of homologous antibody. The test consists of mixing a loopful of colony with equal quantity of specific antiserum and then examining microscopically at 1000X for capsular swelling. Although generally highly specific, cross-

reactivity has been observed between capsular types 2 and 5, 3 and 8, 7 and 18, 13 and 30, and with E. coli, Klebsiella, H. influenzae Type b, and certain viridans streptococci. -hemolysis Hemolysis is the break down of the membrane of red blood cells by a bacterial protein known as hemolysin, which causes the release of hemoglobin from the red blood cell. Many types of bacterial posses hemolytic proteins. These proteins are thought to act by integrating into the membrane of the red blood cell and either punching a hole through the membrane or disrupting the structure of the membrane in some other way. The exact molecular details of hemolysin action is still unresolved.(18) There are three types of hemolysis, designated alpha, beta and gamma. Alpha hemolysis is a greenish discoloration that surrounds a bacterial colony growing on the agar. This type of hemolysis represents a partial decomposition of the hemoglobin of the red blood cells. Alpha hemolysis is characteristic of Streptococcus pneumonia and so can be used as a diagnostic feature in the identification of the bacterial strain.(18)

Bile Solubility Test Bile will selectively lyse colonies of Streptococcus pneumoniae while other strep are immune to bile's activity. When a bile salt such as desoxycholate is added directly to Streptococcus pneumoniae growing on an agar plate or in a broth culture the bacteria will lyse and the area become clear. Other alpha-hemolytic streptococci are resistant to (not lysed by) bile and will stay visible or turbid (cloudy).(19) Addition of a few drops of 10% deoxycholate at 37C lyses the entire culture in minutes. The ability of deoxycholate to dissolve the cell wall, depends upon the presence of the autolytic enzyme, LytA, that causes lysis of organism when grown on artificial medium. (16) The bile salt alters the surface tension of the medium and causes the cell membrane rearrangement. (20) Virtually all clinical isolates of pneumococci harbor the autolysin and undergo deoxycholate lysis.(16) Biochemical Properties S. pneumoniae lacks catalase and ferments glucose to lactic acid, like most other streptococci. However, unlike most other streptococci, it does not display an M protein and it hydrolyzes insulin. (5) S. pneumoniae gets a significant amount of its carbon and nitrogen through extracellular enzyme systems that allow the metabolism of polysaccharides and hexosamines, as well as cause damage to host tissue and enable colonization. (1) S. pneumoniae has the ability to enzymatically disrupt and disintegrate itself. This enzyme, an autolysin, causes a culture of S. pneumonia to undergo an autolysis, which kills the entire culture during the stationary phase (the phase in which growth slows due to exhaustion of

available nutrients and buildup of toxins). With initial growth beginning in optimal conditions, autolysis usually begins within 18-24 hours, with colonies collapsing in the center(1)

Multiplexed Opsonophagocytic Assays Pneumococcal conjugate vaccination is highly efficacious against invasive diseases in young children. Since host protection is mainly mediated by opsonin-dependent phagocytosis, the in vitro measurement of opsonophagocytic activity of the anti-capsular antibodies is assumed to be a reliable correlate of protection to monitor vaccine efficacy. The methods used so far are all tedious to perform and material-consuming. (12) OPA is the viable cell-killing assay described by the CDC in 1997. That assay has several desirable features, such as its micro-scale, its use of standardized target bacteria, and its use of a phagocytic cell line HL-60, which can be standardized and requires no blood donors. However, it has several shortcomings. It is slow and requires relatively large serum volumes. A multilaboratory evaluation of this assay established that the gold standard OPA can be a killing assay based on rabbit complement and HL-60 cells (13) Recently, several multiplex OPA formats have been developed. Two types of multiplexed killing-type OPA were developed using antibiotic-resistant target bacteria. One OPA uses rapid colony counting as the assay readout (7), and the other OPA uses fluorescence as the readout (14). The latter assay measures the fluorescence of alamar blue dye, which reflects the metabolic activity of the surviving living bacteria. In addition to the killing-type OPA, a multiplex uptake OPA has been developed, which uses a flow cytometer to measure the uptake of fluorescent target bacteria or antigen-coated plastic particles(13) In addition, the OPA is converted to a multiplexed OPA by using antibiotic-resistant target bacteria (7, 16). The multiplexing has greatly reduced the amount of serum required for the assay and is very easy to implement for any laboratory performing a conventional killing OPA. A 4-fold multiplexed OPA (MOPA4) has been extensively characterized as showing both specificity and sensitivity. Also, MOPA4 can be routinely performed on a large scale, with the assay throughput of MOPA4 being equivalent to that of ELISA.(13) At present, a method of automated data analysis is being investigated. Instead of providing discrete results (classical titers), the new method would give continuously variable results by interpolation. The continuous results should be more precise and accurate. To improve the long-term stability of MOPA4, acceptance criteria are being developed for assay components such as phagocytes, target bacteria, and complement. In addition, the target bacteria forMOPA4 are readily available and performing MOPA4 requires no special tools. Thus, MOPA4 is ideally suited to be a reference assay. (13) The fourfold multiplexed killing-type OPA can be performed using pneumococcal strains resistant to four clinically irrelevant antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Under development is a panel of antibiotic-resistant target strains that would be useful in evaluating 11- or 13-valent conjugate vaccine(15)

Based on the same principle, another laboratory has developed a sevenfold multiplexed OPA. The seven strains represent the serotypes included in the currently available 7-valent conjugate vaccine. This method uses seven antibiotic-resistant pneumococcal strains that are either from clinical cases or are laboratory derived. The assay conditions are different from those of the Romero-Steiner assay conditions. It uses 30 l of serum sample, 15 l of a mixture of 7 pneumococcal strains (3,000 bacteria/serotype, 2 105 bacteria/ml), 15 l of complement, and 20 l of 1.4 106 HL-60 cells. The OPA titer was determined as the reciprocal of the serum dilution killing 90% or more. The results compared well with conventionally determined OPA titers. The assay, however, has not been fully standardized nor validated. Nevertheless, this study has demonstrated the adaptability and flexibility of an antibiotic-based, multiplexed, killing-type OPA. (15) HL-60 All in vitro OPAs require phagocytes, which are commonly obtained from one of two sources. The first source is peripheral blood of normal donors, which provides the most biologically granulocytes but presents several shortcomings. The genetic or clinical status of individual donors will vary, with clear implications for the standardization of an OPA. It is also inconvenient to perform phlebotomy by routine schedules, as donors need to be screened for health conditions and granulocytes must be purified prior to the OPA being performing. In addition, an assay may require a large number of granulocytes, necessitating phlebotomy of large volumes of blood from an individual or smaller volumes pooled from many donors. For these reasons, a promyelocytic cell line has been used to provide phagocytic cells for the OPA.(21) Recently, a number of laboratories have used HL-60 cells, subjected to conditions that promote differentiation towards granulocyte morphology, as phagocytes with varying degrees of success.(21) The HL-60 cell line was derived from peripheral blood leukocytes of a 36-year-old Caucasian female with acute promyelocytic leukemia (9). It was among the first long-term suspension cultures of human myeloid leukemic cells to be established and has been extensively characterized during the past decades. The original wild-type HL-60 cell line had several properties of malignant cells and expressed various oncogenes.(21) DIFFERENTIATION OF HL-60 Critical to the successful adoption of a cell line for bioassay is the reproducibility of the function of the cell within the assay itself. In the MOPA, the effector cell must first be induced to differentiate to a phagocyte. Size, morphology, and the extent of heterogeneity among cultured HL-60 cells suggest that they may spontaneously differentiate in vitro (9). Approximately 5 to 10% of HL-60 cells appear to be mature myelocytes; many resemble mononuclear phagocytes, metamyelocytes, banded cells, or fully segmented neutrophils. Similarly, about 5 to 10% of cultured HL-60 cells have properties of differentiated granulocytes, such as phagocytic ability and the ability to respond to chemotactic peptides (e.g., N-formylmethionyl-leucine) (33).The effector HL-60 cells for MOPA can also be regulated by induction of differentiation towards three distinct myeloid cell lineages (monocytic, eosinophilic, and granulocytic), depending on the environmental conditions and the chemical inducers used (9,21).

The multipotentiality of this cell line to differentiate into various cell lineages has been a research subject since its discovery.Environmental conditions such as pH and multiple chemical inducers can greatly facilitate the differentiation of HL-60 cell lines into various myeloid lineages . These chemicals generally arrest the cell cycle at the time of induction of differentiation. For example, sodium butyrate induces monocytic differentiation (8) and N,Ndimethylformamide (DMF) and other polar compounds induce granulocytic differentiation (21). Additional inducers such as retinoic acid, dimethyl sulfoxide (DMSO), and dibutryl cyclic AMP have been also reported to induce granulocytic differentiation. Many factors can affect optimal differentiation; conditions for differentiation depend primarily on the concentration of the inducer, the time of exposure, and the relative proportion of cells in different segments of the cell cycle (9,21). Continuous exposure to these inducers is not necessary ; even a short exposure may be sufficient (21). The observations of spontaneous differentiation, selection of sublines, and changes in colonyforming tendencies with increasing passage enforce the requirement for robust control of the cell line and its processes. Bioassays by their very nature require standardization, and unnecessary variation must be avoided. Therefore, for a standardized bioassay it is critical to standardize and optimize differentiation conditions to provide reproducible yields of granulocytes suitable for use as effector cells within the MOPA. Optimized conditions have been described by various authors for differentiation into neutrophils and these include 1.25% (vol/vol) of DMSO in a period of 5 to 7 days (10, 19), 100 mM DMF in a period of 5 days or 0.1 M all-trans-retinoic acid (ATRA) in a period of 5 days (21). In some cases, comparisons between inducers indicate they can be synergistic and are more effective when they are used in combination (9,21). For instance, HL-60 cells widely available in Europe (ECACC 98070106) differentiate into functional neutrophils better when they are exposed to a mixture of ATRA, 1,25 dihydroxyvitamin D3 (vitamin D3), and granulocyte colony-stimulating factor (21). Antibiotics