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CELLS OF THE IMMUNE SYSTEM

Published in Immunology
Saturday, 15 July 2017 11:00
The major cells of the immune system are lymphocytes. Lymphocytes that are critical for immune reactions are of two types namely B-cells and T-cells. Both cells develop from stem cells located in the liver of the foetus and in bone marrow cells of adults.
 
The lymphocytes which are differentiated in the bone marrow are B-cells. The lymphocytes that migrate to thymus and differentiate under its influence are called T-cells. The young lymphocytes migrate to lymphoid tissues such as spleen, lymph nodes and tonsils where they undergo final maturation. Matured lymphocytes circulate in the body fluids. T-cells are responsible for cellular immunity and B-cells produce antibodies about 20 trillion per day.
 
Both components require antigens to trigger them into action but they respond differently.
 
Antigens
 
An antigen is a substance when introduce into an individual, stimulates the production of an antibody with which it reacts. Antigens are large molecules of proteins or polysaccharides. Some of the antigens are the parts of microorganisms others include pollen, egg white, certain fruits, vegetables, chicken, feathers etc.

Antibodies
 
Antibodies are protein molecules called immunoglobulin (Ig). They are produced by lymphocytes. The antibodies inactivate antigens. An antibody  consists of four amino acid chains bounded together by disulphide bonds. Of the four chains two are long, heavy chains and two are short, light chains. All of them are arranged in the shape of the letter ‘Y’. The tail portion of antibody having two heavy chains is called constant fragment (Fc). On the tip of each short arm, an antigen- binding fragment (Fab) is present which specifically hold antigen.
 
Antibody immunity10
 
Based upon the five types of heavy chains, the immunoglobulin's are classified into five major types. Light chains are similar in all types of Immunoglobulin's.
 
TYPES OF IMMUNOGLOBULIN'S
 
lgG is the most important long acting antibody representing about 80% of the antibodies. The second important antibody is IgM. IgA is called secretory antibody, found in tears, saliva and colostrum, (the first milk secreted by mother). IgD serves as a receptor site at the surface of B cells to secrete other antibodies. IgE plays an important role in allergic reactions by sensitizing cells to certain antigens.
 
iimmunoglobulin types12

Scientists develop new antibiotic for gonorrhea

Published in News
Friday, 03 March 2017 15:11

Scientists at the University of York have harnessed the therapeutic effects of carbon monoxide-releasing molecules to develop a new antibiotic which could be used to treat the sexually transmitted infection gonorrhoea.

The infection, which is caused by the bacteria Neisseria gonorrhoeae, has developed a highly drug-resistant strain in recent years with new cases reported in the north of England and Japan.

There are concerns that gonorrhoea, which is the second most common sexually transmitted infection in England, is becoming untreatable.

Almost 35,000 cases were reported in England during 2014, with most cases affecting young men and women under the age of 25. The interdisciplinary team, from the University of York's Departments of Biology and Chemistry, targeted the "engine room" of the bacteria using carbon monoxide-releasing molecules (CO-RMs).

CO is produced naturally in the body, but there is increasing evidence that carbon monoxide enhances antibiotic action with huge potential for treating bacterial infections.

The scientists found that Neisseria gonorrhoeae is more sensitive to CO-based toxicity than other model bacterial pathogens, and may serve as a viable candidate for antimicrobial therapy using CO-RMs.

The CO molecule works by binding to the bacteria, preventing them from producing energy.

Scientists believe the breakthrough, published in the journal MedChemComm, could pave the way for new treatments.

Professor Ian Fairlamb, from the University's Department of Chemistry, said: "The carbon monoxide molecule targets the engine room, stopping the bacteria from respiring. Gonorrhoea only has one enzyme that needs inhibiting and then it can't respire oxygen and it dies.

"People will be well aware that CO is a toxic molecule but that is at high concentrations. Here we are using very low concentrations which we know the bacteria are sensitive to.

"We are looking at a molecule that can be released in a safe and controlled way to where it is needed."

The team say the next stage is to develop a drug, either in the form of a pill or cream, so that the fundamental research findings can be translated on to future clinical trials.

Professor Fairlamb added: "We think our study is an important breakthrough. It isn't the final drug yet but it is pretty close to it." "People might perceive gonorrhoea as a trivial bacterial infection, but the disease is becoming more dangerous and resistant to antibiotics."

The team worked with Professor James Moir from the University's Department of Biology. He added: "Antimicrobial resistance is a massive global problem which isn't going away. We need to use many different approaches, and the development of new drugs using bioinorganic chemistry is one crucial way we can tackle this problem, to control important bacterial pathogens before the current therapies stop working."

Blood Eosinophil Level Predicts Severity of Asthma

Published in Immunology
Tuesday, 18 October 2016 21:28

Asthma represents a significant clinical and economic burden to the US healthcare system. Along with other clinical manifestations of the disease, elevated sputum and blood eosinophil levels are observed in patients experiencing asthma exacerbations. The aim of this study was to evaluate the association between blood eosinophil levels and asthma severity defined using Expert Panel Report 3 guidelines.

Among 1,144 patients with an asthma diagnosis, 60 % were classified as having moderate-to-severe asthma. Twenty four percent of patients with moderate-to-severe asthma and 19 % of patients with mild asthma had an elevated peripheral eosinophil count (p = 0.053). Logistic regression showed that moderate-to-severe asthma was associated with 38 % increased odds of elevated eosinophil level (OR 1.38, 95 % CI: 1.02 to 1.86, p = 0.04).

Patients with moderate-severe asthma are significantly more likely to have an elevated peripheral eosinophil count than patients with mild asthma

Read More: Value of Peripheral Blood Eosinophil Markers to Predict Severity of Asthma

Status of laboratory testing for HIV

Published in Immunology
Monday, 17 October 2016 10:41

Two years have passed since the CDC finally published guidelines addressing HIV laboratory testing and officially endorsed the “new” HIV laboratory testing algorithm. Although many had become aware of the algorithm in the four years prior, and had adopted it to various degrees, this was the final word on this long-awaited guidance. The algorithm gained visibility prior to the official endorsement mainly because it had been heavily referenced in CDC publications and numerous scientific articles.

Advantages of the new algorithm

Why is the new algorithm superior to the old algorithm? First, the new algorithm emphasizes the use of an antigen/antibody (Ag/Ab) combination assay to screen for HIV infection, as the first step. The use of this more advanced technology (fourth generation) provides improved detection of acute HIV-1 infection because antigen/antibody combination assays not only detect established infection in those who have seroconverted, but can also diagnose HIV infection prior to seroconversion by detecting p24 antigen. Fourth generation assays detect acute HIV infections, on average, five to seven days earlier than the third generation, antibody-only assays.

Second, substituting the HIV-1/HIV-2 differentiation assay for the Western blot in the second step allows for correct identification of HIV-2 infection and earlier detection of HIV-1 infection, compared to the Western blot.

Third, the official addition of nucleic acid testing (NAT) is used to rule out acute HIV-1 infection, which is necessary because although HIV-1/HIV-2 differentiation assays can detect HIV infection on average a few days earlier than the Western blot, none of these can detect HIV infection prior to seroconversion.

There is ample evidence that the new algorithm has increased detection of acute HIV-1 infections, due to the use of Ag/Ab combination assays. This is important both for the patient, who can receive prompt treatment that improves health outcome, and also from a public health perspective, because it reduces disease transmission. Many laboratories now have access to a fourth generation assay, since they are offered by multiple vendors on a variety of automated platforms.

The data are not yet in as to whether the new algorithm has resulted in a significant increase in yield of HIV-2 diagnoses; this would provide critical information regarding prevalence and transmission of HIV-2 infections in the United States.

Challenges of the new algorithm

The new algorithm, however, has presented some real challenges for the laboratory. The biggest adjustment to adopting the new algorithm has been replacing the Western blot with an HIV-1/HIV-2 differentiation assay. The only assay with this capability until recently was the Multispot (Bio-Rad). However, the Multispot is no longer available and will be replaced with Bio-Rad’s Geenius. Although the Geenius is also a single use test (FDA-cleared) for confirming reactive HIV screen results and differentiating between HIV-1 and HIV-2 antibodies, it differs from the Multispot in a number of important aspects. The test uses either recombinant or synthetic peptides corresponding to four HIV-1 antigens, gp160, gp41, p31 and p24, and two corresponding to HIV-2 antigens, gp140 and gp36. There are eight possible interpretations based on the pattern observed. Performance characteristics are comparable to Multispot. Sensitivity is 100 percent for both assays, and specificity values are 99.1 percent and 96.3 percent for the Multispot and Geenius, respectively. The results can be read within 30 minutes and are interpreted using an automated cassette reader, therefore eliminating inter-observer subjectivity. The cassette system also allows for placement of a bar code label on each specimen, improving sample tracking. Additionally, because software is necessary for interpretation, the results are digitally captured, automatically recorded, and stored.

However, because the new HIV-1/HIV-2 differentiation assay requires an additional investment in the reader/software component, beyond the cost of the reagents, there is some concern that some small hospital laboratories will revert to sending out supplemental HIV testing to a reference laboratory. It should also be noted that, although adoption of the new algorithm has grown significantly, there is still substantial demand for Western blot testing. Importantly, when a third or fourth generation assay was used for screening, an indeterminate or negative Western blot should also be followed up with NAAT.

There is also much confusion regarding appropriate use of the fourth generation rapid HIV test. Although at first glance it would appear that this assay can be used in lieu of the laboratory based Ag/Ab combination assay and serve as the entry point into the algorithm, that is not the current CDC recommendation. Citing insufficient evidence for such an approach, the CDC suggests that a preliminary positive result obtained with any rapid test, including an antigen/antibody combination rapid test, must be followed up with a laboratory-based antigen/antibody combination assay.

Fifth generation testing

The horizon appears even more complicated now that the “fifth generation” HIV testing is available. This technology is currently offered only by one vendor, but it has the ability to differentiate between antigen, HIV-1 and HIV-2 antibody-positive specimens. While this simplifies the answer with regard to HIV infection status for the patient, there are no guidelines as to how to proceed with follow-up testing. For example, if the sample is positive for antigen only, then the logical follow-up would be to send out for NAT testing, as there is no reason to test with the supplemental HIV-1/HIV-2 differentiation assay that only detects antibodies. If the sample is positive for HIV-2 only, is it appropriate to follow up with the HIV-1/HIV-2 differentiation assay, because the fifth generation test is FDA-approved as a screen only and a supplemental test is needed? Fifth generation technology presents further complications to the algorithm and more complexity for the laboratory in terms of appropriate follow-up and interpretation for clinicians.

Last, one unintended consequence of the new algorithm is the effect on HIV surveillance programs. Ideally for the purpose of HIV surveillance, public health departments would like to have the final answer as to whether a patient has HIV-1, HIV-2, or acute HIV-1 infection, once the HIV testing algorithm is complete. The problem is that this is almost impossible because testing is almost always fragmented and different steps of the algorithm are performed in different laboratories. Often primary institution laboratories have the ability to perform the screening, even with a fourth generation Ag/Ab combination assay, but cannot complete the remainder of the algorithm. The sample is then sent to the reference laboratory, and that laboratory has to determine how to interpret the results without having the screen results. How to report a partial result and make it clear to the clinician that additional testing is needed and also satisfy public reporting needs is much more difficult in the context of the new algorithm, for both the primary and reference laboratory.

In summary, many technological advances have been made that importantly improve detection of HIV-2 and acute HIV-1 infections. These advances are beneficial for both the patient and society. Although most clinicians and laboratories are now familiar with and support the implementation of the algorithm, laboratories are challenged more than ever to provide appropriate test result interpretation and utilization as well as adequate public health reporting for HIV.

References

  1. "Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations". BioScience.pk Digital Library Database. Centers for Disease Control and Prevention (CDC). Published June 27, 2014.

    About the author: Patricia Slev, PhD, DABCC, is Associate Professor of Pathology (Clinical), University of Utah and Medical Director of the Serologic Hepatitis and Retrovirus Laboratory, Core Immunology Laboratory and Co-Director Microbial Immunology Laboratory,  at ARUP. Board certified by the American Board of Clinical Chemistry, Dr. Slev’s research interests are immunogenetics and pathogen interactions, particularly HIV and viral hepatitis.

Source: Medical Laboratory Observer: The status of laboratory testing for the diagnosis of HIV infection

Scientists have finally figured out how cancer spreads through the bloodstream

Published in Immunology
Wednesday, 05 October 2016 19:16
In what could be a major step forward in our understanding of how cancer moves around the body, researchers have observed the spread of cancer cells from the initial tumour to the bloodstream.

The findings suggest that secondary growths called metastases 'punch' their way through the walls of small blood vessels by targeting a molecule known as Death Receptor 6 (no, really, that's what it's called). This then sets off a self-destruct process in the blood vessels, allowing the cancer to spread.

According to the team from Goethe University Frankfurt and the Max Planck Institute in Germany, disabling Death Receptor 6 (DR6) may effectively block the spread of cancerous cells - so long as there aren't alternative ways for the cancer to access the bloodstream.
 
"This mechanism could be a promising starting point for treatments to prevent the formation of metastases," said lead researcher Stefan Offermanns.
 
Catching these secondary growths is incredibly important, because most cancer deaths are caused not by the original tumour, but by the cancer spreading.
 
To break through the walls of blood vessels, cancer cells target the body's endothelial cells, which line the interior surface of blood and lymphatic vessels. They do this via a process known as necroptosis - or 'programmed cell death' - which is prompted by cellular damage.
 
According to the researchers, this programmed death is triggered by the DR6 receptor molecule. Once the molecule is targeted, cancer cells can either travel through the gap in the vascular wall, or take advantage of weakening cells in the surrounding area.

Sabotaging flagella of bacteria to halt infections

Published in Microbiology
Wednesday, 28 September 2016 14:33
Some bacteria have the ability to ‘swim’ in a controlled fashion through the use of appendages called flagella. Researchers think that disabling these flagella is a key step towards infection control.

Motile bacteria move through the function of flagella. These appendages rotate, which propels an organism forwards. This is a little like the propellers on a boat. Some bacteria have one flagellum, others have many, and some possess none at all. Some of the bacteria regarded as human pathogens have flagella. An example of a flagellate bacterium is the ulcer-causing Helicobacter pylori, which uses multiple flagella to propel itself through the mucus lining to reach the stomach epithelium. Some flagella also serve a function in environmental detection, sensing different conditions and signalling to a bacterium to move to or away from a given niche.

Read more: Sabotaging bacteria to halt infections

Neutrophil disorders and their management

Published in Immunology
Tuesday, 27 September 2016 13:49
Neutrophil disorders are an uncommon yet important cause of morbidity and mortality in infants and children. This article is an overview of these conditions, with emphasis on clinical recognition, rational investigation, and treatment.

Neutrophil disorders
  • Disorders of neutrophil number (neutropenia)
  • Disorders of neutrophil function
Neutrophil disorders are an uncommon, yet important, cause of morbidity and mortality in infants and children and should be considered when investigating children for immunodeficiency. They are especially likely when the clinical presentation includes features such as oral ulcers and gingivitis, delayed separation of the umbilical cord, uncommon infections such as hepatic or brain abscesses, uncommon organisms such as S marcescens or Pseudomonas spp, or when the individual has features of syndromic conditions associated with neutropenia or neutrophil dysfunction. All patients with recurrent oral infections, skin abscesses, perianal and perirectal abscesses, poor wound healing, sinopulmonary infections, or deep visceral abscesses should be evaluated for defects in phagocyte function. Appropriate investigations can lead to specific diagnoses, and general and specific management measures can reduce both mortality and morbidity and permit genetic counselling and antenatal diagnosis in some cases.

Read more: Neutrophil disorders and their management

Why people with type O blood more likely to die of cholera

Published in Microbiology
Tuesday, 27 September 2016 12:00
Cholera sickens 3 million to 5 million people around the world every year, leading to 100,000 to 120,000 deaths, many of them in the Indian subcontinent, where cholera has been endemic for centuries.
 
People with blood type O often get more severely ill from cholera than people of other blood types. In people with blood type O, scientists found that cholera toxin hyperactivates a key signaling molecule in intestinal cells. High levels of that signaling molecule lead to excretion of electrolytes and water – in other words, diarrhea. Cholera is marked by severe diarrhea that can lead to dehydration, shock and even death.

The researchers confirmed their results in an intestinal cell line originally derived from a person with blood type A. The cell line was modified to produce the type O antigen instead. They found that cholera toxin induced roughly double the amount of the key signaling molecule in cells with type O antigen than in those with type A.
 
Fleckenstein isn’t sure why cholera toxin induces different responses in cells with different blood group antigens on their surfaces.
 
“The cholera toxin is known to bind weakly to the ABO antigens, so they may be acting as decoys to draw the toxin away from its true target,” Fleckenstein said. “It may be that the type O antigen just isn’t as good of a decoy as the type A antigen.”

Cross Match Procedure in Blood Bank (Manual Method)

Published in Microbiology
Sunday, 26 June 2016 07:26
When the recipient’s ABO and Rh blood groups are determined, the donor blood unit that is ABO and Rh compatible is selected, and compatibility test is carried out. The purpose of compatibility test is to prevent the transfusion of incompatible red cell units and thus avoidance of hemolytic transfusion reaction in the recipient. Compatibility test detects (i) major ABO grouping error, and (ii) most clinically significant antibodies reactive against donor red cells.

There are two types of cross-match: major cross-match (testing recipient’s serum against donor’s red cells) and minor cross-match (testing donor’s serum against recipient’s red cells). However, minor cross-match is considered as less important since antibodies in donor blood unit get diluted or neutralized in recipient’s plasma. Also, if antibody screening and identification is being carried out, minor cross-matching is not essential. Therefore, only the red cells from the donor unit are tested against the recipient’s serum and the name compatibility test has replaced the term cross-matching.

For transfusion of platelets or fresh frozen plasma, cross-matching is not required. However, fresh frozen plasma should be ABO-compatible.
A full cross-matching procedure consists of:
 
  • Immediate spin cross-match at room temperature, and
  • Indirect antiglobulin test at 37°C.

IMMEDIATE SPIN CROSS MATCH
 
The purpose of this test is to detect ABO incompatibility. Equal volumes of 2% saline suspension of red cells of donor and recipient’s serum are mixed, incubated at room temperature for 5 minutes, and centrifuged. Agglutination or hemolysis indicates incompatibility.
 
Causes of False-negative Test
 
  1. A2B donor red cells and group B recipient serum.
  2. Rapid complement fixation of potent ABO antibodies with bound complement interfering with agglutination.
 
Causes of False-positive Test
 
  1. Rouleaux formation
  2. Cold-reactive antibodies: If agglutination disappears by keeping the tube at 37°C for 10 minutes, presence of cold agglutinins is confirmed.
 
INDIRECT ANTIGLOBULIN TEST
 
Saline-suspended red cells of the donor after being incubated in patient’s serum are washed in saline and antiglobulin reagent is added. Following re-centrifugation, examine for agglutination or hemolysis. This test detects most of the clinically significant IgG antibodies.

If agglutination or hemolysis is not observed in any of the above stages, donor unit is compatible with recipient’s serum. Agglutination or hemolysis at any stage is indicative of incompatibility.
 
EMERGENCY CROSS-MATCH
 
If blood is required urgently, ABO and Rh grouping are carried out by rapid slide test and immediate spin cross match (i.e. the first stage of cross match) is performed (to exclude ABO incompatibility). If the blood unit is compatible, then after issuing it, remaining stage of the cross-match is completed. If any incompatibility is detected, the concerned physician is immediately informed about the incompatibility detected.
 
ANTIBODY SCREENING AND IDENTIFICATION
 
Screening for unexpected or irregular antibodies is done during pre-transfusion testing in recipient’s serum and in donor’s blood. In this test, serum of the recipient is tested against a set of three group O screening cells of known antigenic type. If unexpected antibodies are detected, then they are identified and blood unit that lacks the corresponding antigen is selected for compatibility test.
 
REFERENCE
Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Hematology (9th Ed). London: Churchill Livingstone, 2001.

Activated Partial Thromboplastin Time (APTT)

Published in Microbiology
Monday, 13 June 2016 17:01
APTT is a measure of coagulation factors in intrinsic pathway (F XII, F XI, high molecular weight kininogen, prekallikrein, F IX, and F VIII) and common pathway (F X, F V, prothrombin, and fibrinogen).
 
Principle
Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured.
 
Equipment
This is same as for Prothrombin Time test. (Click here to see)
 
Reagents
(1) Kaolin 5 gm/liter: This is a contact activator.
(2) Phospholipid: Various APTT reagents are available commercially, which contain phospholipids.
(3) Calcium chloride 0.025 mol/liter.
 
Specimen
Method
(1) Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a waterbath at 37°C.
(2) Deliver 0.1 ml of plasma in another test tube and add 0.1 ml of kaolin solution. Incubate at 37°C in the waterbath for 10 minutes.
(3) After exactly 10 minutes, add 0.2 ml of phospholipidcalcium chloride mixture, start the stopwatch, and note the clotting time.
 
Normal Range
30-40 seconds.
 
Causes of prolongation of APTT
(1) Hemophilia A or B.
(2) Deficiencies of other coagulation factors in intrinsic and common pathways.
(3) Presence of coagulation inhibitors
(4) Heparin therapy
(5) Disseminated intravascular coagulation
(6) Liver disease
 
Uses of APTT
(1) Screening for hereditary disorders of coagulation: Since deficiencies of F VIII (hemophilia A) and F IX (hemophilia B) are relatively common, APTT is the most important screening test for inherited coagulation disorders. APTT detects deficiencies of all coagulation factors except F VII and F XIII. PT is also performed along with APTT. Prolongation of both PT and APTT is indicative of deficiency of coagulation factors in common pathway. Normal PT with prolongation of APTT is indicative of intrinsic pathway deficiency (particularly of F VIII or IX).
(2) To monitor heparin therapy: Heparin potentiates the action of natural anticoagulant antithrombin III which is an inhibitor of thrombin and activated factors IX, X, and XI. Full dose heparin therapy needs monitoring by APTT to maintain the dose in the therapeutic range (1.5 to 2.5 times the upper reference limit of APTT).
(3) Screening for circulating inhibitors of coagulation: APTT is prolonged in the presence of specific inhibitors (which are directed against specific coagulation factors) and non-specific inhibitors (which interfere with certain coagulation reactions).
 
Mixing experiment for detection of inhibitors: Mixing studies are used to distinguish between factor deficiencies and factor inhibitors (specific coagulation factor inhibitor or non-specific inhibitor such as lupus anticoagulant). If APTT is prolonged, patient’s plasma is mixed with an equal volume of normal plasma (called as a 50:50 mix) and APTT is repeated. In coagulation factor deficiency, prolongation of APTT gets corrected by more than 50% of the difference between the clotting times of control and test plasma. In the presence of lupus anticoagulant, there is no such correction. With lupus anticoagulant, APTT remains prolonged after mixing and for 2 hours following incubation. With F VIII inhibitor (which is time- and temperature-dependent), prolong-ed APTT gets immediately corrected after mixing, but becomes prolonged after incubation.
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