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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 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."

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

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

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.
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 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
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.”

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.

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.
PT assesses coagulation factors in extrinsic pathway (F VII) and common pathway (F X, F V, prothrombin, and fibrinogen).
 
Principle
Tissue thromboplastin and calcium are added to plasma and clotting time is determined. The test determines the overall efficiency of extrinsic and common pathways.
 
Equipment
(1) Water bath at 37°C
(2) Test tubes (75 × 12 mm)
(3) Stopwatch
 
Reagents
(1) Thromboplastin reagent: This contains tissue factor and phospholipids and is available commercially.
(2) Calcium chloride 0.025 mol/liter.
 
Specimen
Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.
 
Method
(1) Deliver 0.1 ml of plasma in a glass test tube kept in water bath at 37°C.
(2) Add 0.1 ml of thromboplastin reagent and mix.
(3) After 1 minute, add 0.1 ml of calcium chloride solution. Immediately start the stopwatch and record the time required for clot formation.
 
Normal Range
11-16 seconds.
 
Causes of prolongation of PT
(1) Treatment with oral anticoagulants
(2) Liver disease
(3) Vitamin K deficiency
(4) Disseminated intravascular coagulation
(5) Inherited deficiency of factors in extrinsic and common pathways.
 
Uses of PT
(1) To monitor patients who are on oral anticoagulant therapy: PT is the standard test for monitoring treatment with oral anticoagulants. Oral anticoagulants inhibit carboxylation of vitamin K-dependent factors (Factors II, VII, IX, and X) and make these factors inactive.
In patients receiving oral anticoagulants, PT should be reported as a ratio of PT of patient to PT of control; it should not be reported as percentage. Various types of thromboplastin reagents obtained from different sources (like ox brain, rabbit brain, or rabbit lung) are available for PT test. These differ in their responsiveness to deficiency of vit. K-dependent factors. Technique of PT is also different in different laboratories. For standardization and to obtain comparable results, it is recommended to report PT (in persons on oral anticoagulants) in the form of an International Normalized Ratio (INR).

INR =  PT of Patient ISI
          PT of Control
 
International Sensitivity Index (ISI) of a particular tissue thromboplastin is derived (by its manufacturer) by comparing it with a reference thromboplastin of known ISI.
INR should be maintained in the therapeutic range for the particular indication (INR of 2.0-3.0 for prophylaxis and treatment of deep venous thrombosis; INR of 2.5-3.5 for mechanical heart valves). Therapeutic range provides adequate anticoagulation for prevention of thrombosis and also checks excess dosage, which will cause bleeding.
(2) To assess liver function: Liver is the site of synthesis of various coagulation factors, including vitamin Kdependent proteins. Therefore PT is a sensitive test for assessment of liver function.
(3) Detection of vitamin K deficiency: PT measures three of the four vitamin K-dependent factors (i.e. II, VII, and X).
(4) To screen for hereditary deficiency of coagulation factors VII, X, V, prothrombin, and fibrinogen.

BLEEDING TIME

The bleeding time test is dependent on appropriate functioning of platelets blood vessels and platelets and evaluates earliest hemostasis (platelets components and vascular).

In this test, incision (a surgical cut made in skin) or a superficial skin puncture is made and the time is measured for bleeding to stop.

There are three methods most commonly used to measure bleeding time:

  1. Duke’s method
  2. Ivy’s method
  3. Template method

In Duke’s method, ear lobe is puncture, and the time is measured for bleeding to stop.  This method is not recommended and cannot be standardized because it can cause a large local hematoma. In Ivy’s method, on the volar surface of the forearm, three punctures are made with a lancet (cutting depth 2-2.5 mm) under normal pulse pressure (between 30-40 mm Hg). A disadvantage of Ivy’s method is closure of puncture wound before stoppage of bleeding. In Template method, a special surgical blade is uses to make a larger cut of about 1 mm deep and 5 mm long. Although Template method is better than other methods, it may produce large scar and even form a keloid (irregular fibrous tissue formed at the site of a scar) in predisposed individuals. Ivy’s method for the measurement of bleeding time is described below.

Ivy’s Method

Principle: On the volar surface of forearm, three normal punctures are made with the help of a lancet under normal pulse pressure (between 30-40 mm Hg).  The average time is measured for bleeding to stop from the puncture sites.

Equipment

  1. Disposable sterile lancets
  2. Sphygmomanometer
  3. Filter paper
  4. Stopwatch

Method

  1. Blood pressure of the patient is measured with the help of sphygmomanometer. The blood pressure of the patient should be normal before going to the further process.
  2. The volar surface of the forearm is cleansed with ethanol 70% and allowed to dry.
  3. With the help of a lancet, in quick succession, three punctures are made about 5 cm apart. Note that scars and superficial veins should be avoided.
  4. Start the stopwatch as soon as puncture made on the volar surface of the forearm.
  5. With the help of the filter paper, blood oozing from the puncture wound is gently absorbed with intervals of 15 seconds.
  6. The timer is stopped when blood no more mark the filter paper.
  7. Time measured for bleeding to stop from all the three puncture wound is recorded. The average time is calculated and reported as the bleeding time.

Reference Ranges

  • Normal range: 2 -7 minutes.
  • The greater numbers of individuals have bleeding time less than 4 minutes. The bleeding time should be reported in minutes or nearest half minute. If the bleeding continues more than twenty minutes, the test is stopped and the bleeding time should be reported as >20 minutes (more than 20 minutes).

Cause of extend of duration of bleeding time

  1. Disorders of blood vessels
  2. Thrombocytopenia: This term is uses when the platelet count is less than its normal value. The bleeding time test should not be performed if the platelet count is less than 1,00,000/ml. It may be difficult to control the bleeding if the platelet count is very low.
  3. Von Willebrand disease
  4. Disorder of platelet function
  5. Afibrinogenemia

CLOTTING TIME

In this test, required time is measured for the blood to clot in a glass test tube, kept at 37° C. Extend of duration of clotting time occurs only if severe deficiency of a clotting factor exists and is normal in moderate or mild deficiency.

Thrombin time assesses the final step of coagulation i.e. conversion of fibrinogen to fibrin by thrombin.
 
Principle
Thrombin is added to patient’s plasma and time required for clot formation is noted.
 
Equipment
(1) Water bath at 37°C
(2) Test tubes (75 × 12 mm)
(3) Stopwatch
 
Reagent
Thrombin solution.
 
Specimen
Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.
 
Method
Take 0.1 ml of buffered saline in a test tube and add 0.1 ml of plasma. Note clotting time after addition of 0.1 ml of thrombin solution.
 
Normal Range
± 3 seconds of control.
 
Causes of Prolongation of TT
(1) Disorders of fibrinogen: Prolongation of TT occurs in afibrinogenemia (virtual absence of fibrinogen), hypofibrinogenemia (fibrinogen less than 100 mgs/dl), and dysfibrinogenemia (dysfunctional fibrinogen).
(2) Heparin therapy: Heparin inhibits action of thrombin.
(3) Presence of fibrin degradation products (FDPs): These interfere with fibrin monomer polymeri-zation. TT is repeated using a mixture of normal plasma and patient’s plasma. If TT remains prolonged, then FDPs are present (provided patient is not receiving heparin).
This is a newly introduced screening test for platelet function that assesses both platelet adhesion and aggregation. This method uses an instrument called as PFA-100 in which anticoagulated whole blood is passed at a high shear rate through small membranes that have been coated with either collagen and epinephrine or collagen and ADP. Platelets adhere to each membrane and gradually occlude an aperture at the centre of the membrane. The time required for complete occlusion of the aperture is called as closure time. Normal closure time is 1-3 minutes. The PFA-100 test is performed initially with the collagen/epinephrine membrane; if closure time is normal, there is no significant platelet function defect. If closure time with collagen/epinephrine is prolonged, test with collagen/ADP is carried out; if normal, aspirin-induced platelet dysfunction is the probable cause; if prolonged, other platelet function defect (acquired or inherited) is likely. This test is more sensitive than bleeding time to assess primary hemostasis, sensitive for detection of von Willebrand disease and easy to perform. However, in the presence of thrombocytopenia and anemia, closure time is prolonged. Also, in the presence of a strong clinical suspicion of a platelet function defect and normal PFA-100 result, further testing is still necessary.
 
REFERENCES
1. Evatt BL, Gibbs WN, Lewis SM, McArthur JR. Fundamental Diagnostic Hematology: The Bleeding and Clotting Disorders (2nd ed), 1992. US Dept. of health and Human Services, Atlanta, Georgia and World Health Organization, Geneva, Switzerland.
2. Shrikhande AV, Warhadpande MS, Kawthalkar SM. A laboratory manual of coagulation (1994). Dept. of Pathology. Govt. Medical College, Nagpur.
3. Wallach J. Interpretation of Diagnostic Tests (7th ed). Philadelphia: Lippincott Williams and Wilkins, 2000.
4.  David J. Kuter, MD, DPhil. Laboratory Tests for Blood Disorders. The Merck™ Manuals, (2014).
Hemostais and Bleeding Disorders

Hemostasis or haemostasis (from the Ancient Greek: αἱμόστασις haimóstasis "styptic (drug)") is a process which causes bleeding to stop, meaning to keep blood within a damaged blood vessel (the opposite of hemostasis is hemorrhage). It is the first stage of wound healing. This involves blood changing from a liquid to a gel. Intact blood vessels are central to moderating blood's tendency to clot. The endothelial cells of intact vessels prevent blood clotting with a heparin-like molecule and thrombomodulin and prevent platelet aggregation with nitric oxide and prostacyclin. When endothelial injury occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor which initiate the maintenance of hemostasis after injury. Hemostasis has three major steps: 1) vasoconstriction, 2) temporary blockage of a break by a platelet plug, and 3) blood coagulation, or formation of a fibrin clot. These processes seal the hole until tissues are repaired.
     Bleeding disorders are the result of a generalized defect in hemostasis due to abnormalities of blood vessels, platelets, or coagulation factors.
     Initial tests, which should be performed in a suspected bleeding disorder, are complete blood count including blood smear, platelet count, bleeding time, clotting time, prothrombin time, and activated partial thromboplastin time. Depending on the results of these screening tests, one or more specific tests are carried out for definitive diagnosis (e.g. platelet function studies, assays of
coagulation factors, and test for fibrin degradation products). Abnormalities of blood vessels are usually not detectable by laboratory tests for hemostasis, and their diagnosis requires correlation of clinical and other investigations.
 
SCRENNING TESTS FOR HEMOSTASIS
(1) Complete Blood Count including Blood Smear
A complete blood count and a blood smear can provide information in the form of:
• Presence of cytopenia (anemia, leukopenia, thrombocytopenia)
• Red cell abnormalities (especially fragmented red cells which may indicate disseminated intravascular
coagulation)
• White cell abnormalities (like abnormal cells in leukemias)
• Abnormalities of platelets: thrombocytopenia (normally there is 1 platelets per 500-1000 red cells), giant platelets (seen in myeloproliferative disorders and Bernard-Soulier syndrome), and isolated discrete platelets without clumping in finger-prick smear (seen in uremia, Glanzmann’s thrombasthenia).
(2) Platelet Count
(3) Bleeding Time (BT)
(4) Clotting Time (CT)
(5) Prothrombin Time (PT)
(6) Activated Partial Thromboplastin Time (APTT)
(7) Thrombin Time (TT)
(8) Platelet Function Analyzer-100
 
LICENSE: This article uses material from the Wikipedia article "HEMOSTASIS", which is released under the Creative Commons Attribution-Share-Alike License 3.0.
Description: Immunoassays is the first practical volume designed to help any biologist develop an immunoassay of any common format for any suitable analyte. The basic principles are described and the choices of assay types and formats listed. Methods for raising and for characterizing antibodies, for preparing radioactive, enzymatic, fluorescent and other labels, are described. Solid-phase reagents, standards and immunogens are explained, with procedures for their preparation. But most components can be obtained commercially and comprehensive lists of suppliers are given. Approaches to combining the reagents and optimizing the required assay are described, including how to eliminate common interferences, and thorough validation procedures are given. Manual and computer methods for the calculation of concentrations are explained. Since only assays that are within control give valid results, a graded and comprehensive approach to internal and external quality assurance methods is detailed, including how to prepare control samples.

Variations of leukocyte count occur in many infectious, hematologic, inflammatory, and neoplastic diseases.

Therefore, laboratory evaluation of almost all patients begins with the examination of the patient’s blood for total leukocyte count and examination of the peripheral blood smear for the differential leukocyte count as well as blood cells picture. Usually, some clinical interpretations may be made from the total leukocyte count and differential leukocyte count.

From total leukocyte count and differential leukocyte count, the absolute count of each leukocyte type can be calculated. The absolute leukocyte count provides a more accurate picture than the differential leukocyte count. (For example, a chronic lymphocytic leukemia patient has a total leukocyte count of 100 x 103 cells µI and a differential leukocyte count of 7 percent neutrophils and 90 percent lymphocytes. By looking at the differential leukocyte count of 7 percent neutrophils alone one get the impression of very low neutrophils in this patient But if the absolute count of neutrophils is calculated, one will be surprised to see a normal neutrophil number in this patient.

Absolute neutrophil count = Total leukocyte count x neutrophil percent
=100 x 103 x 7/100 = 7 x 103 = 7000 / µl

At times of acute bacterial infection enormous numbers of neutrophils are required. Therefore, large number of neutrophils is released from bone marrow to cope with this requirement. Consequently, the number as well as the percentage of neutrophils in the blood increases several fold. Hence, an increase in total leukocyte count with increase in percentage of neutrophil is taken as an important indication of acute bacterial infection.

Leukocytosis

The terms leukocytosis and leukopenia indicate increase or decrease in the total number of leukocytes, respectively. Increase in numbers of neutrophils, eosinophils, lymphocytes, and monocytes are known as neutrophilia, eosinophilia, lymphocytosis, and monocytosis respectively.

i. Neutrophilia is the increase in peripheral blood absolute neutrophil count, above the upper limit of normal of 7.5 x 109/L (in adults). Bacterial infection is one of the common causes of neutrophilia. Neutrophilia is not a feature of viral infections (However, the development of neutrophila late in the course of a viral infection may indicate the emergence of secondary bacterial infection). There is a storage pool of mature neutrophils in the bone marrow (Such storage pool does not occur for other leukocytes).

In response to stress, such as infections the neutrophils from storage pool are released into circulation, resulting in a rise in total leukocyte count and neutrophil percentage. Moreover, there is increased neutrophil production in the bone marrow. Increased neutrophil production during bacterial infection is usually associated with the entry of less mature neutrophils from bone marrow into blood. This is indicated by the appearance of neutrophils with lesser number of nuclear segmentation in the peripheral blood picture. Band cells may also be seen in the peripheral blood smear.

This is referred to as a ‘shift to the left’. The leukemoid reaction is a reactive and excessive leukocytosis, wherein the peripheral blood smear shows the presence of immature cells (e.g. myeloblasts, promyelocytes, and myelocytes). Leukemoid picture occurs in severe or chronic infections and hemolysis. Another most common change that occurs in neutrophils during infection is the presence of toxic basophilic inclusions in the cytoplasm.

Eosinophilia is an increase in peripheral blood absolute eosinophil count beyond 0.4 x 109/L (in adults). Eosinophilia is usually associated with allergic conditions such as asthma and hay fever and parasitic infections. Eosinophilia may also occur in reactions to drugs.

Leukopenia

Decrease in total leukocyte count is known as leukopenia. Reduction in the number of neutrophils is the most frequent cause of leukopenia.

i. Neutropenia is the decrease in peripheral blood absolute neutrophil count below the lower limit of normal of 2 x 109/L (in adults). Neutropenic patients are more vulnerable to infection.

ii. Lymphopenia in adults is the decrease in peripheral blood absolute lymphocyte count below the lower limit of normal of 1.5 x 109/L. Lymphopenia is common in the leukopenic prodromal phase of many viral infections. A selective depletion of helper T lymphocytes (CD4+) with or without absolute lymphopenia occurs in acquired immunodeficiency disease (AIDS).

Cholinesterase testing has two main uses:
  • It can be used to detect and diagnose organophosphate pesticide exposure and/or poisoning. It may also be used to monitor those who may be at increased risk of exposure to organophosphate compounds, such as those who work in agricultural and chemical industries, and to monitor those who are being treated for exposure. Typically, tests for red blood cell acetylcholinesterase (AChE) and serum pseudocholinesterase (PChE) are used for this purpose.
  • It can be used several days prior to a surgical procedure to determine if someone with a history of or family history of post-operative paralysis following the use of succinylcholine, a common muscle relaxant used for anesthesia, is at risk of having this reaction. In these cases, the test for pseudocholinesterase is usually used. A second test, referred to as a dibucaine inhibition test, may be done to help determine the extent to which the activity of the enzyme is decreased.
 
When is it ordered?
People who work with organophosphate compounds in the farming or chemical industries may be routinely monitored to assess any adverse exposure, once baseline levels have been established. Cholinesterase testing can also be used to assess any acute exposure to these compounds, which can cause neuromuscular damage. Toxicity can follow a rapid absorption of the compound in the lungs, skin, or gastrointestinal tract. The symptoms of toxicity are varied depending on the compound, quantity, and the site of exposure. Early symptoms may include:
  • Headache, dizziness
  • Nausea
  • Excessive tearing in the eyes, sweating and/or salivation
 
As the effects of the poisoning worsen, some additional symptoms may appear:
  • Vomiting, diarrhea
  • Dark or blurred vision due to constricted pupils
  • Muscle weakness, twitching, lack of coordination
  • Slowed breathing leading to respiratory failure, requiring lifesaving ventilation
  • In serious cases, seizures, coma, and death
 
Pre-operative screening for pseudocholinesterase activity is advised if a person or a close relative has experienced prolonged paralysis and apnea after the use of succinylcholine for anesthesia during an operation.
 
What does the test result mean?
In monitoring for occupational pesticide exposure
Following exposure to organophosphate compounds, AChE and PChE activity can fall to about 80% of normal before any symptoms occur and drop to 40% of normal before the symptoms become severe. Those who are regularly exposed to these compounds may be monitored for toxic exposure by establishing a baseline activity level and then testing on a regular basis to watch for a significant reduction on activity of acetylcholinesterase or pseudocholinesterase.
 
In testing for acute pesticide exposure/poisoning
Significantly decreased cholinesterase activity levels usually indicate excessive absorption of organophosphate compounds. Pseudocholinesterase and RBC acetylcholinesterase activity are usually decreased within a few minutes to hours after exposure. Pseudocholinesterase activity may regenerate in a few days to weeks, while acetylcholinesterase activity will remain low for as long as one to three months. Both plasma and RBC activities are immediately affected by pesticide exposure but, upon removal from exposure, AChE and PChE regenerate at different rates since AChE is produced in blood cells, which have a lifespan of 120 days, whereas PChE is produced in the liver, with a half-life of about two weeks.
 
In testing for succinylcholine sensitivity
About 3% of people have low activity levels of pseudocholinesterase due to an inherited deficiency and will have prolonged effects from the muscle relaxant succinylcholine. Total quantitative pseudocholinesterase levels will be evaluated prior to surgery for patients with a history or family history of prolonged apnea after use of this drug. Low activity levels of pseudocholinesterase levels indicate that these people may be at increased risk of experiencing prolonged effects of the muscle relaxant. A second test, the dibucaine inhibition test, may also be performed to help characterize the degree of a person's sensitivity to the drug. The lower the result from a dibucaine inhibition test, the greater the risk of drug sensitivity.
Reduced cholinesterase levels can also be caused by chronic liver disease and malnutrition. Total cholinesterase activity can be lowered in a number of other conditions, including pregnancy, renal disease, shock, and some cancers.
 
Is there anything else I should know?
If someone unexpectedly has prolonged apnea after surgery, testing for succinylcholine sensitivity may be performed; however, the sample should be obtained after 24 to 48 hours have elapsed following the surgery to avoid interference by any drugs given during the surgery that could affect the results. Drugs called cholinesterase inhibitors may have a moderate benefit in those with early diagnosed Alzheimer's disease.
An acetylcholine receptor (AChR) antibody test is used to help diagnose myasthenia gravis (MG) and to distinguish it from other conditions that may cause similar symptoms, such as chronic muscle fatigue and weakness.
 
AchR antibodies hinder the action of acetylcholine, a chemical (neurotransmitter) that transmits messages between nerve cells. The antibodies do this in three major ways:
  • "Binding" antibodies attach to the acetylcholine receptors on nerve cells and may initiate an inflammatory reaction that destroys them.
  • "Blocking" antibodies may sit on the receptors, preventing acetylcholine from binding.
  • "Modulating" antibodies may cross-link the receptors, causing them to be taken up into the muscle cell and removed from the neuromuscular junction.
Three different types of tests are available to determine which of these may be the problem in a particular individual. However, the test that measures "binding" antibodies is most commonly used because it is generally rare for the other two tests to be positive without the "binding" test being positive as well. These other tests may be used when a doctor strongly suspects myasthenia gravis and the "binding" test is negative.
 
One or more of the AChR antibody tests may be ordered as part of a panel of tests that may also include a striated muscle antibody test to help establish a diagnosis. Depending upon results, an anti-MuSK (muscle-specific kinase) antibody test may also be ordered. The AChR antibody test may be ordered initially as a baseline test and then as indicated to evaluate MG disease activity and/or response to therapy.
 
People with MG often have an enlarged thymus gland and may have thymomas (typically benign tumors of the thymus). Located under the breastbone, the thymus is an active part of the immune system during childhood but normally becomes less active after puberty. If a thymoma is detected, such as during a chest computed tomography (CT) scan done for a different reason, then an AChR antibody test may sometimes be used to determine whether the person has developed these antibodies.
 
When is it ordered?
The AChR antibody test may be ordered when a person has symptoms that suggest MG, such as:
  • Drooping eyelid
  • Double vision
  • Decreased eye movement control
  • Difficulty swallowing, chewing, with choking, drooling and gagging
  • Slurred speech
  • Weak neck muscles
  • Trouble holding up head
  • Difficulty breathing
  • Difficulty walking and an altered gait
  • Specific muscle weakness but normal feelings/sensations
  • Muscle weakness that worsens with sustained effort and improves with rest
When a person has been diagnosed with MG, an AChR antibody test may be ordered occasionally to evaluate MG disease activity and/or response to therapy.
 
An AChR antibody test may sometimes be ordered when a thymoma is detected.
 
What does the test result mean?
AChR antibodies are not normally present in the blood. They are autoantibodies and their presence indicates an autoimmune response.
 
If a person has AChR antibodies and symptoms of MG, then it is likely that the person has this condition.
 
AChR antibodies may be seen with some thymomas, in people who are being treated with drugs such as penicillamine, with some small cell lung cancers, with autoimmune liver disease, and with Lambert-Eaton myasthenic syndrome (a condition associated with interference with the release of acetylcholine from the nerve ending).
 
A negative test result does not rule out MG. Up to 50% of those with ocular MG (affecting only eye-related muscles) and about 10-15% of those with generalized MG will be negative for AChR antibodies.
 
In general, the greater the quantity of AChR antibody, the more likely a person is to have significant symptoms, but the test results cannot be used to evaluate the severity of symptoms in a specific person.

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