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.

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

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.

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.

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).