Friday, December 12, 2008

Warm and Cold Ischemic Time

There are in fact 2 warm ischemic times. We usu mean the donor warm ischemic time (ie Ischemia during organ retrieval, from the time of cross clamping (or of asystole in non-heart-beating donors), until cold perfusion is commenced.

Cold Ischemic Time: Ischemia when the organ is cooled with a cold perfusion solution after ORGAN PROCUREMENT surgery, and ends after the tissue reaches physiological temperature during implantation procedures.


The following article addresses the issue of warm ischemic time:

Warm ischemia in transplantation : Search for a consensus definition
Auteur(s) / Author(s)
HALAZUN K. J. (1) ; AL-MUKHTAR A. (1) ; ALDOURI A. (1) ; WILLIS S. (1) ; AHMAD N. (1) ;
Affiliation(s) du ou des auteurs / Author(s) Affiliation(s)
(1) Department of Organ Transplantation, St. James University Hospital, Leeds, ROYAUME-UNI
Résumé / Abstract
"Warm ischemia" is a term used to describe ischemia of cells and tissues under normothermic conditions. In the transplant setting, this term is used to describe two physiologically distinct periods of ischaemia: (1) Ischemia during implantation, from removal of the organ from ice until reperfusion, and (2) Ischemia during organ retrieval, from the time of cross clamping (or of asystole in non-heart-beating donors), until cold perfusion is commenced. These periods of warm ischemia differ in their nature and the magnitude of their pathophysiologic consequences. In much transplant literature, however, the term "warm ischaemia" is used to describe both of these periods indiscriminately. This paper attempts to produce a definition to distinguish between the two periods of warm ischemia. Methods. We conducted a questionnaire survey of all UK transplant surgeons. The definitions proposed in the survey were: (a) warm ischemia and re-warm ischemia; (b) first warm ischemia and second warm ischemia; (c) in-situ warm ischemia and ex-vivo warm ischemia; (d) warm ischemia in donor and warm ischemia in recipient; (e) no opinion or other opinion. Results. There was a 64% response rate among 134 consultants with no consensus definition being reached. The majority of consultants (31.4%) preferred the terms "warm ischemia in donor", and "warm ischemia in recipient" to distinguish the two periods. Conclusions. This paper highlights the need to adopt uniform terms to avoid confusion between different types of warm ischemia in transplantation.
Revue / Journal Title
Transplantation proceedings ISSN 0041-1345 CODEN TRPPA8
Source / Source
2007, vol. 39, no5, pp. 1329-1331 [3 page(s) (article)] (15 ref.)

Thursday, December 11, 2008

Interpreting Hepatitis C results

Hepatitis C detection

What are the diagnostic tests for hepatitis C virus and how are they used to diagnose hepatitis C virus infection?

A number of diagnostic tests are currently available for hepatitis C virus. They are categorized below according to the function of the specific tests.

What about screening tests?

Screening tests are done to determine the presence of antibodies to hepatitis C virus in the blood. The enzyme immunosorbent assay (EIA) is the conventional, initial screening test to diagnose hepatitis C infection. The EIA measures specific antibodies to small pieces of the hepatitis C virus proteins (antigens). This test, therefore, is referred to as the anti-hepatitis C virus antibody test. Patients who have elevated liver enzymes (ALT/AST) and/or any of the risk factors for hepatitis C virus can be diagnosed to have hepatitis C virus with a greater than 95% certainty when the EIA is positive.

On the other hand, certain patients whose immune systems are impaired (suppressed) may not have detectable anti-hepatitis C virus antibodies even if they are actually infected with hepatitis C virus. Such immunosuppressed patients include those who are on renal dialysis, suffer from cancer and are receiving chemotherapy (drugs to kill cancer cells), or have active HIV infection. These patients cannot produce enough anti-hepatitis C virus antibodies necessary to generate a positive EIA test.

When there is a low likelihood (risk) of hepatitis C infection, individuals who test positive for hepatitis C by EIA should undergo confirmatory testing using a specialized assay that likewise tests for antibodies against the hepatitis C virus proteins. This assay is called the Recombinant Immunoblot Assay (RIBA).

Both the EIA and RIBA tests, however, do not distinguish among acute, chronic, and resolved hepatitis C virus infections because the anti-hepatitis C virus antibodies are in the blood in all three of these situations. Although EIA and RIBA are tests that measure antibodies against hepatitis C virus, these antibodies do not confer protection to the patient against acquiring hepatitis C virus. Rather, they only indicate exposure of the patient to the virus.

What are molecular tests for hepatitis C virus?

As previously described, hepatitis C virus is an RNA virus. The code of the genetic material, hepatitis C virus RNA, is unique to this virus. Several types of tests (assays) are available to measure the hepatitis C virus RNA in a person's blood. These tests are referred to as molecular tests because they examine the virus at the molecular level. The two most common systems for measuring hepatitis C virus RNA are the reverse transcription polymerase chain reaction (RT-PCR) assay and the branched chain DNA (bDNA) assay. Recently, a third type of assay, called transcription-mediated amplification (TMA), has been released.

First of all, it is important to put in perspective the relative amount of virus in an individual infected with hepatitis C virus as compared to some other types of chronic viral infection. The average number of virus particles/milliliter of blood in an individual with chronic hepatitis C virus is hundreds of thousands to several million. In contrast, someone with active hepatitis B infection has several hundred million to billions of copies (virus particles) per milliliter of blood. The relatively low concentration of the hepatitis C virus in the blood is one of the reasons it took so long for scientists to characterize the hepatitis C virus.

RT-PCR is a very powerful tool for detecting relatively low amounts of genetic material (RNA or DNA). The basis of this technique is the amplification of a target piece of nucleic acid several million times so that this target becomes measurable. Due to the extreme sensitivity of this technique, however, the slightest contamination can lead to a false positive result. On the other hand, RNA is relatively unstable (degrades easily), so that blood and tissue samples need to be handled with special precautions. If not, this instability would lead to a false negative result, that is, a negative result in someone who has hepatitis C virus.

In the early 1990's, each laboratory had its own in-house technique for the RT-PCR assay and the reliability of these assays was quite variable. Even as of now, the FDA has not approved any of the RT-PCR assays. However, most laboratories currently use one of the several available diagnostic kits that are automated and designed to reduce the likelihood of contamination. There are two types of RT-PCR, qualitative and quantitative. Qualitative hepatitis C virus RT-PCR provides the greatest sensitivity, meaning that it can measure as few as 100 copies (viral particles) of hepatitis C virus/ml of serum. As the name implies, however, qualitative RT-PCR provides only a positive (presence of hepatitis C virus) or negative (absence of hepatitis C virus) result.

By contrast, quantitative RT-PCR measures the amount of virus. These tests, however, are only accurate within a certain range of viremia (circulating virus in the blood). This means that quantitative assays are not as sensitive as qualitative assays and can only detect as few as 500 copies/ml. Moreover, these assays are less accurate at extremely high viral levels (over 2 million copies/ml). In the past year, there has been an attempt to standardize these various quantitative assays so that the levels of virus that are measured by different assays can be compared. As a matter of fact, results of quantitative RT-PCR are now reported in standard International Units/ml (IU/ml).

Branched chain DNA (bDNA) is the other quantitative technique. It is based on the amplification of the detection signal rather than of the nucleic acid itself. As a result, this test is less prone to contamination and is more accurate when measuring higher levels of the virus as compared to RT-PCR. However, the bDNA assay is not as sensitive as the RT-PCR and is unable to measure levels of virus below 200,000 copies/ml.

Finally, transcription mediated amplification (TMA) is a qualitative technique that is distinct from PCR. This test can measure as few as 2 to 5 copies of virus/ml.

What is the role of the qualitative molecular tests?

Qualitative RT-PCR is a useful test in determining whether or not a patient has circulating virus in the blood (viremia). Hence, it can be used to confirm that a reactive (positive) anti-hepatitis C virus result reflects active hepatitis C virus infection. However, confirmatory testing is usually not necessary in someone who tested reactive (positive) for anti-hepatitis C virus and also has risk factors and abnormal liver tests. In this situation, the RT-PCR most certainly would be positive. On the other hand, an individual who is anti-hepatitis C virus reactive and has risk factors but normal liver tests should undergo confirmatory testing with RT-PCR. This person may have cleared the viral infection some time ago, leaving the anti-hepatitis C virus as a marker of past exposure.

Qualitative hepatitis C virus RNA testing should also be done in individuals who may have been recently exposed to hepatitis C. Hepatitis C virus RNA is more sensitive (that is, will detect more cases) than the conventional anti-hepatitis C virus (EIA) testing in this setting. The reason for this greater sensitivity is that it may take a person as many as six to eight weeks after exposure to hepatitis C virus to develop the antibodies, whereas hepatitis C virus RNA becomes detectable five to ten days after exposure. Finally, qualitative hepatitis C virus RNA testing may be helpful to assess the patient's virologic response at certain time points during antiviral therapy (see treatment of hepatitis C virus below).