Answering your questions on routine VDRL on CSF, Zeta Sedimentation Ratio, hemoconcentration due to tourniquet, Kleihauer-Betke stain, cholesterol tests for health fair, and sweat test utilization.

Daniel M. Baer

Routine VDRL on CSF 

Q VDRL tests are done routinely on all cerebrospinal fluid (CSF). If this more sensitive test is done to test for syphilis antibodies that are leaked into the CSF from the spinal tap, why cannot an Rapid Plasma Reagin (RPR) screen be done on the patent's serum and thus save resources and time on a very low yield procedure? Most times patients have already been tested using the RPR test. Should routine VDRL testing be limited?

A The diagnosis of neurosyphilis is difficult because the clinical symptoms are nonspecific, the disease may occur in nearly all stages of syphilis, and we lack a highly sensitive and specific laboratory rest. The CSF-VDRL test is the only accepted laboratory test for CSF specimens. However, the sensitivity of the test varies from 20% to 70% and a nonreactive result does not rule out neurosyphilis. Therefore, the diagnosis of neurosyphilis is based on a combination of clinical and laboratory data. When the laboratory and ordering physicians agree to use a set of guidelines to determine when a CSF specimen is tested, the result is a more cost-effective approach to screening patients for neurosyphilis. In general, a CSF specimen should be tested by the VDRL method if the patient has (a) clinical symptoms consistent with syphilis or a history of syphilis, (b) a reactive serum rapid plasma reagin or fluorescent treponemal antibody absorption test, and (c) an abnormal spinal fluid (elevated protein and leukocyte c ount). Because neurosyphilis is infrequently diagnosed in the United States, efficient use of the CSF-VDRL test requires that the patient be seropositive. The use of these guidelines can significantly decrease the number of inappropriate CSF-VDRL tests performed. 1

Reference:

(1.) Albright RE, Christianson RH, Emlet JL, et al. Issues in cerebrospinal fluid management: CSF Venereal Disease Research Laboratory Testing. Am J Clin Pathol. 1991, 95:397-401.

-David Sewell, PhD, ABMM 

Director of Microbiology

Veterans Affairs Medical Center

Portland, OR

Zeta Sedimentation Ratio 

Q A few years ago, I was told that the FDA had removed their approval for the Zeta Sedimentation Ratio test and the Zetafuge instrument (Coulter Electronics, Miami, FL). Is this true? Is it still legal to run the test and use the Zetafuge?

A The Zeta Sedimentation Ratio (ZSR) is a micro-sedimentation rate test described in 1972 by Bull and Brailsford [1]. The test uses microhematocrit capillary tubes (without additives) that are spun on a special low-speed centrifuge, the Zetafuge. This instrument was made by Coulter, but has not been available for many years.

The method had some popularity, but apparently not enough for Coulter to continue manufacturing the Zetafuge, because it was a micro method, and more importantly, because it was not affected by anemia, and did not require a correction for low hematocrits.

Since the method predates FDA oversight of test methods, the ZSR is not subject to FDA regulation. The test and method are not listed in the CLIA test categorization listings; therefore, it is classified as a high complexity test.

Is it legal to run this or any other obsolete test? The answer is yes, if the following conditions are met:

*It can only be performed by personnel qualified to run high complexity tests;

*It must be run according to the published method;

*The reliability of the test must be validated;

*The lab's clinical consultant must be able to demonstrate the clinical relevance of the test;

*The manufacturer's instructions for operation and maintenance of the system must be followed;

*the capillary tubes must be un-coated, have no additives, and be the same size as described in the original method;

*The instrument must be in good working condition; and

*As there is no quality control material manufactured for this test system, some other means of demonstrating day-to-day reliability must be used.

Finally, it may not be worth the trouble to satisfy the conditions for validating this old test. The lab should consider newer alternatives, and assess whether the ZSR still meets the lab's clinical needs.

Reference

(1.) Bull BS and Bralisford JD. The zeta sedimentation ratio. Blood 1972;40:550-559

--Daniel M. Baer, MD

Professor Emeritus

Oregon Health Sciences University

Portland, OR

Hemoconcentration due to tourniquet 

Q Do you have any information on how hemoconcentration affects a CBG? I would like to know how leaving the tourniquet on for an extended period of time would affect the white blood count (WBC) and platelet counts. I know the red cell counts and hemoglobin will be elevated.

A Prolonged constriction results in an increase in the intervascular blood pressure forcing an efflux of small molecules and fluid from the capillaries into the tissue. This results in a decrease in blood volume and, hence, an increase in the concentration of anything left behind, i.e., large protein-bound nondiffusible analytes and cells. [1-6] Consider excessive tourniquet time to be similar conceptually to pulling a net full of fish Out of the sea. Cells and large molecules become disproportionate in concentration downstream from the tourniquet, hence the term hemoconcentration. I am not aware of any study that quantifies the effect on CBCs, but it is considered significant enough for the National Committee for Clinical Laboratory Standard to set the limit on tourniquet time to be one minute and that the tourniquet should be released as soon as the vein is accessed. [6]

In addition to this efflux of molecules, biochemical changes take place in the trapped blood that can significantly alter chemical analytes in your specimens. When blood is unable to circulate it invites glycolysis, which lowers the pH of the pooled blood. This drives potassium out of the cell (which has 25 times the amount of potassium as plasma) and increases its concentration in the backlogged blood, blood that will soon be collected and sent for testing. [5,7,8] Ionic calcium and magnesium are also elevated because the low pH causes them to disassociate from albumin. So, you can see that excessive tourniquet time affects more than just the CBC.

References

(1.) Becan-McBride K, Garza D. Phlebotomy Handbook. Stamford, CT: Appleton & Lange; 1999.

(2.) Dale J, Preanalytical variables in laboratory testing. Lab Med 1998;29(9):540-545.

(3.) Becan-McBride K. Preanalytical phase an important requisite of laboratory testing. Adv Med Lab Prof. 1998;10(201:12-17.

(4.) Narayanan, S. The preanalytic phase: an important component of laboratory medicine. Am J Clin Pathol 2000;l13:429-452.

(5.) Ernst D, Ernst C. Phlebotomy for Nurses and Nursing Personnel. Ramsey, IN: HealthStar Press; 2001.

(6.) National Committee for Clinical Laboratory Standards. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture. Approved Standard, H3-A4, Wayne, Pa, 1998.

(7.) Statland, B. Tourniquet application and elevated potassium. Tips on Specimen Collection. Montvale, NJ: Medical Economics; 1997. P5

(8.) Statland BE, Bokelund H and Winkel R Factors contributing to intra-individual variation of serum constituents: Effects of posture and tourniquet application on variation of serum constituents in healthy subjects. Clin Chem 1974;20:1513-1519.

--Dennis Ernst, MT(ASCP)

Director

Center for Phlebotomy Education

Ramsey, IL

Kleihauer-Betke stain

Q In our laboratory we offer the Kleihauer-Betke stain for the determination of fetal hemoglobin in red blood cells. But the procedure is difficult and time-consuming, and we are looking for an alternative. Can you help us?

A The Kleihauer-Betke (more correctly named the Kleihauer-Braun-Betke to include the third co-author) stain has been used to differentiate normal adult hemoglobin-containing red cells from red cells containing fetal hemoglobin. [1]

If there is a fetal to maternal bleed during pregnancy, which could trigger the production of maternal antibodies directed against the fetal cells resulting in hemolytic disease of the newborn, the early discovery can lead to the prevention of immunization by giving immune gamma globulin to the mother.

In the past, the transfusion service tested the mother's blood for the presence of Rh D-positive red cells from the fetus. Now a positive result requires confirmation with a Kleihauer-Betke stain or flow cytometric quantitation of fetal hemoglobin. This is because the required dose of Rh immune globulin depends on the volume of the fetal-maternal bleed, larger bleeds requiring larger doses of immune globulin.

Also there can be increased levels of fetal hemoglobin-containing red cells in thalassemia, sickle cell, and other anemias and in such cases the determination of fetal hemoglobin-containing cells is a necessary diagnostic test.

As you know, the test can be somewhat daunting especially if it is requested infrequently. A blood film is reacted with a weakly acidic solution which preferentially elutes adult hemoglobin from the red cells. The film is stained with hematoxylin and eosin and carefully examined under the microscope for the presence of so-called ghost red cells that have lost their content of hemoglobin. Especially low concentrations of such cells require counting larger numbers of red cells in order to ensure the precision necessary to determine the proper dose of immune globulin. Commercial kits (e.g., Sigma Diagnostics) are marketed and could help solve your problem.

Recently, flow cytometric procedures have been developed which are quite accurate and precise, but may not be available in many laboratories.

The NCCLS has recently published a comprehensive guideline for fetal red cell detection and you will find a wealth of information on all of these methods in that document.

John A. Koepke, MD

Professor Emeritus of Pathology

Duke University Medical Center

Durham, NC

References

(1.) Kleihauer, E, Braun, H & Betke, K. Demonstration of fetal hemoglobin in erythrocytes of a blood smear. Kiln Wochenschr 35: 637-638, 1957

(2.) Fetal Red Cell Detection; Proposed Guideline H52-P. NCCLS, Wayne, PA 2601.

Cholesterol tests for health fair

Q My hospital is getting ready to do a health fair. Do you know of any cholesterol tests that would be appropriate to use at this health fair?

A There are several factors to consider in selecting the appropriate technology for use at a health fair:

* Throughput and turnaround time: Large numbers of people attend successful health fairs. The test system should be capable of producing test results while the attendees are at the fair, without a long wait to provide a specimen. Point of care, unitized single test systems tend to require the technologist to do one test at a time, completing one test before the next one can be run.

* Cost Point of care, unitized single test systems are expensive as compared to other simple strip or slide technologies.

* Simplicity: The test should be simple to run, preferably on a whole blood specimen, and without much manipulation of instruments or reagent additions.

* Regulatory issues: Remember that health fairs are covered by some regulatory requirements. For instance, in some states, a laboratory site license must be obtained for a health fair. It is best to consider a health fair testing site as just another lab location, and to follow all of the CLIA rules regarding procedure documentation, operator training and authorization to perform testing, records of those tested, and quality control.

Having said all this, where should you look for appropriate testing systems? Tests appropriate for a point of care or physician's office are ideal for a health fair. Generally, these will be amongst tests classified for CLIA in the Waived Complexity Category. A current list of these, sorted by analyte, can be seen at the CDC website, www.cdc.govlphppo/dls/testcat.him. It is sometimes possible to borrow an instrument for use at a health fair from a manufacturer, vendor, or from a health organization such as the American Heart Association.

Daniel M. Baer, MD

Professor Emeritus

Oregon Health Sciences University

Portland, OR

Sweat test utilization

Q Respiratory allergies and asthma are common here in southwest Missouri, and some technologists feel that sweat testing is over utilized. This spring we are performing 4 to 6 sweat tests per week. Are there published indications for sweat testing that we can send to our pediatricians? Any suggestions would be appreciated on how to encourage our physicians to show some restraint and order sweat testing only for chronic and not seasonal respiratory problems.

A There are a variety of clinical presentations which suggest cystic fibrosis (CF) as a possible diagnosis. These are listed in Appendix A of NCCLS's guideline, Indications for Sweat Testing of Sweat Testing. Because the majority of patients with cystic fibrosis (CF) present with acute or chronic respiratory symptoms, it is highly appropriate that a sweat test be ordered on a patient demonstrating symptoms of asthma or respiratory allergies. The most common clinical presentations are recurrent or chronic respiratory infections, failure to thrive, and chronic diarrhea, the three of which comprise the majority of illnesses in infants and young children who are diagnosed with cystic fibrosis. Unfortunately these presentations are also frequent findings in normal infants so that a large number of sweat tests must be performed in order to rule out cystic fibrosis and to properly identify the few among the many who indeed have the disease.

Most sweat tests performed will be negative for CF; for example, at our hospital, over 90% of the sweat tests are negative. Our sweat testing workload increases during the late winter and early spring corresponding to an increase in respiratory infections. In conclusion, physicians should be encouraged, rather than discouraged, to order a sweat test in patients with symptoms suggestive of CF.

Dr. Vicky A. LeGrys

Professor

School of Medicine Division of Clinical

Laboratory Science

University of North Carolina at Chapel Hill 

Reference

(1.) Indications for Sweat Testing of Sweat Testing: Sample Collection and Quantitative Analysis; Approved Guideline C34-A2, Second Edition. Wayne, PA. NCCLS 2060.

Daniel M. Beer is professor emeritus of laboratory medicine at Oregon Health Sciences University in Portland, OR, and a member of MLO's editorial advisory board.