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Answering your questions.

White blood cell clumping?

Q What is the reason for the clumping of white blood cells? Is it significant to certain disorders? What is the best laboratory practice for resolving this problem?

A Leukocyte agglutination, or clumping of white blood cells, is an uncommon but well-recognized phenomenon that mostly involves neutrophils. (1) Cases with lymphocyte, basophil, and lymphoma cell involvement, however, have also been reported.

Neutrophil aggregate is most commonly induced by EDTA, but it has also been reported rarely after the use of lithium heparin and buffered sodium citrate. The incidence rate ranges from 1:7,500 to 1:32,500, according to various published data. There is no specific disease process associated with leukocyte agglutination. It may be related to acute and chronic inflammation, intravenous gamma-globulin administration, alcoholic liver diseases, autoimmune diseases, or conditions associated with cold agglutinin generation.

Lymphoagglutination is also caused by EDTA but is a far less frequent phenomenon than neutrophil agglutination. It has been reported in patients with lymphoproliferative disorders, non-Hodgkin lymphoma, and chronic lymphocytic leukemia.

Although the underlying mechanism for leukoagglutination remains unclear, it has been suggested that it is related to an EDTA-dependent antibody, since mixing plasma with anti-IgM antibody or dithiothreitol (DTT) can abolish or decrease the size of the neutrophil aggregate. In another study, high integrin expression on the neutrophil membrane has been implicated in neutrophil agglutination. Additionally, low temperature alone or combined with EDTA can cause neutrophil agglutination.

Leukoagglutination can cause spurious results on hematology analyzers, which results in a pseudoneutropenia or generates flags, most commonly as immature granulocytes or band cells. Such false-positives can potentially be clinically significant since they can be mistakenly identified as neutropenia and may lead to unnecessary investigative procedures or therapies.

To overcome the leukocyte agglutination, several methods can be used. (2) First, specimens can be warmed up to 37[degrees]C to rule out cold agglutinin, although it is much less common than EDTA-induced leukocyte agglutination. Second, collecting another specimen in sodium citrate usually will correct the problem. Others suggest that immediate dilution of the blood samples after a finger prick can prevent the agglutination. Finally, aminoglycosides (kanamycin) has been reported to be effective in resolving or preventing leukoagglutination. (3)

Leukoagglutination is mainly caused by EDTA, can cause pseudo-leukopenia, and, potentially, can be clinically significant. It is important to check peripheral blood smears to rule out an inaccurate WBC count caused by leukoagglutination.

--Gang Xu, MD, PhD

--Guang Fan, MD, PhD

Oregon Health and Science University

Portland, OR

References

(1.) Dalai Bl, Brigden ML. Interpretation of the peripheral blood film. In: Pierre RV, ed. Clinics in Laboratory Medicine. 2002:22(1);81-100.

(2.) Zandecki M, Genevieve F, Gerard J, et al. Spurious counts and spurious results on haematology analysers: a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes. Int J Lab Hematol. 2007:29(1);21-41.

(3.) Hoffmann JJ. EDTA-induced pseudo-neutropenia resolved with kanamycin. Clin Lab Haematol. 2001:23(3);193-196.

Prion-disease specimen precautions

Q We handle a lot of cerebrospinal fluid (CSF) specimens; and recently, a few technologists have expressed concern about handling suspected Creutzfeldt-Jakob disease (CJD) specimens. We do take some extra precautions when the specimens are being sent out for CJD testing; however, we know that we have handled some specimens that were later tested and found to be positive for the CJD protein. Our extra precautions include soaking most items that come in contact with the sample in bleach; however, I have read that bleach has no affect on the protein. Is this true? Can CSF become airborne via the use of a cytofuge? Will CSF remain present in our chemistry instrumentation forever? What precautions are recommended so we can protect ourselves? What is the likelihood that we could contract CJD from CSF?

A CSF has a very low infectivity rate for prion diseases, and simple skin contact cannot transmit the disease. The fluid or tissue would have to come in contact with other brain material, spinal fluid, or dura mater.

The most authoritative review of infection-control procedures related to prion disease is found at www.who.int/csr/resources/publications/bse/WHO_CDS_CSR_APH_2000_3/en in the World Health Organization's (WHO) infection control guidelines for transmissible spongiform encephalopathies, which lists the following decontamination methods for instruments and materials:

Incineration:

* Use for all disposable instruments, materials, and wastes.

* Preferred method for all instruments exposed to high-infectivity tissues.

Autoclave/chemical methods for heat-resistant instruments:

* Immerse in sodium hydroxide (IN NaOH) and heat in a gravity-displacement autoclave at 121[degrees]C for 30 minutes; clean; rinse in water; and subject to routine sterilization.

* Immerse in NaOH or sodium hypochlorite (undiluted, 20,000 ppm available chlorine) for one hour; transfer instruments to water; heat in a gravity-displacement autoclave at 121[degrees]C for one hour; clean; and subject to routine sterilization.

* Immerse in NaOH or sodium hypochlorite for one hour; remove and rinse in water; then transfer to open pan and heat in a gravity displacement (121[degrees]C) or porous load (134[degrees]C) autoclave for one hour; clean; and subject to routine sterilization.

* Immerse in NaOH and boil for 10 minutes at atmospheric pressure; clean; rinse in water; and subject to routine sterilization.

* Immerse in sodium hypochlorite (preferred) or NaOH (alternative) at ambient temperature for one hour; clean; rinse in water; and subject to routine sterilization.

* Autoclave at 134[degrees]C for 18 minutes. Cytocentrifugation can release aerosols; although, if the infectivity of CSF is low, this may not be a realistic concern. Some cytocentrifuges have forced airflow designed to prevent aerosols. Otherwise, the centrifuge can be operated in a biological safety hood.

Although the infectivity of prions persists for a long time, the infectivity potential is low; and in a chemistry instrument where there is constant cleaning and dilution, the risk is negligible.

The CDC also has extensive information about precautions for handling specimens suspected for CJD and other similar prion diseases at www.cdc.gov/ncidod/dvrd/cjd/qa_cjd_infection_control.htm.

The CDC's and WHO's recommendations for a hospital or clinical laboratory that has exposure to prion-disease specimens are no different from the usual good laboratory practices using universal precautions for biological specimens.

--Daniel M. Baer, MD (Deceased)

Proper Lp(a) measurement

Q How do I properly report Lp(a) mass in units of nmol/L?

A The accurate analytical measurement and reporting of Lp(a) poses a significant analytical challenge to the laboratory. Lp(a) is the only lipoparticle known to contain a single but highly heterogeneous apo(a) protein. Approximately 30 different isoforms of apo(a) have been documented to exist among individuals. In fact, a single individual may often possess a heterogeneous mix of apo(a) isoforms. The heterogeneity of apo(a) proteins may be largely attributed to the number of Kringle 4 (K4) type 2 domain repeats present within apo(a), and these K4 domain repeats vary from three to 40 copies per apo(a) protein isoform. This is equivalent to a range in apo(a) molecular weight of 187 kDa to 662 kDa (1), (2) which significantly impacts the molecular weight of Lp(a).

Conversion of Lp(a) mass from units of weight (mg/dL) to molar units (nmol/L) cannot be directly calculated for individuals when the laboratory is relying on a Lp(a) standard curve expressed in units of weight. This is due to the fact that the exact molecular weight of each individual's apo(a) isoform(s)--and by implication Lp(a)--is unknown.

In 2003, the World Health Organization/International Federation of Clinical Chemistry and Laboratory Medicine working group provided the first reference material with the aim of harmonizing methods of Lp(a) immunochemical mass measurement. This Lp(a) reference material, termed SRM 2B, was generated from a pool of donors and was characterized as containing apo(a) isoforms with 13, 19, 24, 32, and 38 Kringle 4 type 2 repeats. (5) SRM 2B was rigorously evaluated and assigned a molar concentration of 107 nmol/L upon reconstitution. (5), (6) Lp(a) manufacturers should elect to use the SRM 2B reference material to set an accurate molar concentration for their calibrators. Thus, the laboratory must ask its Lp(a) assay manufacturer for the theoretical concentrations of the calibrators in molar units and then generate a separate calibration curve if it wishes to report Lp(a) in nmol/L.

When 22 commonly used Lp(a) mass assays were calibrated with SRM 2B, the CV among assays was 2.8%. Despite achieving uniform calibration to SRM 2B, these 22 assays demonstrated 6% to 31% CV when used to measure the Lp(a) concentration of 30 designated fresh-frozen serum samples of variable apo(a) isoform content (three to 40 Kringle repeats). (5) This persistence in the variability of Lp(a) mass measurement among methods was attributed to the common use of anti-apo(a) polyclonal detection antibodies that are sensitive to K4 type 2 repeat number. (1-6) Serum samples containing Lp(a) isoforms containing a greater number of K4 type 2 repeats than a manufacturer's calibrator will be more reactive, thus producing an overestimation of the Lp(a) mass concentration and vice versa.

In order to produce the most accurate Lp(a) mass measurement that is independent of apo(a) size, thus allowing an accurate report of Lp(a) mass in units of molar concentration (i.e., particle number), it is recommended that Lp(a) mass assays eventually update their methodology by using a monoclonal detection antibody directed against a constant Lp(a) domain such as apo(a) Kringle 4 type 8 or 9. (3-6)

In summary, avoid converting an Lp(a) value in mg/dL directly to nmol/L for individual patients, as this will yield an inaccurate value. Instead, request the assigned calibrator values expressed in nmol/L from the assay manufacturer in order to generate a calibration curve that will allow the accurate calculation and reporting of Lp(a) in nmol/L. Despite using calibrators with SRM2B assigned molar concentrations, some degree of inaccuracy still persists in measuring Lp(a) mass because of the common use of polyclonal antibodies recognizing K4 type 2 domains.

--Susan Wurster, PhD

Clinical Chemistry Fellow

Department of Laboratory Medicine and Pathology

Mayo Clinic

Rochester, MN

References

(1.) Rifai N, Warnick G. Lipids, Lipoproteins, Apolipoproteins, and Other Cardiovascular Risk Factors. In: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, Fourth Edition. St. Louis, MO: Elsevier Saunders, 2006, Chapter 26.

(2.) Marcovina S, Koschinsky M. Lipoprotein (a): Structure, Measurement, and Clinical Significance. In: Handbook of Lipoprotein Testing. Washington DC: AACC Press, 1997, Chapter 15.

(3.) Albers J, Marcovina S. Lipoprotein (a) quantification: Comparison of Methods and Strategies for Standardization. Curr Opinion in Lipidology. 1994; 5:417-421.

(4.) Kostner G, Steinmetz A. Standardization of Lp(a) Measurements. Clin Genet. 1997; 52:393-397.

(5.) Marcovina S, et al. Use of a Reference Material Proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to Evaluate Analytical Methods for the Determination of Plasma Lipoprotein(a). 2000;46(12): 1956-1967.

(6.) Dati F, et al. First WHO/ IFCC International Reference Reagent for Lipoprotein (a) for Immunoassay-Lp(a) SRM 2B. Clin Chem Lab Med. 2004; 42(6):670-676.

MLO's "Tips from the Clinical Experts" provides practical, up-to-date solutions to readers' technical and clinical issues from a panel of experts in various fields. Readers may send questions to Brad S. Karon, MD, PhD, by e-mail at tips@mlo-online.com.

Edited by Brad S. Karon, MD, Phd

Brad S. Karon, MD, PhD, is assistant professor of laboratory medicine and pathology, and director of the Hospital Clinical Laboratories, point-of-care testing, and phlebotomy services at Mayo Clinic in Rochester, MN.

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Title Annotation:using agglutination tests to treat leukocyte disorders; treating creutzfeldt-Jakob disease
Author:Karon, Brad S.
Publication:Medical Laboratory Observer
Geographic Code:1USA
Date:Nov 1, 2009
Words:1949
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