Answering your questions

Nov. 1, 2009

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?

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 37^0C 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


  1. Dalal BI, Brigden ML. Interpretation of the
    peripheral blood film. In: Pierre RV, ed. Clinics in Laboratory
  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

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?

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
the World Health Organization's (WHO) infection control guidelines for
transmissible spongiform encephalopathies, which lists the following
decontamination methods for instruments and materials:


  • 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 (1N NaOH) and heat in a
    gravity-displacement autoclave at 121^0C 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 121^0C 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 (121^0C) or porous load (134^0C) 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
  • 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 134^0C 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

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

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

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 kDa1,2 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


  1. Rifai N, Warnick G. Lipids, Lipoproteins,
    Apolipoproteins, and Other Cardiovascular Risk Factors. In: Tietz
    Textbook of Clinical Chemistry and Molecular Diagnostics, Fourth
    . 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.

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.