Page 54 - Williams Hematology ( PDFDrive )

30 Part I: Clinical Evaluation of the Patient Chapter 3: Examination of the Marrow 31

longitudinally. Such preparations may contain an increased number of material. Isolated DNA or RNA can be stored for long periods at −70°C,
broken cells if too much pressure is applied, but they provide a large whereas viable, intact cells can be reliably preserved only by controlled
number of particles from which marrow cellularity can be estimated rate freezing in dimethylsulfoxide (DMSO) and storage in liquid
and which are useful for estimating the amount of hemosiderin present. nitrogen.
Broken cells may be minimized using a squash technique with cover-
slips instead of glass slides, which compared favorably to push (wedge) HISTOLOGIC SECTIONS
films in achieving representative distribution of intact cells derived A variety of techniques for preparing aspirated material for histologic
from marrow particles. 22 study have been advocated. All of the techniques are designed to col-
The EDTA-anticoagulated sample may be centrifuged (1500 g for lect a sufficient number of marrow particles in a small volume so that
10 minutes) in a Wintrobe tube to concentrate the cellular elements adequate sections can be prepared. This goal can be accomplished by
of the marrow. After centrifugation, the fatty layer and plasma are discharging the marrow aspirate onto a glass slide, allowing the particles
removed, and the “buffy coat” is mixed with an equal amount of plasma. to settle for a few seconds, and then gently tilting the slide so that the
Multiple films of this preparation are made. All smears should be thor- excess blood runs off. The particles then are pushed together with an
oughly air-dried, which can take longer in a humid environment, before applicator stick, and the remaining blood is allowed to clot. The clot is
staining to avoid artifacts.
promptly fixed, typically in buffered formalin, for tissue processing and
sectioning. An alternative method using filtration of the anticoagulated
TOUCH PREPARATIONS aspirate specimen has been described. 30
After a biopsy specimen is obtained using the Jamshidi needle, the The core marrow biopsy specimen is processed for histologic
specimen should be extruded through the hub of the needle and then examination typically by fixation in neutral buffered formalin, followed
gently rolled across a glass slide (using an applicator stick to move the by decalcification and embedding in paraffin. Decalcification can be
specimen) before it is placed in fixative, taking care to avoid crushing. accomplished with acid reagents or EDTA, the latter of which is pre-
The touch preparations are allowed to dry and are stained in the same ferred as it provides better preservation of nucleic acid and protein anti-
manner as films. gens. Sections of high quality cut at 3 μm and stained with hematoxylin
and eosin are satisfactory for routine work. Refinements in fixation and
SPECIAL STUDIES embedding techniques have enabled use of many immunologic markers
in decalcified paraffin-embedded marrow biopsy specimens. Fixation
It is essential to formulate the diagnostic question before performing a in neutral-buffered formalin and embedding without decalcification in
marrow aspiration to ensure an adequate sample is obtained for all the plastic resin has the advantage of superior morphology, but is less fre-
31
special studies that may be needed to make the correct diagnosis, while quently used as the potential for immunostaining and molecular assays
avoiding aspiration of more marrow than is needed, which can lead to is more restricted.
23
dilution of the sample. A sterile anticoagulated sample containing via-
ble unfixed cells in single-cell suspension is the best substrate for nearly
all special studies. Specifically, flow cytometry is best performed on MORPHOLOGIC INTERPRETATION OF
EDTA- or heparin-anticoagulated aspirate specimens, which are stable MARROW PREPARATIONS
for at least 24 hours at room temperature. For cytogenetic or cell cul-
ture analysis, preservative-free heparin-anticoagulated marrow should OVERVIEW
be added to tissue culture medium and analyzed as soon as possible to The Wright-Giemsa–stained direct marrow aspirate film should be
maintain optimal cell viability. Cytogenetic samples are generally not examined as quickly as possible to provide a preliminary assessment of
24
adversely affected by overnight incubation. In cases where the marrow the marrow morphology and allow setup of specialized testing based on
aspirate is dry, a duplicate biopsy specimen can be disaggregated to pro- this preliminary evaluation while the sample is fresh. Final interpreta-
duce a cell suspension for morphology, flow cytometry, and cytogenetic tion of the marrow biopsy and aspirate should be integrated with results
25
studies. FISH for detection of chromosomal deletions, duplications, from the clinical history, blood film, cell counts, laboratory data, cell
and translocations can be performed in marrow biopsies when EDTA- marker studies, and molecular or cytogenetic data. No other histologic
based decalcification protocols are used. 26 specimen exists in which a state-of-the-art interpretation is dependent
For molecular analysis of fresh specimens, sample storage should on such an array of supportive data. The challenge for the hematopa-
be minimized, and storage at 4°C is preferable. EDTA is the preferred thologist and hematologist is to understand the advantages and lim-
anticoagulant because heparin can interfere with some molecular assays. itations of each diagnostic approach so that results can be reconciled
DNA is relatively stable, but RNA has a variable half-life in an intact cell and placed into perspective. Some common pitfalls in preparation and
and is degraded rapidly (on the order of seconds to minutes) in a cell interpretation of marrow aspirates and biopsies have been reviewed.
4
5
lysate by ubiquitous ribonucleases. Sample storage prior to RNA isola-
27
tion should be minimized. Collection tubes have been designed for
stabilization of RNA, but for maximal RNA recovery, samples should ADEQUACY OF THE MARROW SAMPLE
be transported to the laboratory immediately, where cell suspensions The first question in interpreting the marrow is whether the sample is
(typically buffy coat or mononuclear cell preparations) can be prepared adequate for diagnosis. At the time of the procedure, the presence of
and nucleic acids extracted under conditions that inhibit ribonucleases. marrow particles in the aspirate is the best indicator that the needle
DNA and messenger RNA can be extracted and analyzed from paraf- entered the medullary cavity and marrow was successfully withdrawn.
fin-embedded tissue sections and dried stained films, with variable Marrow particles are bony with a glistening appearance caused by fat
28
29
degree of degradation dependent on the length of sequence required. in the particles. Specimens containing cortical bone, muscle, or other
Archival storage of marrow specimens is important in light of tissue with little or no medullary bone are inadequate for marrow inter-
advances in molecular diagnosis that may necessitate validation stud- pretation. Samples with extensive crush artifact or hemorrhage are sub-
ies using samples of known origin or retrospective testing of diagnostic optimal, underscoring the importance of proper technique in obtaining

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