2 Examination of Blood and Marrow Cells Flashcards
Red cell parameters that are directly measured
- Mean cell volume [MCV]
- Red cell number
- Hemoglobin concentration
- Red cell distribution width [RDW]
Red cell parameters that are derived
- Hematocrit
- Mean cell hemoglobin [MCH]
- Mean cell hemoglobin concentration [MCHC]
Formula for hematocrit
(Hct [L/100 L] =RBC [× 10−12/L] × MCV [fL]/10)
TRUE OR FALSE
The hemoglobin determination now is preferred to the hematocrit, because it is measured directly and is the best indicator of the oxygen-carrying capacity of the blood
TRUE
The hemoglobin determination now is preferred to the hematocrit, because it is measured directly and is the best indicator of the oxygen-carrying capacity of the blood
Measurement of Hemoglobin
To determine hemoglobin concentration in the blood, red cells are lysed and hemoglobin variants are converted to the stable compound cyanmethemoglobin for quantification by absorption at _______
540 nm
All forms of hemoglobin are readily converted to cyanmethemoglobin except_____________________, which is rarely present in significant amounts.
Sulfhemoglobin
In practice, the major interference with hemoglobin measurement is__________________, but newer instruments identify and minimize this interference.
Chylomicronemia
After the first week or two of extrauterine life, the hemoglobin falls from levels of approximately 17 g/dL to levels of approximately 12 g/dL by 2 months of age; with most of the decline occurring within first week of life, likely by__________ mechanism
Neocytolysis
Any child with a hemoglobin level below ______ should be considered anemic
11 g/dL
TRUE OR FALSE
When it comes to gender variation in hemoglobin, it has been found that adult men have significantly lower red blood cell counts, hemoglobin levels, and hematocrits than women
FALSE
When it comes to gender variation in hemoglobin, it has been found that adult women have significantly lower red blood cell counts, hemoglobin levels, and hematocrits than men
An estimate of the variance in volume within the population of red cells, which is expressed as 1 SD of red cell volume measurements divided by the MCV.
Red Cell Distribution Width
A biomarker predicting morbidity and mortality in a broad variety of clinical settings, such as angina/myocardial infarction; heart failure; trauma; pneumonia; sepsis;intensive care treatment; renal and liver disease; and in the general population.
Red Cell Distribution Width
It is at least as sensitive as the MCV in detecting iron-deficiency states
An advantage is the consistency across different analyzer types, as it is derived from two of the most accurately measured parameters: hemoglobin and red cell count.
Mean Cell Hemoglobin
Measures the concentration rather than amount of hemoglobin per red blood cell
Not used much diagnostically, and is primarily useful for quality control purposes, such as detecting sample turbidity
MCHC
Permits an estimate of marrow erythrocyte production, which is useful in evaluating the pathogenesis of anemia by distinguishing inadequate production from accelerated destruction
Reticulocyte Count
An early sign of marrow recovery from cytotoxic therapy or treatment for nutritional anemias
Increase in the immature (highest RNA content) reticulocyte fraction
May be a better predictor of depleted marrow iron stores than traditional serum iron parameters in nonmacrocytic patients, and is a more sensitive predictor of iron deficiency than hemoglobin for screening infants and adolescents for iron deficiency.
Reticulocyte-specific hemoglobin content (CHr)
Conditions wherein automated leukocyte counts may be falsely elevated
- Cryoglobulins or cryofibrinogen
- Clumped platelets or fibrin from an inadequately anticoagulated or mixed sample
- Ethylenediaminetetraacetic acid (EDTA)-induced platelet aggregation,
- Nucleated red blood cells,
- Nonlysed red cells
Conditions wherein automated leukocyte counts may be falsely decreased
EDTA-induced neutrophil aggregation
Causes of falsely decreased platelet counts
- Incomplete anticoagulation of the sample (sometimes accompanied by small clots in the specimen or fibrin strands on the stained film)
- Platelet clumping (pseudothrombocytopenia) or “satellitism” (adherence of platelets to neutrophils)
- Aggregation induced by nonpathogenic antibodies recognizing platelet adhesion molecule epitopes exposed as a result of chelation of divalent cations in the anticoagulated sample
Compared to sodium EDTA anticoagulant, is reported to more effectively inhibit platelet aggregation in these patients and provide an accurate platelet count
Magnesium EDTA anticoagulant
Causes of falsely elevated platelet count
- Severe microcytosis
- Cryoglobulins
- Leukocyte cytoplasmic fragmentation
Platelet count may be roughly estimated (FORMULA)
2000 times the number of platelets in 10 consecutive oil immersion (1000×) fields
A marker of marrow megakaryocytopoiesis and is proposed as a way of differentiating decreased production of platelets from circulatory destruction or removal as a cause of thrombocytopenia, in an analogous fashion to the use of the reticulocyte count
Reticulated platelets or immature platelet fraction
The percentage of reticulated platelets is increased in destructive thrombocytopenias, but remains within the reference range in hypoproductive states.
Reticulated platelet number is correlated with risk of death in patients with acute coronary syndrome and DIC, and with hyporesponsiveness to platelet function inhibitors or aspirin.
The most common error in blood film interpretation
Selection of a portion of the blood film for analysis that is too thick or too thin for proper morphologic evaluation
Magnification
The optimal portion of the film is examined at ______________ to systematically assess the size, shape, and morphology of the major cell lineages.
High magnification (×1000, oil immersion)
TRUE or FALSE
The normal-sized erythrocyte is about the diameter of the nucleus of a small lymphocyte.
TRUE
The normal-sized erythrocyte is about the diameter of the nucleus of a small lymphocyte.
Mean diameter of approximately 7.5 μm
A more sensitive measure of red cell volume than of the red cell diameter
MCV
Term that describes variation in erythrocyte size, and is the morphologic correlate of the RDW.
Anisocytosis
Early (“shift” or “stress”) reticulocytes (ie, those with the most residual RNA) appear in stained films as large, slightly bluish cells, referred to as
Polychromatophilic cells
Term used to describe variations in the shape of erythrocytes
Poikilocytosis
Difference between a platelet that overlie an erythrocyte vs an inclusion body or a parasite
The differentiation depends on the observation of a halo around the platelet, determination that it lies above the plane of the erythrocyte, and observation of the characteristics of a normal platelet in the “inclusion.”
The nucleus of 1% to 16% of the neutrophils from females may have an appendage that is shaped like a drumstick and is attached to one lobe by a strand of chromatin.
It represents the _______________________
Inactive X chromosome of the pair
The predominant type of lymphocyte in normal blood, is round and contains a relatively large, round, densely stained nucleus
The cytoplasm is scanty and stains pale to dark blue.
Small lymphocyte
Contain azurophilic granules and relatively abundant cytoplasm, and generally represent cytotoxic T or natural killer (NK) cells
Large granular lymphocytes
Caused by Epstein-Barr virus, cytomegalovirus, adenovirus, or other organisms, are large with indented nuclei and abundant blue cytoplasm
Nuclear chromatin condensation is variable, and nucleoli may be evident.
Reactive lymphocytes
A low nuclear-to-cytoplasmic ratio and greater degree of chromatin condensation distinguishes reactive lymphocytes from neoplastic cells
In a systemic inflammatory reaction, neutrophil granules may appear larger than normal and stain more darkly, often assuming a dark blue-black color
Toxic granulation
Huge misshapen granules are found in the neutrophils, and giant azurophilic granules are present in the lymphocytes
Chédiak-Higashi anomaly
Are sharply outlined, red-staining rods found in the cytoplasm in blast cells, and occasionally in more mature leukemic cells
Auer rods
Light blue round or oval inclusions seen in the cytoplasm of neutrophils of patients with infections, burns, and other inflammatory states
Caused by RNA of the rough-surfaced endoplasmic reticulum
Döhle bodies
Leukocytic inclusions, pale blue-stained, irregularly shaped inclusions are precipitates of nonmuscle myosin heavy chains with macrothromobytopenia
May-Hegglin anomaly
Leukocytes may be damaged, resulting in an enlarged nucleus with homogeneous, slightly reddish chromatin strands with a large blue nucleolus.
No specific association with disease other than chronic lymphocytic leukemia
Damaged (“Smudge,” “Basket”) and Apoptotic Cells
TRUE or FALSE
Aspiration is always attempted because of the superior morphology offered by examination of the aspirate smear.
TRUE
Aspiration is always attempted because of the superior morphology offered by examination of the aspirate smear.
The gold standard for diagnosis of hematologic malignancy and allows construction of a good differential diagnosis for nonmalignant disorders.
Morphology of marrow cells
Allows study of almost any surface or intracellular protein, with the added ability to detect important quantitative changes in cellular proteins and simultaneous determination of multiple proteins within the same cell
Flow cytometry
Requires that cells be viable and dissociated from tissue
Molecular assays include:
- Metaphase cytogenetics
- Fluorescence in situ hybridization (FISH)
- Reverse transcriptase polymerase chain reaction (PCR), and
- Massively parallel sequencing (also known as next-generation sequencing) of a targeted gene panel or the whole exome/genome
**For most molecular assays, fresh tissue (blood or marrow aspirate) is preferred.
** Many molecular assays may be performed on formalin-fixed, paraffin-embedded tissue as long the tissue has not been decalcified with hydrochloric acid
Fat cells begin to replace hemopoietic marrow in the extremities in the_________ year.
Fifth to seventh
By adulthood, the hemopoietic marrow is limited to the ______ skeleton and the _______ portions of the extremities
Axial skeleton
Proximal portions of the extremities
The preferred site for marrow aspiration and biopsy
Posterior iliac crest
In adults, the anterior iliac crest and rarely the sternum have been used
The sternum should be used for aspiration only.
The _____________________________ is an option for infants younger than 1 year old (particularly newborns), but the posterior iliac crest is still the preferred site.
Anteromedial surface of the tibia
Serious adverse outcomes after marrow aspiration or biopsy are rare, occurring in less than __________
Morbidity most frequently involved ______________
0.05%
Hemorrhage
Infection and reactions to anesthetic agents are other infrequent complications.
Adequate infiltration of the anesthetic at the periosteal surface is important to minimize severe pain during the procedure, but no more than __________ lidocaine should be used in an adult.
20 mL of 1% lidocaine
The most common cause of failure to obtain marrow is
Faulty positioning of the needle
The bloody fluid that is aspirated contains light-colored particles (fragments) of marrow approximately 0.5–1 mm in diameter, which are referred to as
Marrow spicules
Preparation that is the best for evaluating cellular morphology and differential counts of the marrow
Direct marrow aspirate film
Best for estimating marrow cellularity and megakaryocyte abundance, but morphology is obscured in the thicker parts of the film
Marrow aspirate particle film
Prepared from a concentrate of nucleated cells (marrow buffy coat) achieved bycentrifugation of a small volume of anticoagulated marrow, is sometimes used for detecting low-abundance cells when the marrow is hypocellular
Marrow aspirate concentrate film
Can be used for evaluating cellular morphology when the aspirate is hypocellular
Touch imprint
Flow cytometry is best performed on _______________-anticoagulated aspirate specimens, which are stable for at least 24 hours at room temperature.
EDTA- or heparin
TRUE OR FALSE
Cytogenetic samples are generally not adversely affected by overnight incubation.
TRUE
Cytogenetic samples are generally not adversely affected by overnight incubation.
TRUE OR FALSE
In cases where the marrow aspirate is dry, a duplicate biopsy specimen can be disaggregated to produce a cell suspension for morphology, flow cytometry, and cytogenetic studies.
TRUE
In cases where the marrow aspirate is dry, a duplicate biopsy specimen can be disaggregated to produce a cell suspension for morphology, flow cytometry, and cytogenetic studies.
For molecular assays,__________ is the preferred anticoagulant because heparin can interfere with some molecular assays.
EDTA
Advantage of clotted marrow aspirate
Absence of decalcification artifact
The best indicator that the needle entered the medullary cavity and marrow was successfully withdrawn
The presence of marrow particles in the aspirate
A biopsy specimen should contain at least a _______ length of marrow cavity.
0.5-cm
For detection of lymphoma or metastatic tumor, current recommendations suggest a biopsy length of ________ cm
1.6–2.0 cm
TRUE OR FALSE
The normal cellularity percentage of marrow space occupied by hematopoietic cells as opposed to fatty and nonhematopoietic tissue of iliac crest marrow decreases from a mean of 80% in early childhood to 50% by age 30 years, with further decreases after age 70 years.
TRUE
The normal cellularity percentage of marrow space occupied by hematopoietic cells as opposed to fatty and nonhematopoietic tissue of iliac crest marrow decreases from a mean of 80% in early childhood to 50% by age 30 years, with further decreases after age 70 years.
The M:E cell ratio should be between ______and _____
2:1 and 4:1
The relative proportions of cell types should be assessed only on the direct marrow film, biopsy imprint, or particle preparation, not a _________, which has been manipulated by centrifugation.
Concentrate film
A decreased M:E cell ratio can be interpreted as either ______________or _____________, depending on the overall marrow cellularity.
Myeloid hypocellularity or erythroid hyperplasia
Megakaryocyte numbers can be assessed from the direct marrow aspirate film, where at least______megakaryocytes should be present in the optimal portion of the film.
5 megakaryocytes
The most sensitive diagnostic tests for disseminated leishmaniasis.
Microscopic examination and culture of the marrow
Characterized by loss of normal marrow architecture, indistinct cellular margins, and a background of amorphous eosinophilic material
Seen in sickle cell disease and neoplastic processes involving the marrow
Marrow necrosis
Characterized by amorphous extracellular material (proteoglycans), fat atrophy, and marrow hypoplasia
Seen in patients with severe weight loss
Gelatinous transformation of the marrow
The marrow differential count may require examination of ________ nucleated cells.
300–500
They are round or oval. The nucleus is small, is coarse and clumped.
Nucleoli are not visible.
The cytoplasm is deep blue, often with a paranuclear clear zone
Plasma Cells
Has dark-blue granules, which usually completely fill the cytoplasm and may obscure the nucleus
The nucleus often is not visible but when seen is round or oval with a vesicular chromatin pattern.
Mast Cells
Large cells and may be larger than 100 μm in diameter
They superficially resemble megakaryocytes but contain multiple separated nuclei that have a moderately fine chromatin pattern with nucleoli.
The cytoplasm varies from slightly basophilic to intensely acidophilic because of the content of acidophilic granules.
Osteoclasts
Oval cells up to 30 μm in the longest diameter.
They often occur in groups. The nucleus usually is quite eccentric and may seem to be spilling out of the cell.
The chromatin pattern is uniform, and 1–3 nucleoli are present.
The cytoplasm is light blue and may contain a few red granules.
Osteoblasts
Stain used in evaluation iron stores in bone marrow
Prussian blue
Characterized by an increased number of iron granules arranged in a ring encircling at least one-third of the nucleus, reflecting accumulation of iron in mitochondria
Pathologic ring sideroblasts
Flow Cytometry
What can be determined by
Low angle or forward scatter:
90-degree angle or side scatter:
Low angle or forward scatter: cell size
90-degree angle or side scatter: cellular granularity and nuclear complexity
Some lineage-specific markers (CD3 in precursor T cells; CD79a and CD22 in B cells; myeloperoxidase in granulocyte lineage; cyclin D1 in mantle cell lymphoma) are expressed only in the cytoplasm at certain stages of development.
TRUE OR FALSE
A primary requirement for flow cytometry analysis is that cells must be viable and in single-cell suspension prior to staining
TRUE
A primary requirement for flow cytometry analysis is that cells must be viable and in single-cell suspension prior to staining
Flow Cytometry
The process, referred to as ____________ , is typically accomplished by a combination of CD45 (common leukocyte antigen) and 90-degree light scatter (side scatter).
Gating
The “blast gate,” defined by dim CD45 expression and low to intermediate side scatter
Applications of Flow Cytometry (Neoplastic)
- Characterizing blast populations in leukemia and myelodysplastic syndromes.
- Detecting abnormal phenotypes of dysplastic myeloid and monocytic populations.
- Detecting clonal B lymphocytes, clonal plasma cells, and aberrant T/ NK lymphocytes.
- Monitoring/detecting residual leukemia, lymphoma, and myeloma cells after treatment.
The gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria
Flow cytometry analysis of glycosylphosphatidylinositol (GPI)– linked proteins
FLAER (fluorescently labeled inactive variant of the bacterial protein aerolysin)
Applications of Flow Cytometry (Non-Neoplastic)
- Diagnosis of paroxysmal nocturnal hemoglobinuria
- Enumerate CD34+ progenitors when evaluating the adequacy of blood stem cell collections
- Lymphocyte subset quantitation: important in acquired and congenital immunodeficiency states as well as to follow up response to immunosuppressive therapy
