2 Examination of Blood and Marrow Cells

Question 1

Red cell parameters that are directly measured

Answer 1

• Mean cell volume [MCV]
• Red cell number
• Hemoglobin concentration
• Red cell distribution width [RDW]

Question 2

Red cell parameters that are derived

Answer 2

• Hematocrit
• Mean cell hemoglobin [MCH]
• Mean cell hemoglobin concentration [MCHC]

Question 3

Formula for hematocrit

Answer 3

(Hct [L/100 L] =RBC [× 10−12/L] × MCV [fL]/10)

Question 4

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

Answer 4

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

Question 5

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 _______

Answer 5

540 nm

Question 6

All forms of hemoglobin are readily converted to cyanmethemoglobin except_____________________, which is rarely present in significant amounts.

Answer 6

Sulfhemoglobin

Question 7

In practice, the major interference with hemoglobin measurement is__________________, but newer instruments identify and minimize this interference.

Answer 7

Chylomicronemia

Question 8

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

Answer 8

Neocytolysis

Question 9

Any child with a hemoglobin level below ______ should be considered anemic

Answer 9

11 g/dL

Question 10

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

Answer 10

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

Question 11

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.

Answer 11

Red Cell Distribution Width

Question 12

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.

Answer 12

Red Cell Distribution Width

Question 13

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.

Answer 13

Mean Cell Hemoglobin

Question 14

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

Answer 14

MCHC

Question 15

Permits an estimate of marrow erythrocyte production, which is useful in evaluating the pathogenesis of anemia by distinguishing inadequate production from accelerated destruction

Answer 15

Reticulocyte Count

Question 16

An early sign of marrow recovery from cytotoxic therapy or treatment for nutritional anemias

Answer 16

Increase in the immature (highest RNA content) reticulocyte fraction

Question 17

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.

Answer 17

Reticulocyte-specific hemoglobin content (CHr)

Question 18

Conditions wherein automated leukocyte counts may be falsely elevated

Answer 18

• 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

Question 19

Conditions wherein automated leukocyte counts may be falsely decreased

Answer 19

EDTA-induced neutrophil aggregation

Question 20

Causes of falsely decreased platelet counts

Answer 20

• 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

Question 21

Compared to sodium EDTA anticoagulant, is reported to more effectively inhibit platelet aggregation in these patients and provide an accurate platelet count

Answer 21

Magnesium EDTA anticoagulant

Question 22

Causes of falsely elevated platelet count

Answer 22

• Severe microcytosis
• Cryoglobulins
• Leukocyte cytoplasmic fragmentation

Question 23

Platelet count may be roughly estimated (FORMULA)

Answer 23

2000 times the number of platelets in 10 consecutive oil immersion (1000×) fields

Question 24

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

Answer 24

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.

Question 25

The most common error in blood film interpretation

Answer 25

Selection of a portion of the blood film for analysis that is too thick or too thin for proper morphologic evaluation

Question 26

# Magnification The optimal portion of the film is examined at ______________ to systematically assess the size, shape, and morphology of the major cell lineages.

Answer 26

High magnification (×1000, oil immersion)

Question 27

# TRUE or FALSE The normal-sized erythrocyte is about the diameter of the nucleus of a small lymphocyte.

Answer 27

TRUE The normal-sized erythrocyte is about the **diameter of the nucleus of a small lymphocyte.** ## Footnote Mean diameter of approximately 7.5 μm

Question 28

A more sensitive measure of red cell volume than of the red cell diameter

Answer 28

MCV

Question 29

Term that describes **variation in erythrocyte size**, and is the morphologic correlate of the RDW.

Answer 29

Anisocytosis

Question 30

**Early (“shift” or “stress”) reticulocytes** (ie, those with the most residual RNA) appear in stained films as large, slightly bluish cells, referred to as

Answer 30

Polychromatophilic cells

Question 31

Term used to describe **variations in the shape** of erythrocytes

Answer 31

Poikilocytosis

Question 32

Difference between a platelet that overlie an erythrocyte vs an inclusion body or a parasite

Answer 32

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.”

Question 33

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 _______________________

Answer 33

Inactive X chromosome of the pair

Question 34

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.

Answer 34

Small lymphocyte

Question 35

Contain azurophilic granules and relatively abundant cytoplasm, and generally represent cytotoxic T or natural killer (NK) cells

Answer 35

Large granular lymphocytes

Question 36

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.

Answer 36

Reactive lymphocytes ## Footnote A low nuclear-to-cytoplasmic ratio and greater degree of chromatin condensation distinguishes reactive lymphocytes from neoplastic cells

Question 37

In a systemic inflammatory reaction, neutrophil granules may appear larger than normal and stain more darkly, often assuming a dark blue-black color

Answer 37

Toxic granulation

Question 38

Huge misshapen granules are found in the neutrophils, and giant azurophilic granules are present in the lymphocytes

Answer 38

Chédiak-Higashi anomaly

Question 39

Are sharply outlined, red-staining rods found in the cytoplasm in blast cells, and occasionally in more mature leukemic cells

Answer 39

Auer rods

Question 40

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

Answer 40

Döhle bodies

Question 41

Leukocytic inclusions, pale blue-stained, irregularly shaped inclusions are precipitates of **nonmuscle myosin heavy chains** with macrothromobytopenia

Answer 41

May-Hegglin anomaly

Question 42

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**

Answer 42

Damaged (“Smudge,” “Basket”) and Apoptotic Cells

Question 43

# TRUE or FALSE Aspiration is always attempted because of the superior morphology offered by examination of the aspirate smear.

Answer 43

TRUE Aspiration is always attempted because of the **superior morphology** offered by examination of the aspirate smear.

Question 44

The **gold standard for diagnosis of hematologic malignancy** and allows construction of a good differential diagnosis for nonmalignant disorders.

Answer 44

Morphology of marrow cells

Question 45

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

Answer 45

Flow cytometry ## Footnote Requires that cells be viable and dissociated from tissue

Question 46

Molecular assays include:

Answer 46

* 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 ## Footnote **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

Question 47

Fat cells begin to replace hemopoietic marrow in the extremities in the_________ year.

Answer 47

Fifth to seventh

Question 48

By adulthood, the hemopoietic marrow is limited to the ______ skeleton and the _______ portions of the extremities

Answer 48

Axial skeleton Proximal portions of the extremities

Question 49

The preferred site for marrow aspiration and biopsy

Answer 49

Posterior iliac crest ## Footnote In adults, the anterior iliac crest and rarely the sternum have been used **The sternum should be used for aspiration only.**

Question 50

The _____________________________ is an option for infants younger than 1 year old (particularly newborns), but the **posterior iliac crest** is still the preferred site.

Answer 50

Anteromedial surface of the tibia

Question 51

Serious adverse outcomes after marrow aspiration or biopsy are rare, occurring in less than __________ Morbidity most frequently involved ______________

Answer 51

0.05% Hemorrhage ## Footnote **Infection** and **reactions to anesthetic agents** are other infrequent complications.

Question 52

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.

Answer 52

20 mL of 1% lidocaine

Question 53

The most common cause of failure to obtain marrow is

Answer 53

Faulty positioning of the needle

Question 54

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

Answer 54

Marrow spicules

Question 55

Preparation that is the best for evaluating **cellular morphology and differential counts** of the marrow

Answer 55

Direct marrow aspirate film

Question 56

Best for estimating **marrow cellularity and megakaryocyte abundance**, but morphology is obscured in the thicker parts of the film

Answer 56

Marrow aspirate particle film

Question 57

Prepared from a concentrate of nucleated cells (**marrow buffy coat**) achieved by*centrifugation of a small volume of anticoagulated marrow*, is sometimes used for detecting low-abundance cells when the marrow is hypocellular

Answer 57

Marrow aspirate concentrate film

Question 58

Can be used for evaluating cellular morphology when the aspirate is hypocellular

Answer 58

Touch imprint

Question 59

**Flow cytometry** is best performed on _______________-anticoagulated aspirate specimens, which are stable for at least 24 hours at room temperature.

Answer 59

EDTA- or heparin

Question 60

# TRUE OR FALSE Cytogenetic samples are generally not adversely affected by overnight incubation.

Answer 60

TRUE Cytogenetic samples are generally not adversely affected by overnight incubation.

Question 61

# 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.

Answer 61

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.

Question 62

For molecular assays,__________ is the preferred anticoagulant because heparin can interfere with some molecular assays.

Answer 62

EDTA

Question 63

Advantage of **clotted marrow aspirate**

Answer 63

Absence of decalcification artifact

Question 64

The best indicator that the needle entered the medullary cavity and marrow was successfully withdrawn

Answer 64

The presence of marrow particles in the aspirate

Question 65

A biopsy specimen should contain at least a _______ length of marrow cavity.

Answer 65

0.5-cm

Question 66

For detection of lymphoma or metastatic tumor, current recommendations suggest a biopsy length of ________ cm

Answer 66

1.6–2.0 cm

Question 67

# 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.

Answer 67

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.

Question 68

The M:E cell ratio should be between ______and _____

Answer 68

2:1 and 4:1

Question 69

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.

Answer 69

Concentrate film

Question 70

A decreased M:E cell ratio can be interpreted as either ______________or _____________, depending on the overall marrow cellularity.

Answer 70

Myeloid hypocellularity or erythroid hyperplasia

Question 71

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.

Answer 71

5 megakaryocytes

Question 72

The most sensitive diagnostic tests for disseminated leishmaniasis.

Answer 72

Microscopic examination and culture of the marrow

Question 73

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

Answer 73

Marrow necrosis

Question 74

Characterized by amorphous extracellular material (proteoglycans), fat atrophy, and marrow hypoplasia Seen in patients with **severe weight loss**

Answer 74

Gelatinous transformation of the marrow

Question 75

The marrow differential count may require examination of ________ nucleated cells.

Answer 75

300–500

Question 76

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

Answer 76

Plasma Cells

Question 77

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.

Answer 77

Mast Cells

Question 78

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.

Answer 78

Osteoclasts

Question 79

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.

Answer 79

Osteoblasts

Question 80

Stain used in evaluation **iron stores** in bone marrow

Answer 80

Prussian blue

Question 81

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

Answer 81

Pathologic ring sideroblasts

Question 82

# Flow Cytometry What can be determined by Low angle or forward scatter: 90-degree angle or side scatter:

Answer 82

Low angle or forward scatter: cell size 90-degree angle or side scatter: cellular granularity and nuclear complexity ## Footnote 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.

Question 83

# TRUE OR FALSE A primary requirement for flow cytometry analysis is that cells must be viable and in single-cell suspension prior to staining

Answer 83

TRUE A primary requirement for flow cytometry analysis is that cells must be viable and in single-cell suspension prior to staining

Question 84

# 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**).

Answer 84

Gating ## Footnote The “**blast gate**,” defined by **dim CD45** expression and **low to intermediate side scatter**

Question 85

Applications of Flow Cytometry (Neoplastic)

Answer 85

1. Characterizing blast populations in leukemia and myelodysplastic syndromes. 2. Detecting abnormal phenotypes of dysplastic myeloid and monocytic populations. 3. Detecting clonal B lymphocytes, clonal plasma cells, and aberrant T/ NK lymphocytes. 4. Monitoring/detecting residual leukemia, lymphoma, and myeloma cells after treatment.

Question 86

The gold standard for diagnosis of **paroxysmal nocturnal hemoglobinuria**

Answer 86

Flow cytometry analysis of glycosylphosphatidylinositol (GPI)– linked proteins FLAER (fluorescently labeled inactive variant of the bacterial protein aerolysin)

Question 87

Applications of Flow Cytometry (Non-Neoplastic)

Answer 87

* 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