Veterinary Clinical Pathology Study Material

2006-09-01T04:55:00.000-07:00

Question of the day

The blue-staining aggregates or "reticulum" seen in reticulocytes does not occur as such in living cells but results from what?

Yesterday's question:

Diff-Quik is superior to Wright or Wright-Giemsa stains in staining what in canine blood cells?

Answer: Distemper inclusions

Technorati Tags: reticulum, reticulocytes, cells, Diff-Quik, Wright, Wright-Giemsa, Giemsa, Distemper, canine, blood cells

2006-08-31T04:58:00.000-07:00

Question of the day

Diff-Quik is superior to Wright or Wright-Giemsa stains in staining what in canine blood cells?

Yesterday's question:

What is one limitation of Diff-Quik stain?

Answer: Does not stain basophil or mast cell granules well

Technorati Tags: Diff-Quik, stain, Wright, Wright-Giemsa, Giemsa, canine, blood

Technorati Tags: Diff-Quik, stain, Wright, Wright-Giemsa, Giemsa, canine, blood

2006-08-30T05:06:00.000-07:00

Question of the day

What is one limitation of Diff-Quik stain?

Yesterday's question:

Blood films are routinely stained with a ___________-type stain.

Answer: Romanowsky

Technorati Tags: Diff-Quik, blood, Romanowsky, stain

2006-08-29T04:35:00.000-07:00

Question of the day

Blood films are routinely stained with a ___________-type stain.

Yesterday's question:

At what age do plasma protein values reach adult ranges?

Answer: 3-4 months

2006-08-28T07:07:00.000-07:00

Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil-specific antibody

Vet Clin Pathol. March 2006;35(1):55-71.

Elizabeth Villiers, Stephen Baines, Anne-Marie Law, Victoria Mallows

I. Introduction

a. Immunophenotyping by flow cytometry is now used routinely in the diagnosis of leukemia in human medicine, and is becoming more widely available in veterinary medicine as increasing numbers of antibodies become available for this purpose

b. Canine lymphocytes can be identified readily using antibodies against:

i. CD3 (expressed by all T cells)

ii. CD4 (expressed by helper T cells, but also by canine neutrophils)

iii. CD5 (expressed by T cells and a subset of B cells)

iv. CD8 (expressed by cytotoxic T cells)

v. CD21 (expressed by B cells)

vi. CD79a (expressed by B cells)

c. Fewer antibodies are available that are specific for cells of monocytic and granulocytic lineage

i. CD11b is expressed principally on granulocytes, monocytes, and dendritic cells, but also may be expressed by subpopulations of lymphocytes

ii. CD11c integrin is expressed by monocytes, neutrophils, dendritic cells, and macrophages

iii. Antibodies recognizing canine CD14 are available and are specific for cells of monocytic lineage

d. The CD34 antigen is expressed by stem cells of any lineage

e. The aim of the study reported here was to evaluate three antibodies:

i. Canine neutrophil-specific antibody (NSA)

ii. Mouse anti-human macrophage antibody (MAC387)

iii. Mouse anti-human myeloperoxidase (MPO) antibody

f. NSA has been shown to label only neutrophils and not other granulocytes, monocytes, or lymphoid cells

i. It is not known whether this molecule is expressed by myeloblasts and other neutrophil precursors or whether expression is confined to more mature neutrophils

g. MAC387 recognizes the intracytoplasmic antigen calprotectin expressed in granulocytes, monocytes, and macrophages, and demonstrates cross-reactivity in the dog

h. MPO recognizes the peroxidase present in azurophilic granules in neutrophils and their precursors

i. It is expressed by cells of monocytic and granulocytic lineages

II. Materials and Methods

a. Animals and samples

i. 6 control dogs

1. Presented for orthopedic conditions

ii. Blood and/or bone marrow samples from dogs with suspected leukemia were sent to the laboratory from private veterinary practices in the UK, or were obtained from patients in the University of Cambridge Veterinary Hospital between January 2002 and June 2003

iii. A diagnosis of acute leukemia was based on the finding of >30% blasts in bone marrow or >30% blasts in peripheral blood, with cytopenias

iv. A diagnosis of AML was based on the morphology of blast cells, together with lack of staining reaction to antibodies recognizing the lymphoid antigens CD3, CD5, CD8, CD21, and CD79a, and by positive staining reaction to CD14 or CD4 with no reaction to other lymphoid markers

v. Staining reaction to a given antibody was regarded as positive if ≥20% of the blast cells stained

vi. The cutoff for positive staining to MPO was set at 10%

b. Flow cytometric analysis

i. Antibody staining was performed in plastic tubes containing 10⁶ nucleated cells in whole blood or anticoagulated bone marrow

ii. The antigens recognized by MPO and MAC387 are intracellular

1. Aliquots of whole blood containing 10⁶ nucleated cells were first permeabilized using a cell permeabilization kit that also lyses the red cells

iii. Cytometric analysis was performed using a FACScan flow cytometer with CellQuest software

iv. Cell populations were identified on the basis of their side light-scatter vs. forward light-scatter characteristics

v. The permeabilization step altered the scatter plot from that for nonpermeabilized cells; in some sample, this made accurate gating of monocytes and neutrophils difficult, as the two regions overlapped by variable amounts

III. Results

a. Control blood

i. Scatter plots for control blood samples indicated three distinct populations:

1. Monocytes

2. Neutrophils

3. Lymphocytes

ii. Neutrophils stain strongly with NSA and MAC387

iii. Monocytes did not stain with NSA

iv. Lymphocytes did not stain with any of the antibodies

b. Leukemic blood

i. In all cases, acute leukemia was suspected based on circulating blast cells in the peripheral blood, bi- or pancytopenia, and clinical signs of disease

ii. Propidium iodide staining revealed <5% of dead cells in all cases except case 7

iii. Case 1

1. Hematologic abnormalities

a. Neutropenia

b. Mild anemia

c. >30% circulating blast cells

2. The blast cells were:

a. Slightly larger than small lymphocytes

b. Had a round nucleus

c. Moderate amount of pale cytoplasm

d. Sometimes contained nucleoli

3. Flow cytometric analysis revealed positive staining with CD4 in 85% of the blast cells

4. The staining reaction to all other lymphoid markers was negative

5. The blast cells also expressed CD11b and CD11c

a. Suggested the cells were of neutrophil lineage

6. The blast cells strongly expressed NSA, MPO, and MAC387, again consistent with AML-M1

iv. Case 2

1. Hematologic abnormalities included:

a. Marked monocytosis

b. Numerous circulating blast cells

2. Bone marrow Cytologic examination revealed 80% blast cells with large indented or convoluted nuclei containing 1-3 nucleoli

3. The cells were considered most consistent with monoblasts

4. The predominance of monoblasts over maturing monocytes was consistent with AML-M5a

5. The cells did not express any lymphoid markers with the exception of CD4

6. There was strong staining reaction to CD14 as well as to CD11a, 11b, and 11c, consistent with AML-M5a

7. The CD34 integrin was expressed by 35% of the smaller cells, consistent with acute leukemia

8. The permeabilization procedure for staining intracellular antigens resulted in a change in the scatter plot such that, aside from the neutrophil population, only one distinct population was visible

a. Within this population, MAC387 stained 56% of the cells positive and MPO staining was negative

v. Case 3

1. Hematologic findings were similar to those in case 2

a. Marked monocytosis

b. Numerous circulating blast cells that had morphologic features suggestive of monoblasts

2. Blasts had highly convoluted nuclei containing one ore two nucleoli

3. Flow cytometric analysis resulted in a scatter plot with a discrete population of blasts/monocytes

4. 71% of cells stained positive for CD14, consistent with monocytic lineage

5. Substantial staining reaction to CD11b and CD11c, consistent with granulocytic or monocytic lineage

6. 31% of the cells stained positive for CD4

a. Some of this positivity may have been due to staining of neutrophils

vi. Case 4

1. Hematologic abnormalities included:

a. Neutropenia

b. Thrombocytopenia

c. Anemia

2. No monocytosis, but a high proportion of blast cells had large, round, or reniform nuclei

3. The blasts were identified in two distinct regions on the scatter plots

a. Did not express lymphoid markers, but did express CD14 and beta-2 integrins, consistent with monocytic leukemia

b. 53% of the cells in R1 expressed CD4

i. This was not thought to be consistent with neutrophil contamination because the dog was neutropenic and because the location of cells in the scatter plot was not consistent with that of neutrophils

c. 50% of the blast cells expressed MAC387, but none expressed MPO

vii. Case 5

1. Severe anemia

2. Thrombocytopenia

3. Neutropenia

4. Numerous circulating blasts

5. Blast cells formed a discrete region on the scatter plot

a. Expressed CD14 (68%) and CD34 (40%)

b. Did not express NSA, MPO, MAC387, or CD4

viii. Case 6

1. Anemia

2. Thrombocytopenia

3. Marked monocytosis

4. Numerous circulating blast cells

5. Blasts appeared in two regions on the scatter plot

a. Cells in R3 expressed only CD34

b. Cells in R1 expressed CD14, CD34, MPO, MAC387, and CD4

6. NSA results were negative

ix. Case 7

1. Hematologic findings included:

a. Anemia

b. Neutropenia

c. Thrombocytopenia

d. Numerous circulating atypical blasts

2. Bone marrow Cytologic examination revealed 99% blast cells

3. On the basis of morphology alone, it was not clear whether these were granulocytic/monocytic or lymphoid cells

4. Flow cytometric analysis of bone marrow revealed two populations of cells

5. Staining with propidium iodide in the isotype control tubes revealed a large number of dead cells in the small region (R3)

a. Only staining beyond this peak was considered positive

6. The population of larger cells in R1 contained few dead cells

a. The cells stained with CD14, MPO, MAC387, and CD34

b. Staining reaction for CD4 was negative

x. Case 8

1. Hematologic abnormalities included:

a. Anemia

b. Thrombocytopenia

c. Mild neutrophilia

d. High percentage of circulating blasts

2. Bone marrow Cytologic evaluation revealed 80% blast cells

3. There was moderate generalized lymphadenopathy due to an infiltrate of blast cells in the lymph nodes

4. Cells expressed CD14, CD4, NSA, MPO, and MAC387, as well as CD11a, CD11b, and CD11c

5. All stained negative for lymphoid-specific markers and CD34

IV. Discussion

a. In human medicine, AML is classified on the basis of blood and bone marrow morphology, and the results of cytochemical, immunophenotypic, and cytogenetic analyses

i. AML with recurrent genetic abnormalities

ii. AML with multilineage dysplasia

iii. Therapy-related AML

iv. AML not otherwise categorized

b. The most significant change is that the requisite blast percentage for a diagnosis of AML should be >20% myeloblasts in the blood or bone marrow (reduced from 30%)

c. In AML, monoblasts and promonocytes are counted as blast equivalents, and in acute promyelocytic leukemia, abnormal promyelocytes are counted as blast equivalents

d. In veterinary medicine, AML is analogous to the human category, AML not otherwise categorized

e. The presence of >20% or 30% myeloblasts or monoblasts in the peripheral circulation can only be due to AML

f. In healthy individuals, CD34 is expressed by hematopoietic stem cells

i. In humans and animals, expression of CD34 in Hematologic malignancies is exclusive to blast cells in acute leukemia and is absent form mature cells in chronic disorders

ii. Expression of CD34 does not indicate cell lineage; it may be expressed in ALL and AML

g. MPO is an important antibody for identifying myeloid leukemia

i. Leukemia is defined as being of myeloid (i.e., granulocytic or monocytic) lineage when ≥3% of blast are positive fro MPO by use of enzyme cytochemistry

ii. Flow cytometry is more sensitive than cytochemistry in detecting the presence of MPO when the lower threshold is used

iii. Cytochemical staining is limited to the contents of MPO granules rather than the perigranular membrane

iv. In flow cytometry, a monoclonal antibody is used to detect the presence of MPO within permeabilized granulocytes and is not dependent on enzyme activity or microscopic resolution

v. MPO is the only myeloid-specific antibody from the first-line human panel that is available for use in dogs and is makes a useful addition to a panel of antibodies for Immunophenotyping canine leukemia

h. Study of normal blood indicated that NSA was specific for neutrophils

i. Study of normal blood indicated that MAC287 stained both monocytes and neutrophils

j. CD4 may be expressed by monocyte precursors

i. This contrasts with findings in humans, in which CD4 is not expressed by mature neutrophils and is absent or weakly expressed on monocyte precursors, but is expressed by neutrophil precursors and mature monocytes

k. CD14 expression develops at an early stage of monopoiesis

l. Positive staining with CD11b is suggestive of myeloid lineage, although it also is expressed in disseminated Histiocytic sarcoma and in a minority of lymphoid leukemias

m. Expression of CD11c is relatively specific for myeloid lineage, although again, it may stain disseminated Histiocytic sarcoma cells in dogs and a small proportion of cells in B-cell lymphoid leukemias

n. Cytoplasmic CD79a is known to stain canine B lymphocytes from and early stage of development through that of mature plasma cells

o. The CD3 integrin is present on the cell surface only in more mature T cells

p. MPO is known to stain canine myeloblasts

q. Results of this case series suggest that CD14, CD4, MPO, MAC387, and NSA are expressed at an early stage of myeloid cell development

r. Immunophenotypic classification of leukemia should never be based on expression/lack of expression of one particular marker, but should be determined by the pattern of expression of a wide panel of antibodies

2006-08-28T04:45:00.000-07:00

Question of the day

At what age do plasma protein values reach adult ranges?

Yesterday's question:

Are plasma protein values in newborn animals higher or lower than adult values?

Answer: Lower

2006-08-27T07:35:00.000-07:00

Question of the day

Are plasma protein values in newborn animals higher or lower than adult values?

Yesterday's question:

What is a hereditary cause of lipemia in miniature schnauzer dogs?

Answer: Idiopathic hyperlipidemia

2006-08-26T05:28:00.000-07:00

Question of the day

What is a hereditary cause of lipemia in miniature schnauzer dogs?

Yesterday's question:

What is a hereditary cause of lipemia in dogs and cats?

Answer: Lipoprotein lipase deficiency

2006-08-25T04:51:00.000-07:00

Question of the day

What is a hereditary cause of lipemia in dogs and cats?

Yesterday's question:

Name three diseases which may result in lipemia in dogs?

Answer: Diabetes mellitus, pancreatitis, hypothyroidism

2006-08-24T04:38:00.000-07:00

Question of the day

Name three diseases which may result in lipemia in dogs?

Yesterday's question:

The presence of lipemia is frequently the result of what?

Answer: A recent meal

2006-08-23T12:48:00.000-07:00

Receiver-operating characteristic curves and likelihood ratios: improvements over traditional methods for the evaluation and application of veterinary clinical pathology tests

Vet Clin Pathol. March 2006;35(1):8-17. 45 Refs

Ian A Gardner, Matthias Greiner

I. Introduction

a. For both continuous and ordinal measurements, a threshold or cutoff value is required to categorize a test result as positive (abnormal) or negative (normal)

b. Cutoff values are required in test evaluation studies fro calculation of sensitivity (Se) and specificity (Sp)

c. The main criticism leveled against the use of cutoff values that give equal weight to all results on the same side of the cutoff is that it fails to adequately capture the magnitude of the test result and associated probability of disease or nondisease

i. These concerns raise the fundamental question as to whether cutoff values are essential to the test evaluator and clinician who makes the decisions and, if they are not, what alternatives are available

d. The paucity of veterinary clinical pathology articles that use these techniques most likely reflects a lack of familiarity with methods and lack of awareness of how readily these calculations can be done with many computer programs

II. What are ROC curves and when should they be used?

a. ROC methodology was developed to differentiate between signal and noise in the context of problems with different radar devices

b. In the medical sciences, ROC analysis has been most widely applied in medical imaging, psychometric research, and clinical chemistry

c. We use the term plot to denote a graph of the empirical data and the term curve to denote a smoothed ROC function where the curve parameters are estimated from empirical data

i. ROC plots (and curves) provide a graphic representation of all possible true positive (Se) and false positive (1-Sp) fractions for an ordinal or continuous test

ii. ROC plots can be used, therefore, as universal tools for test comparison even when the tests are quite different in their cutoff values and in their units and ranges of measurement

III. Why use ROC analysis?

a. Because there is an inverse relationship between Se and Sp as the cutoff value is changed, optimizing Se (Sp) will result in decreased Sp (Se)

b. ROC analysis provides a cutoff-independent method of summarizing the diagnostic accuracy of a test over all possible operating points

c. ROC analysis is especially useful for comparison of the accuracy of two tests for the same disease because there is no need to select cutoff values to compare Se and Sp

d. There may be some situations where presentation of the ROC curve and AUC comparison over all operating points could be misleading

e. Geometrically, the point of the ROC plot with the greatest distance in the northwest direction from the diagonal line represents the maximum combined value of Se and Sp that can be achieved with the test

f. The advantage of TG-ROC is that one can read the measures of accuracy from the graph as a function of cutoff value, and the program can be used to derive two cutoff values that define an intermediate or suspicious category of test results

IV. Plotting ROC curves in practice

a. ROC curves can be generated using spreadsheet programs such as Excel

V. Area under the ROC curve and its interpretation

a. The area under the ROC curve provides a global summary statistic of test accuracy and for useful tests, AUC ranges between 0.5 and 1

b. A perfect test has an AUC of 1 and a useless test has an area of 0.5

c. Two main interpretations of the AUC

i. It can be interpreted as the probability that test results for a randomly selected pair of diseased and nondiseased animals are correctly ordered

1. The probability of a correct answer in this experiment is independent of prevalence because it is fixed at 50% by design

ii. The AUC can be interpreted as the average Se, averaged across the entire range of false-positive fractions between 0 and 1

VI. Estimation of the ROC curve and its associate area

a. Methods of estimation can be classified into 1 of 3 broad categories:

i. Empirical methods

ii. Distribution-free parametric methods

iii. Distribution-modeling methods

VII. Use of ROC curves to compare the accuracy of multiple tests

a. For dichotomous test results, the Se and Sp of pairs of test can be compared using Pearson’s chi-square tests or McNemar’s test for correlated data

b. Testing of the same set of samples in a paired design usually is preferable because confounding by variables such as age, stage of disease, or disease severity will be prevented

c. For comparison of the accuracy of ordinal or continuous tests, the optimal approach is comparison of the test-specific ROC curves and estimation of the difference in the AUC of these curves

d. Two ROC curves can have almost identical areas and even intersect at some points, but have quite different shapes

e. A procedure for comparing two ROC curves based on a permutation has been developed and is useful for paired-sample designs and for comparing the shape of ROC curves with identical AUC’s

VIII. Sample size considerations for ROC studies

a. Sample size is usually considered to be of secondary importance to the representativeness of samples used in an ROC analysis

i. However, the number of samples tested from diseased and nondiseased animals affects the precision of estimation of the AUC of a single test and the ability to detect differences between the AUC’s of two tests, regardless of whether the tests are performed on the same or independent samples

IX. ROC analysis without a gold standard

a. For many animal diseases, a gold standard is not available, is prohibitively expensive to acquire, or is not feasible or ethically possible to obtain

i. In the absence of a gold standard, the problem is not statistically identifiable, unless additional information can be provided about the accuracy of one ore more of the tests

X. Likelihood ratios

a. Usually, the aim of testing is to increase the post-test probability of disease above a threshold of certainty that warrants an intervention, or to reduce the post-test probability below a threshold that warrants exclusion of the given disease as a diagnostic possibility

b. The magnitude of the calculated post-test probability of disease (D+) is dependent on the pretest (prior) probability of disease (P) and the Se and Sp of the tests

c. The pretest odds is the ratio of the number of diseased to nondiseased animals and is calculated as P/(1-P)

d. The LR represents a combination of Se and Sp values into a single estimate for a positive (LR+) and a negative (LR-) test result

e. LR+ (LR-) expresses the odds that a + (-) result is evident in an individual with the disease of interest (compared to without)

XI. Clinical application of likelihood ratios

a. If a test outcome is continuous, the probability of observing a unique value (x) theoretically will be 0 in both diseased and nondiseased animals

XII. Selection of a pretest probability for post-test probability calculations

a. Pretest probabilities for a given disease can vary between 0 and 100% and are uniquely determined based on prior experience of clinicians and local conditions such as the type of practice (primary or referral) and geographic location

b. A recent study found large variation in the estimated pretest probability of disease for doctors presented with identical patient information

c. One disadvantage of interval likelihood ratios is that unless the sample size is large, there is loss of precision when many interval are used

XIII. Advantages of likelihood ratios and misuse in the medical literature

a. They summarize the information in both Se and Sp into a single value

b. Do not vary with prevalence per se

c. Most important, they can be calculated on an interval (continuous data) or a category basis (ordinal data) rather than at a single threshold

d. If the pretest probability of a disease is known, likelihood ratios allow for direct calculation of post-test probabilities based on a modification of Bayes theorem

e. When test are used sequentially, the final post-test probability can be readily obtained by multiplying the post-test odds derived using the first test result in the sequence by the likelihood ratio for the second test

f. Common misuses of likelihood ratios in the medical literature include:

i. Reporting of likelihood ratios without CIs to represent the inherent uncertainty associated with limited numbers of test results in each category

ii. Selection of a cutoff point based on LR+ alone

iii. Not considering the information content in the LR-

iv. The greatest problem in the literature often is the lack of differentiation of likelihood ratios for cutoff points vs. ranges of values

XIV. Relationship between likelihood ratios and ROC curves

a. Likelihood ratios can be calculated from the slope of the ROC curve

b. There are three possible slope definitions depending on whether test results are dichotomous, continuous and categorized into intervals, or continuous but not categorized into intervals

XV. Conclusions

a. Test evaluation studies must be well designed following recommended guidelines to avoid bias in estimation of ROC curves and likelihood ratios

b. We recommend reporting of ROC curves and AUC (and 95% CI) as standard practice in test evaluation and comparison studies involving ordinal and continuous tests

c. The main advantage of these approaches is that they provide a cutoff independent method of test accuracy assessment

d. The website www.anaesthetist.com/mnm/stats/roc contains ROC applets for readers interested in getting started in ROC curves

2006-08-23T04:35:00.000-07:00

Question of the day

The presence of lipemia is frequently the result of what?

Yesterday's question:

_______ is recognized as a white opaque appearance caused by chylomicrons and very low density lipoproteins.

Answer: Lipemia

2006-08-22T04:27:00.000-07:00

Question of the day

_______ is recognized as a white opaque appearance caused by chylomicrons and very low density lipoproteins.

Yesterday's question:

Red discoloration of plasma indicates the presence of what?

Answer: Hemolysis

2006-08-21T05:02:00.000-07:00

Question of the day

Red discoloration of plasma indicates the presence of what?

Yesterday's question:

In species other than the horse, yellow plasma with a normal hematocrit suggests hyperbilirubinemia secondary to what?

Answer: Liver disease

2006-08-20T08:04:00.000-07:00

Question of the day

In species other than the horse, yellow plasma with a normal hematocrit suggests hyperbilirubinemia secondary to what?

Yesterday's question:

Increased plasma bilirubin concentration in horses often occurs secondarily to what?

Answer: Anorexia

2006-08-19T12:24:00.000-07:00

Question of the day

Increased plasma bilirubin concentration in horses often occurs secondarily to what?

Yesterday's question:

Increased yellow coloration of plasma usually indicates what?

Answer: Increased bilirubin concentration

2006-08-18T06:28:00.000-07:00

Classification of Myeloid Neoplasms: A Comparative Review

Vet Clin Pathol. September 2005;34(3):189-212. 265 Refs

Patricia M McManus

I. Introduction

a. The 30% blast threshold for diagnosing AML was originally advocated by FAB-NCI because it was estimated to be the point at which a myeloid neoplasm becomes sensitive to cytoreductive therapy

b. In some leukemias, cytogenetic mutations were determined to be better prognostic indicators than either leukemia phenotype or blast percentage, so a classification system that incorporated genetic status was needed

II. Human myeloid neoplasms

a. Much of the old terminology is retained, although new and more precise definitions of those terms are applied

b. Myelodysplasia is a misnomer for clonal disorders that are more accurately classified as chronic myeloneoplasms

c. WHO recommends that the diagnostic work-up of patients with suspected myeloid neoplasms always include:

i. 200-cell differential counts on peripheral blood

ii. 500-cell differential counts on bone marrow aspirate

iii. A bone marrow biopsy

iv. Genetic analysis

v. A panel of histochemical/cytochemical stains and immunophenotypic markers

d. Historically, lymphocytes, plasma cells, mast cells and macrophages were excluded from all nucleated cell (ANC) counts

e. Blasts include myeloblasts, monoblasts, promonocytes, atypical promyelocytes and megakaryoblasts

f. Erythroblasts are only included in the blast count if the patient has “pure erythroleukemia” with >80% erythroid elements in marrow

g. WHO recommends that morphologic atypia should be evident in ≥10% of cells within a single lineage to warrant designation as “significant lineage dysplasia” for MDS diagnosis and in ≥50% of cells within 2 or more lineages for “AML with multilineage dysplasia”

h. Examination of ≥20% megakaryocytes/marrow is recommended when determining the absence or presence of dysmegakaryocytopoiesis

i. Most emphasis being placed on the presence of micromegakaryocytes as indicators of clonality

j. Atypical morphologies used as indicators of clonality and not likely subject to interobserver variability include ring sideroblasts and pseudo-Pelger-Huet anomaly

k. Acute Myeloid Leukemia

i. AML accounts for 90% of acute leukemias seen in older patients

ii. The marrow blast threshold has been lowered from 30% to 20%

iii. The biologic behavior of dysplastic marrow containing between 20% and 30% blasts was determined to be just as aggressive as AML with multilineage dysplasia, suggesting that these 2 diseases should be treated as a single disease

iv. Cytogenetics is now recognized as the greatest predictor of survival potential

v. Risk groups

1. Favorable (25% relapse, 70% survival to 4 years)

2. Intermediate (50% relapse, 40-50% survival to 4 years)

3. High (70% relapse, 20% survival to 4 years)

vi. De novo AML is not usually associated with dysplastic morphologies, frequently has a single low risk cytogenetic defect, has no age predisposition, and is more responsive to chemotherapy

vii. AML with dysplasia and AML-therapy-related are more likely to have evolved over time, have multiple, inconsistent, high-risk cytogenetic defects, and are usually seen in older patients

viii. Stratification according to:

1. Presence of recurrent, specific cytogenetic abnormalities

2. Associated dysplasia

3. Previous chemotherapy

ix. AML with recurrent genetic abnormalities

1. 20-30% of all myeloid leukemias

2. Most genetic alterations in this group are balanced chromosomal transformations

3. Acute promyelocytic leukemia

a. Addressed therapeutically by induction of terminal differentiation using vitamin A-derived trans-retinoic acid

b. T-RA has fewer dire side effects compared with more conventional chemotherapy

c. APL is the most common of the AMLs, accounting for ~5-8%, and is the most curable of all AML subtypes, because patients with APL have a 70-80% survival rate at 5 years

d. APL is characterized by promyelocytes

i. Variably sized cells with variably shaped, frequently reniform or bilobed nuclei and pleomorphic nucleoli

e. The hypergranular form is more common

x. AML with multilineage dysplasia

1. Subdivided in 2 groups:

a. Those patients in which MDS was previously diagnosed

b. Those in which it was not

2. Dysplastic morphologies must be evident in at least 2 myeloid cell lines and in 50% of the cells

3. AML with dysplasia is generally a disease seen in the elderly rather than in children

4. AML occurring in patients who first presented with MDS is considered a secondary AML

a. Secondary AML accounts for ≥10% to possibly 40% of all AMLs

xi. Therapy-related AML

1. Subdivided according to agents

a. Alkylating and/or radiation

b. Topoisomerase II inhibitor

2. Risk increases as total dose and age of the patient increases and by specific type of drug

3. It should be noted that patients treated for a primary neoplasm are already at increased risk of developing second and even third neoplasms because of an increased susceptibility to cancer independent of therapy-associated risks

4. ALK/RAD t-MDS and ALK/RAD t-AML are classified together, presumably because:

a. Prognoses are equally poor for both

b. Neither responds to chemotherapy

c. Marrow blasts in ALK/RAD t-AML patients are >20% but still a minority cell type

5. Those that present with t-MDS die without progression to t-AML

6. ALK/RAD patients, overt disease generally develops after therapy, whereas with Topo II inhibitors, latency to AML is shorter at 203 years

7. Topo II inhibitor-associated AMLs usually have a very strong monocytic component and show a conventional response to antileukemic therapy predicable by their genetics

xii. AML, not otherwise categorized

1. Most closely reflect the previous French-American-British system

2. Alpha numeric designations have been discontinued

3. These leukemias are grouped by what they lack rather than what they have:

a. Multilineage dysplasia is not apparent

b. There is no consistent cytogenetic abnormality

c. There is no history of previous chemotherapy

4. Acute myeloblastic leukemia without maturation

a. Generally associated with:

i. Severe anemia

ii. Neutropenia

iii. Thrombocytopenia

iv. Significant leukocytosis consisting of mostly blasts

b. Blasts generally are easily identifiable as myeloblasts because of the presence of fine cytoplasmic granules

c. Blasts constitute most cells within the marrow

d. Blasts are ≥3% myeloperoxidase (MPO) and Sudan Black B (SBB) positive

5. Acute myeloblastic leukemia with maturation

a. ≥10% of cells have evidence of neutrophilic differentiation beyond the myeloblast stage

b. Myeloblasts must be ≥20% of ANC

6. Acute myeloblastic leukemia, minimally differentiated

a. Blasts are negative or <3% positive for cytochemistries

b. Blasts tend to be:

i. Medium-sized with finely dispersed to condensed chromatin

ii. Round or Slightly indented nuclei

iii. 1-2 variably distinct nucleoli

iv. Agranular variably blue cytoplasm

c. Blasts constitute most cells within the marrow

7. Acute myelomonocytic leukemia

a. Has evidence of maturation in both granulocytic and monocytic lineages

b. Myeloblasts, monoblasts and promonocytes collectively must be ≥20% of ANC

c. Neutrophils and neutrophilic precursors and monocytes and monocytic precursors must each account for ≥20% of ANC

d. Blasts should be ≥30% MPO positive

8. Acute monoblastic leukemia and acute monocytic leukemia

a. Contain ≥80% monocytic cells in the marrow with at least 20% monoblasts and promonocytes

b. If the majority (>50%) of marrow monocytic cells are monoblasts, the diagnosis is acute monoblastic leukemia

c. If promonocytes are the majority, the diagnosis is acute monocytic leukemia

d. Frequency associated with:

i. Hyperleukocytosis

ii. DIC

iii. Extramedullary solid tumors in the skin, oral cavity, and CNS

e. A recent study using current treatment modalities could not establish a significant difference between monocytic AML and nonmonocytic AML in complete remission rates, survival without recurrence, or overall long term survival

9. AML with predominantly erythroid phenotypes

a. The first FAB proposal made a diagnosis of erythroleukemia nearly impossible

b. Synonyms for M6-b include: acute erythremic myelosis, Di Guglielmo’s disease, and in the veterinary literature M6Er

c. Synonyms for cases with significant nonerythroid components are M6a and Di Guglielmo’s syndrome

d. The blast threshold has been lowered to 20%

e. Most cells in these cases are proerythroblasts (rubriblasts)

f. M6a is now called acute erythroleukemia or acute erythroid/myeloid leukemia

g. M6b is called pure erythroid leukemia

h. Primary signs are the result of anemia

i. As many as 50% of erythroid/myeloid leukemias and erythroleukemias diagnosed in human medicine are therapy related

j. Have uniformly poor prognoses

10. Acute megakaryoblastic leukemia

a. Blast population consisting of ≥50% blast of megakaryocytic lineage

b. Formerly AML-M7

c. The blast population ranges from mostly minimally differentiated cells that require immunophenotyping for lineage identification to megakaryoblasts with obvious, albeit abnormal, megakaryocytic traits

d. Marrows are severely fibrotic

e. Marrow aspiration may result in a “dry tap”

f. Present with cytopenias, including thrombocytopenis, although thrombocytosis is possible with AMegL

g. Fulminant diseases with poor prognoses

11. Acute leukemias of ambiguous lineage

a. Those that have 2 distinct blast populations, one myeloid and one lymphoid (bilineal acute leukemias), lack markers for either myeloid or lymphoid lineages, or have blasts that coexpress myeloid and lymphoid markers

l. Chronic myeloproliferative diseases

i. Rare chronic productive neoplasms characterized by peripheral blood increases in mature nondysplatic cells of myeloid lineage, including granulocytes, platelets, and erythrocytes

ii. MPD blast cell counts must be <20%

iii. Liver and spleen are generally enlarged because of sequestration of cells within these organs or extramedullary hematopoiesis

iv. Hypercellular marrows seen early in these diseases are virtually identical to reactive hyperplasias, which are primary differentials to consider during diagnostic evaluation

v. Mild to moderate anemia frequently accompanies all these diseases

vi. The final event is marrow failure, either as a result of clonal evolution to an acute blastic crisis or sever myelofibrosis

vii. The molecular basis for leukemogenesis in these diseases is poorly understood

viii. Chronic myelogenous leukemia

1. Most common and most thoroughly documented MPD is CML

2. Diagnosis requires detection of either the Philadelphia chromosome or the BCR-ABL fusion gene

3. The early phenotype is an unexplained, often asymptomatic, markedly elevated neutrophil count

4. Neutrophilia is not an isolated finding because it is always accompanied by basophilia and, frequently, eosinophilia

5. Examination of a marrow biopsy indicates a hypercellular state with increased granulopoiesis of all 3 cell types

6. Atypical megakaryocytes are usually 80% smaller in both cellular and nuclear size compared with normal

7. Dysgranulopoiesis is not a distinctive feature of CML

8. Scoring is based on a patient’s age, spleen size, and blast cell, eosinophil, basophil, and platelet counts to determine placement in 3 risk groups – low, intermediate, and high – that predict survival to 96, 65, or 42 months

9. Today’s treatment regimens include recently approved Imatinib

ix. Chronic neutrophilic leukemia

1. A rare disorder that is diagnosed by exclusion

2. Virtually identical to a leukemoid response in that it is characterized by leukocytosis of only neutrophilic granulocytes

3. Circulating neutrophils are variably toxic and mostly mature, with a left shift largely limited to band forms

4. Extramedullary neutrophilic granulopoiesis is present in the spleen and can be present in the liver

5. Clonal proliferation of neutrophils that are resistant to apoptosis, resulting in an accumulation of mature cells

6. Differential diagnosis includes other MPDs with a concurrent neutrophilic component, infection, inflammatory disease, and paraneoplastic response to malignant Neoplasia

7. In human medicine, a common cause for paraneoplastic Neutrophilia is multiple myeloma

x. Chronic eosinophilic leukemia

1. A clonal proliferation of eosinophils in which internal organs are infiltrated with eosinophils, and there is persistent unexplained eosinophilia for >6 months at >1500 cells/μL

2. Clinical signs are similar for CEL and HES, and both occur in men far more frequently than women

3. Morphologically, the only clue useful indistinguishing the two is an increase in blast cells in CEL

4. Both disorders are multisystemic diseases with infiltrates detected in multiple organs

5. Cytokine release by eosinophils results in damage to involved organs

6. Overall survival is good but variable

xi. Polycythemia vera

1. Characterized by clonal erythropoiesis that is independent of normal control process, an elevation of hemoglobin concentration >25% above the reference interval mean, and associated vascular disturbances

2. The mutation is within the multipotential stem cell

3. Panmyelosis is a common finding

4. Peripheral blood neutrophilia, basophilia, and thrombocytosis are common, and marrow megakaryocytic hyperplasia is striking

5. Two phases in PV progression

a. The proliferative or “polycythemic phase”

i. Marrow is frequently hypercellular and organ enlargement is primarily reflective of congestion

b. Followed by the spent or “postpolycythemic phase”

i. Typified by cytopenias

ii. Severe marrow fibrosis

iii. Splenomegaly secondary to prominent extramedullary hematopoiesis

iv. Erythrocytes have dacryocyte poikilocytosis

6. Repeated phlebotomy remains the most typical treatment regimen

7. Most patients die from thrombotic complications or hemorrhage

xii. Chronic idiopathic myelofibrosis

1. Monoclonal proliferative disease of a multilineage stem cell, manifesting heightened granulopoiesis and megakaryocytopoiesis, massive splenomegaly, and severe reactive marrow fibrosis

2. Proliferation of fibroblasts is likely influenced by neoplastic hematopoietic cells

3. Disease is stage as either prefibrotic, which is when 20-30% of patients are first seen, or postfibrotic, which is the more common presentation, but development of fibrosis is progressive rather than abrupt

4. As marrow fibrosis worsens, a leukoerythroblastic peripheral blood profile develops, and erythrocytes display dacryocyte poikilocytosis

5. A biopsy generally indicates:

a. Decreased cellularity

b. Increased reticulin and collagen fibers

c. Increased adipocytes

d. Osteosclerosis

e. Prominent atypical megakaryocytic proliferation

6. Death is usually tied to marrow failure

xiii. Essential thrombocythemia

1. Involves primarily the megakaryocytic lineage

2. Thrombocytosis is common (>600,000 cells/µL) and marrow megakaryocytes are increased, but there is no marrow fibrosis, extramedullary hematopoiesis is minimal, and megakaryocyte atypia is limited to an increase in cell size

3. Disease is often detected when a CBC is requested for unrelated reasons

4. Signs, when present, usually reflect vascular occlusion or GI hemorrhage

5. Diagnosis is often one of exclusion

6. Treatment of ET is not always needed

xiv. Chronic myeloproliferative disease, unclassified

1. Reserved for cases that do not fit the WHO criteria for any other category or that have features that overlap 2 disorders

m. Myelodysplastic syndromes

i. Characterized by cytopenias secondary to ineffective, and usually morphologically atypical, hematopoiesis, and also are termed dysplastic hematopoiesis or dyshematopoiesis

ii. Fairly common in people >70 years old

iii. Associated with significant morbidity and a high risk of progression to acute myeloid leukemia

iv. The classification system recognizes that some MDS are unclassifiable

v. Of importance is recognition of the difficulty in distinguishing between MDS and transient, nonclonal, dysplastic morphologies secondary to other processes, e.g., nutritional deficiencies, toxins, or drug exposure

vi. A biopsy is considered an important tool in the diagnosis of MDS

vii. Histologic detection of abnormal localization of immature precursors (ALIP) is considered a critical determinant of a poor prognosis

1. ALIP refers to detection of cluster of immature myeloid cells, e.g., ≥5-8 myeloblasts and promyelocytes in central regions rather than in their typical location, i.e., adjacent to sinusoid walls and end steal surfaces

n. Myelodysplastic syndromes/myeloproliferative diseases

i. MDS are thought to be proliferative disease characterized by heightened apoptosis so that hematopoiesis is ineffective, and the syndromes result in cytopenias

ii. Chronic MPDs are thought to be effective proliferations of cells that have heightened sensitivity to growth factors and/or have feedback loop failures, resulting in high peripheral blood counts

iii. Some lineages, usually granulocytic and/or monocytic, are effectively proliferative, whereas others, usually erythroid and megakaryocytic, are ineffectively proliferative

iv. Patients eventually die from evolution to AML or complications secondary to severe anemia and/or thrombocytopenia

v. Chronic myelomonocytic leukemia (CMML)

1. Patients with proliferative CMML have more pronounced splenomegaly and higher peripheral WBC counts, but other peripheral blood counts, age at diagnosis, marrow blast counts, and survival times are virtually identical to dysplastic CMML

2. Better predictors of short survival are immature myeloid cells in circulation, >10% blasts in marrow, anemia, lymphocytosis >2500 cells/µL, and thrombocytopenia

3. Subgrouping is now based on blast percentages in the blood or marrow or the presence of Auer rods

4. CMML-1 has <5% blasts in the blood and <10% in the marrow

5. CMML-2 has 5-19% blasts in peripheral blood or 10-19% blasts in marrow or Auer rods

6. Diagnosis of CMML requires an unexplained monocytosis of >1000 monocytes/µL and dysplasia of one or more myeloid lineages

7. If dysplasia is minimal or absent, CMML is diagnosed based on the persistence of monocytosis for >3 months or detection of a clonal cytogenetic abnormality within the marrow cells

8. CML is easiest to identify distinctly because it:

a. Is defined by its genetics

b. Has the least granulocytic dysplasia

c. Has the lowest percentage of monocytes

d. Has the highest frequency of basophils

9. CMML is distinguished from CML and aCML by its monocytosis, less pronounced granulocytic left-shifting in the peripheral blood, and higher percentage of erythroid cells in marrow

vi. Atypical chronic myeloid leukemia (aCML)

1. Characterized by significantly dysplastic and left-shifted granulopoiesis

2. Atypical traits include:

a. Acquired Pelger-Huet anomaly

b. Atypical nuclear segmentation

c. Atypical chromatin clumping

d. Abnormal cytoplasmic granularity

3. Leukocyte counts may exceed 300 x 10³ cells/µL

4. Erythroid dysplasia may manifest as elliptical macrocytes

vii. Juvenile myelomonocytic leukemia (JMML)

1. Rare disease

2. Pronounced splenomegaly because of increased extramedullary hematopoiesis

3. Anemia with circulating nRBC

4. Thrombocytopenia

5. >10% fetal hemoglobin

6. Diagnostic criteria include monocytes of >1000 cells/µL in peripheral blood and <20% blasts in blood or marrow

7. Prognosis is poor

III. Myeloid neoplasms in animals

a. Acute myeloid leukemia

i. APL in dogs, cats, horses, cattle, or sheep has yet to be reported

ii. The lack of animal cases, perhaps, reflects chromosomal differences in location of PML and RARα loci in animals so that breakage and translocation in a similar manner cannot occur readily

iii. In all but AML with erythroid phenotype, blast cells should be enumerated relative to ANC, with ANC defined as excluding plasma cells, lymphocytes, mast cells, and macrophages

iv. An MDS-Meg diagnosis has never been applied in animals or people

v. ALSG recommended a blast threshold of ≥30% in marrow and/or blood, similar to NCI guidelines, but assessment of blood for blast percentages is often left out of published guidelines

vi. The inclusion of a diagnosis based on the percentage of blast cells in blood was meant to clarify occasional cased in which the percentage of blast cells in marrow is high but still<30%, whereas the blood blast percentage is ≥30%

vii. The most common types of acute leukemia were myeloblastic forms M1 and M2

viii. Erythroid leukemias accounted for 20%, with 21 cases of M6 and 10 of M6Er

ix. Least common were AUL, AMML, and acute monocytic and acute megakaryoblastic leukemia

x. Dyserythropoiesis was significant in M6 and M6Er patients

xi. The highest marrow blast cell percentage of ANC were in AUL, AML-M1, and AML5a, whereas the lowest were in AML-M6Er

xii. High peripheral WBC counts did not predict high peripheral blast counts

1. Although cats with monocytic leukemias had the highest leukocyte counts, they were least likely to have circulating blasts because two thirds were noted to be aleukemic

xiii. 80% of the cats required bone marrow evaluation to confirm the diagnosis

xiv. Anemia was noted in 91.3% of the cats

xv. Thrombocytopenia was noted in 51.2% of the cats with known platelet counts

xvi. Most studies of AML in animals report that splenic involvement is common

xvii. Anemia and thrombocytopenia are common

xviii. The course of the disease is short, usually only days to weeks

xix. The majority of feline AML patients are positive for FeLV

xx. The majority of canine patients are large-breed dogs

xxi. Most animals are in their early to middle years

b. Chronic myeloid neoplasms

i. MDS is the result of a mutagenic event in the multipotential stem cell

ii. Has been shown to be monoclonal in the majority of feline patients and, therefore, is neoplastic

iii. Is not reversible and is nonresponsive to chemotherapy

iv. Patients can survive for several months and, occasionally, several years with supportive care

v. In cats, MDS is strongly associated with FeLV infection

vi. MDS is defined by marrow blast cells that may be increased, but are <30% of ANC in marrow with <50% erythroid precursors

1. Peripheral blood blasts are also <30%

vii. Düsseldorf scoring was a better prognostic index than MDS classification

viii. Blast percentages for most feline MDS patients reported in the literature are <20%

ix. Idiopathic hypereosinophilic syndrome and CEL are difficult to distinguish

1. Both characterized by persistently elevated peripheral eosinophil counts

2. Hypereosinophilia is polyclonal, whereas CEL is monoclonal

IV. Diagnostic tools

a. Cytochemical staining augments cytomorphology by identifying cytoplasmic chemical content generally associated with specific lineages

b. Staining must be present in ≥3% of blasts based on a 200-cell count to be considered positive

c. Cytochemical and histochemical staining

i. Standard cytochemical panels typically include:

1. Peroxidase (PO)

2. Chloroacetate esterase (CAE)

3. α-naphthyl-butyrate esterases (α-NBE)

4. α-naphthyl-acetate esterases (α-NAE)

5. Leukocyte alkaline phosphatase (LAP)

ii. PO and SBB are generally positive in cells of the neutrophilic and, in some species, the eosinophilic lineages

iii. CAE is usually positive in primary and secondary granules of neutrophilic, basophilic, and mast cell lineages

iv. α-NAE and α-NBE are usually positive in monocytic cells, but lymphocytes, platelets, and megakaryocytes also are potentially positive

1. Positivity in all but monocytes is resistant to sodium fluoride (NaF), whereas positivity in monocytes is inhibited by NaF

v. Lymphocyte α-NBE positivity corresponds to lysosomal granules found in granulated lymphocytes, i.e., cytotoxic T cells and NK cells; therefore, positivity is localized to the Golgi zone

vi. There are no enzymes specifically used to identify erythroid precursors

vii. Plastic-embedding generally results in less shrinkage of cells

viii. EM is required for distinguishing AUL from minimally differentiated AML and sometimes for identification of AMegL

ix. SBB stain cannot be adapted for use on plastic-embedded sections

d. Immunophenotyping

i. Based on using monoclonal antibodies (MAbs) to detect lineage-specific and lineage-associated antigens

ii. Most commercially available MAbs are designed for human or murine studies

1. Cross-reactivity has been demonstrated

iii. Immunophenotyping can be done on smears, tissue sections, or fluids

iv. Most, but not all, MAbs can be adapted for flow cytometry

e. Cytogenetics

i. Required in the diagnostic work-up of myeloid neoplasms because of its value in determining treatment and prognosis

ii. Karyotyping of canine patients is difficult because of almost identical chromosome morphologies, high canine diploid number (2n=78 in dogs), and resistance to banding

1. No association of a specific abnormality with leukemia type or outcome

iii. Karyotyping is easier in cats

V. Recommendations

a. Myelofibrosis is common in certain diseases and results in difficulty in obtaining marrow aspirations

b. AMLs with dysplasia are often secondary leukemias

c. Most cases of AML, but not all, continue to be poor responders to medical intervention compared with lymphoid neoplasms

2006-08-18T04:29:00.000-07:00

Question of the day

Increased yellow coloration of plasma usually indicates what?

Yesterday's question:

In some species, certain leukocytes may be present in the top portion of the packed erythrocyte column in a spun hematocrit tube, such as neutrophils in what species?

Answer: Cattle

2006-08-17T04:37:00.000-07:00

Question of the day

In some species, certain leukocytes may be present in the top portion of the packed erythrocyte column in a spun hematocrit tube, such as neutrophils in what species?

Yesterday's question:

What is the aggregation or clumping of erythrocytes together in clusters?

Answer: Agglutination

2006-08-16T04:29:00.000-07:00

Question of the day

What is the aggregation or clumping of erythrocytes together in clusters?

Yesterday's question:

Significant methemoglobinemia has been associated with clinical cases of what toxicity in horses?

Answer: Red Maple

2006-08-15T04:46:00.000-07:00

Question of the day

Significant methemoglobinemia has been associated with clinical cases of what toxicity in horses?

Yesterday's question:

Significant methemoglobinemia has been associated with clinical cases of what toxicity in sheep and goats?

Answer: Copper

2006-08-14T04:53:00.000-07:00

Question of the day

Significant methemoglobinemia has been associated with clinical cases of what toxicity in sheep and goats?

Yesterday's question:

Significant methemoglobinemia has been associated with clinical cases of what toxicity in cattle?

Answer: Nitrite

2006-08-13T06:30:00.000-07:00

Question of the day

Significant methemoglobinemia has been associated with clinical cases of what toxicity in cattle?

Yesterday's question:

Significant methemoglobinemia has been associated with clinical cases of what three toxicities in cats and dogs?

Answer: Benzocaine, acetaminophen, and phenazopyridine

2006-08-12T04:59:00.000-07:00

Question of the day

Significant methemoglobinemia has been associated with clinical cases of what three toxicities in cats and dogs?

Yesterday's question:

Does methemoglobin contain iron in the ferrous (+2) or ferric (+3) state?

Answer: Ferric (+3) state

2006-08-11T04:33:00.000-07:00

Question of the day

Does methemoglobin contain iron in the ferrous (+2) or ferric (+3) state?

Yesterday's question:

___________ is a protein consisting of four polypeptide globin chains, each of which contains a heme prosthetic group within a hydrophobic pocket.

Answer: Hemoglobin