Leukocyte Data and WBC Pattern Analysis

• Examination of leukocyte data from a CBC involves describing/categorizing abnormalities and analyzing them to fit a pattern that assists in patient diagnosis.
• Though it doesn't typically lead to specific disease diagnoses, evaluation of the WBC pattern, or leukogram, can aid in identifying diseases or physiological processes.
• Classifying a leukogram involves evaluating the total WBC count, which could be normal (within normal limits or WNL), increased (leukocytosis), or decreased (leukopenia).
• A normal total WBC count does not necessarily indicate a normal leukogram, morphologic abnormalities may still be present.
• Following the overall count, describe abnormalities within specific cell lines, including numerical and morphologic abnormalities.

Leukocyte Numerical Abnormalities

• Evaluate specific WBC lines for abnormal numbers, remembering that absolute cell counts are more useful to evaluation than the percentage.
• Percentages are a factor to calculate the absolute cell numbers.
• "*philia" or "*osis" indicates increased cell numbers, with "*philia" used for granulocytes and "*osis" for mononuclear cells.
• "*penia" indicates decreased cell numbers.
• Granulocytes include neutrophils (neutrophilia/neutropenia), heterophils (heterophilia/heteropenia), eosinophils (eosinophilia/eosinopenia), and basophils (basophilia or basocytophilia).
• Mononuclear cells include lymphocytes (lymphocytosis/lymphopenia) and monocytes (monocytosis/monocytopenia).
• Basophilia can describe a color change, so basocytophilia may be used instead for increased basophils, to avoid confusion.
• Basopenia doesn't really exist.

Absolute Cell Number Key Point

• Use absolute numbers and not percentages when looking for increases or decreases in cell numbers.

Neutrophil Responses in Leukograms

• Neutrophilia is seen with increased neutrophil production/release or demargination of neutrophils.
• Neutropenia is associated with increased loss due to tissue demand/destruction, increased margination, or decreased production.

Neutrophilia Mechanisms

• Neutrophilia can arise from increased neutrophil production in response to inflammation or myeloproliferative disorders.
• Shifting neutrophils from the marginal to the circulating pool can be caused by corticosteroids (stress leukogram), catecholamines/epinephrine (physiologic leukocytosis), or rare leukocyte adhesion molecule mutations.
• "Mature neutrophilia" describes neutrophilia with no left shift with only mature neutrophils present.
• An increase in segmented neutrophils plus an increase in bands is "neutrophilia with a left shift".

Neutropenia Mechanisms

• Increased tissue demand (margination/depletion) can lead to neutropenia, seen in acute inflammation (especially in species with small storage pools), acute endotoxemia, or severe inflammation.
• In marrow issues, neutropenia can arise from decreased production or from neutrophil destruction (immune-mediated or hemophagocytic syndrome).
• Infectious causes of neutropenia include viral diseases (parvovirus, panleukopenia, FeLV, FIV, equine herpesvirus-1) and Ehrlichiosis/anaplasmosis.
• Can occur because of generalized marrow hypoplasia/aplasia, myelophthisic disease (which can lead to pancytopenia with neutropenia developing first, prior to thrombocytopenia and anemia).
• Other causes of neutropenia:
  • Drugs/toxins like cytotoxic drugs or idiosyncratic reactions to trimethoprim-sulfa, phenylbutazone etc.
  • Long-term rG-CSF treatment from another species
  • Estrogens (endogenous or therapeutic)
  • Bracken fern in cattle

Immune-Mediated Neutropenia

• Destruction of mature cells or precursors can cause immune-mediated neutropenia, which is difficult to diagnose.
• It should only be diagnosed if infections have been ruled out.
• The causes of immune-mediated neutropenia include idiopathic causes, response to infectious organisms (Anaplasma phagocytophilum), or drug reactions.
• Combinations of causes may occur, such as parvovirus causing marrow suppression and inflammation.
• Neutropenia results in decreased immune response, which can lead to secondary infections.
• Ruminants often show neutropenia with acute inflammation because of their small neutrophil storage/maturation pools.
• Ruminants may also show a degenerative left shift as neutrophil numbers recover (neutrophilia after neutrophil production increases), and does not imply a poor prognosis.

Absolute Cell Numbers

• Example: Goat CBC Results
  • WBC: 2.600 x10³/ul (low), normal range (4.000-13.000 x10³/ul)
  • Seg N: 0.104 x10³/ul (low), normal range (1.200 - 7.200 x10³/ul)
  • Bands N: 0.026 x10³/ul
  • Lymphs: 2.366 x10³/ul, normal range (2.000 - 9.000 x10³/ul)
  • Monos: 0.104 x10³/ul, normal range (0.000 - 0.550 x10³/ul)
• The above are not lymphocytosis: lymphocyte % is elevated, but lymphocyte count is normal.
• The goat in this example has leukopenia caused by neutropenia.
• The pattern is typical of acute inflammation in ruminants.

Lymphocyte and Monocyte Responses in Leukograms

• Lymphocytosis can occur because of epinephrine release, antigenic stimulation, infections (viral diseases, Ehrlichia canis infection, Leishmaniasis, Trypanosomiasis), hypoadrenocorticism, lymphoid neoplasia, or non-lymphoid neoplasia (thymoma).
• Lymphopenia is caused by increased corticosteroid levels, acute inflammation/infection/endotoxemia, loss of lymph, or immunosuppression/hereditary immunodeficiency.
• Monocytosis is a relatively nonspecific finding that can occur in situations similar to neutrophilia (inflammation, corticosteroids), and may occur in acute or chronic inflammation.
• Monocytopenia isn't often clinically relevant.
• Monocyte responses can be used to monitor bone marrow activity/recovery due to their lack of a storage pool.

Eosinophil and Basophil Responses in Leukograms

• Eosinophilia can be caused by parasitic disease, allergic disease/hypersensitivity, eosinophilic infiltrative or proliferative diseases, paraneoplastic disease, hypoadrenocorticism, or hyperthyroidism treated with methimazole.
• Eosinopenia is not always diagnosed due to low reference intervals, and can show with increased corticosteroid levels or epinephrine use in cattle.
• Basophilia is associated with respiratory disease in dogs/cats and neoplasia in dogs/cats, and certain associations may aid clinicians in narrowing down underlying causes.
• Basopenia doesn't really occur, given reference intervals typically have 0/ul as a lower limit.

Common Types of Leukograms

• Inflammatory, stress/corticosteroid, physiologic leukocytosis, decreased production/pancytopenia, leukemic.
• Combinations of these patterns can certainly occur.

Inflammatory Leukograms

• WBC findings vary with different causes of inflammation, patient species, and the duration/severity of inflammation.
• Classic findings include neutrophilia (or but neutrophils may be normal or decreased), left shift, toxic change, monocytosis, and variable lymphocyte counts (normal, increased, or decreased).
• A left shift may not be present in mild or chronic inflammation.
• Not all of these changes may be present.
• The number of neutrophils in circulation is dependent on production vs. utilization.

Neutrophilic Left Shift in Inflammation

• Left shifting indicates the recruitment of neutrophils from the maturation pool in the bone marrow.
• Myeloid/granulocytic leukemia can also cause immature neutrophils to appear in blood, but is much less common than inflammation.
• Mild inflammation may not deplete the storage pool, while chronic inflammation can result in increased bone marrow neutrophil production that "catches up" with tissue demand causing no left shift.

Neutropenia Caused by Inflammation

• Acute, severe inflammation can cause neutropenia.
• Endotoxemia and/or inflammatory mediators cause neutrophils to shift from the circulating to marginal pool and exit the vessels to migrate to the site of inflammation.
• Bone marrow releasing neutrophils from the storage/maturation pool and from increased production can counter this.
• Increased neutrophil production should compensate for tissue demand, resulting in neutrophil counts progressing to WNL or neutrophilia.

Evidence of Inflammation with Normal or Decreased Neutrophil Count

• A left shift can be seen as well as toxic neutrophils, increased acute phase proteins, and direct evidence of inflammation (lesions, fever).
• Commonly evaluated acute phase proteins that increase in patients with inflammation include:
  • Fibrinogen
  • C-Reactive Protein (CRP) in dogs
  • Serum Amyloid A (SAA) in cats and horses
• Negative acute phase protein that tends to decrease with in inflammation is albumin.

Example Leukogram Case: Trenton

• A 3-year-old Thoroughbred mare presented with colic
• Laboratory Data on Day 1:
  • WBC: 12.9 x 103/ul (normal)
  • Seg: 10.965 x 103/ul (high)
  • Band: 0.387 x 103/ul (high)
  • All other values were within the normal range, and the fibrinogen 200(normal)
• The leukogram showed leukocytosis and neutrophilia with a left shift (inflammatory leukogram).
• Laboratory Data on Day 3:
  • Total leukocyte count was within the normal range, with increasing fibrinogen concentration (hyperfibrinogenemia).
• Laboratory Data on Day 4:
  • Developed profuse diarrhea with severe leukopenia (WBC 1.8 x 103/ul)
  • There was neutropenia, a degenerative left shift, toxic change, and lymphopenia with hyperfibrinogenemia.
  • This indicated severe/acute inflammation with stress
• Laboratory Data on Day 7:
  • Diarrhea improving
  • Total leukocyte count in reference interval.
  • Low segmented neutrophils
  • Lymphopenia improves
  • Still Hyperfibrinogenemia, with a final interpretation of being severe inflammation (but improving).
• Laboratory Data on Day 11:
  • Diarrhea resolved.
  • Total leukocyte count within reference interval.
  • Mature neutrophil count within reference interval (w/left shift).
  • Resolution of toxic change in neutrophils.
  • Monocytosis; with hyperfibrinogenemia (but with a decreasing trend)
• Final diagnosis: Fecal PCR + for salmonellosis.

Summary of Inflammatory Leukograms

• Neutrophilia may occur (especially above 2x the upper reference interval limit), but neutrophil count may be normal or low.
• Neutrophilia may also occur with stress/physiologic leukograms, but without a left shift or toxic change.
• Other findings include a significant left shift and toxic change in neutrophils, and combinations of the above.
• Monocytes may be inconsistently increased.
• Very high cell counts or extreme left shifting may be seen with leukemias.
• Inflammatory disease may not always show an inflammatory leukogram and is thus not 100% sensitive.

Corticosteroid-Induced ("Stress") Leukocytosis

• This is caused by elevated levels of endogenous or exogenous corticosteroids, or similar changes may occur in horses with sustained exercise.
• It can be seen with pain, chronic stress, corticosteroid therapy, or hyperadrenocorticism, while the stress leukogram pattern is common in dogs, but less common in cats/horses.

Distinguishing Types of "Stress"

• "Stress" may describe two different types of physiological reactions:
  • Chronic "stress" or glucocorticoid therapy shows Corticosteroid responses.
  • Acute stress and fear from “fight or flight," shows Epinephrine responses.

Corticosteroid-Induced/Stress Leukocytosis – Typical Pattern

• Lymphopenia, due to redistribution of lymphocytes to tissues and possibly cell lysis occurs and neutrophils decreased selectin causes neutrophil demargination that shift to circulating pool.
• Monocytosis may be present as well as Eosinopenia, but may be difficult to detect.
• Example: Casey, a 6-year-old Golden Retriever had the following results
  • WBC: 17,500/μΙ Η (high)
  • Segs: 14,350/μΙ Η (high)
  • Lymphs: 525/µl L(low)
  • Monos: 2,625/μΙ Η (high) with no toxic change or other abnormal morphology.
• Diagnosis: consistent with stress/corticosteroid leukogram.

Stress Leukogram Considerations

• All of the typical cell changes may not occur, with just lymphopenia sufficient for diagnosis.
• It is most typical to be present, and cells may shift within the reference interval "high normal" to "low normal.”
• Pattern may be superimposed on another pattern (i.e., acute inflammation + corticosteroid release) such as that of stress vs. inflammatory leukograms:
  • Lymphopenia is associated with both patterns, the interpretation is if present findings that suggest inflammation as well but is not expected in a stress alone:
  • Severe neutrophilia (>2x increase is unlikely with corticosteroid effect alone).
  • A significant left shift may be seen as well as toxic change in neutrophils with an inflamed lesion.

Epinephrine ("Physiologic") Leukocytosis

• This is caused by epinephrine release (fight or flight response).
• Is often seen in young cats with lymphocyte counts up to 36,000/ul and total WBC counts up to 26,000/ul seen in young horses.
• This pattern is less common in dogs.

Physiologic Leukocytosis Findings

• Neutrophilia (up to 2x normal count) with no left shift (caused by demargination of neutrophils) occurs, in addition to lymphocytosis.
• The leukocytosis is short-lived, lasting about 30 minutes.
• Example: Mia, a 6-month-old Siamese cat. She had Leukocytosis/ Mature neutrophilia/Mild lymphocytosis and Slight polycythemia from an increased Hematocrit..
• The interpretation was consistent with physiologic leukocytosis due to excitement/fear (epinephrine) response/ and Polycythemia from splenic contraction.

Review of Leukogram Interpretations

• Inflammation: Neutrophilia (+/- left shift and/or toxic alteration), +/- monocytosis, lymphocytes may be decreased/increased/WNL.
• Corticosteroid/stress: Lymphopenia, neutrophilia (+/- eosinopenia, monocytosis).
• Epinephrine/physiologic leukocytosis: Neutrophilia and lymphocytosis
• Eosinophilia for parasitic infections of lungworm
• Pancytopenia includes Anemia, thrombocytopenia, and leukopenia/neutropenia.
• Leukemic occurs if there are extremely high numbers of a single cell line.

Pancytopenia – Suppression or Aplasia

• Pancytopenia is a decrease in all three major cell types in blood, suggestive of the bone marrow suppression or non-regenerative anemia
• Bone marrow sampling is usually indicated for diagnosis.

Causes of Pancytopenia

• Myelophthisis replacing normal bone marrow from e.g., neoplasia
• Toxins or hormone usage such as estrogen
• Infectious agents such as parvovirus
• Immune-mediated destruction of hematopoietic cells
• Combination effeects from blood loss, anemia and infection

Example Case: Byron

• A 4-year-old MC Golden Retriever, had a history of lethargy and lymphadenopathy.
• The CBC showed;
  • WBC low - 1,225/µl
  • LOW neutrophils and band count WNL - approx. 30%
  • Lymphs- High and Monocytopenia
    • Note: The note states that the band count is WNL, approx. 30% of all neutrophils are bands, making this a significant left shift.
• Hematology description of non0regenerative anemia and leukopenia
• Byron has anemia (nonregenerative), leukopenia/neutropenia and thrombocytopenia
• The interpretation was that this was pancytopenia.
• Final diagnosis was Stage V lymphoma due to bone marrow metastasis
• Stage V lymphoma occurs as Pancytopenia is the result of myelophthisis (replacement of hematopoietic tissue with neoplastic cells).

Leukemic Leukograms

• Leukemia results from the proliferation of hematopoietic cells.
• Patients with these conditions have high WBC and single cell type.
• Some patients are Aleukemic with only neoplastic cells or no neoplastic cells (stage V).
• Often occurs as in the other hematopoietic cell lines.

Understanding Further of Leukemia

• Proliferation occurs in the hematopoietic tissues and in both myeloid cells.
  • Lymphocytic cells: acute leukemia
  • Maturity of blast: chronic leukemia
  • High blood cell count/Neoplastic: sub-leukemic
  • Circulating ells: aleukemic

Other Leukogram notes

• High neutrophil count is inflammatory response: or "leukemoid"

Case Example: Camilla the Cat

• Camilla, a 7-month-old cat who disappeared and returned weak, dehydrated, and with diarrhea, had sequential CBCs:
  • Day of presentation: Leukopenia, severe neutropenia, lymphopenia.
  • Four days later: Intensified neutropenia, left shift (bands almost as numerous as segs), near-normal lymphocytes.
  • Two days later: WBC within normal limits, degenerative left shift, normal lymphocytes, increased monocytes (indicates bone marrow recovery).
  • Four days later: Leukocytosis, neutrophilia, left shift (no longer degenerative), increased monocytes.
• Diagnosed with feline panleukopenia (FELV/FIP negative), demonstrating bone marrow recovery and overproduction of neutrophils.

Case Example: Bjorn the Dog

• Bjorn, a 9-year-old German Shepherd presented with non-weight-bearing lameness:
  • Mild neutrophilia, lymphopenia, mild monocytosis, normal WBC morphology, and a normal overall WBC, initially suggestive of a stress leukogram.
• Diagnosis was an aggressive lesion of the right femoral neck in joint fluid as well as a septic joint.
• Synovial fluid sample indicated septic joint, despite initial CBC not pointing to an inflammatory cause.

Leukogram Tips

• Monocytosis can occur in acute inflammation as well as chronic.
• When classifying a leukogram look not just for absolute values, but also at combinations to narrow down differentials.
• Even if the CBC appears normal, there can still be underlying inflammatory lesions.
• Inflammation will tend to cause stress.