Impedance Particle Counters

1.     counts particles & IDs by size based on electrical impedance

a)     Cons – may ID large platelets as RBCs, etc.

Optical/Laser Flow Cell Cytometers

1.     looks at how light is scattered by diff’t cells

a)     Pros – a little better than impedance

1.     QBC Analysis (centrifugation) –

 add float to tube of blood & spin, forces buffy coat to expand

a)     Older method – gives you Hct, [platelets], [WBC]

1.     Hemocytometer= 

put fluid on glass plates, counts cells/area

a)     Uses – examine CSF, sperm counts

iSTAT –

1.     Idexx ProCyte –

Erythrogram: Qualitative

-       size, shape, color, organisms, inclusions, cell ixns

a)     MCV–  

mean RBC volume (avg volume of red cell in patient)

a.     Micro/Normo/Macrocytic  indices

mean cell Hb concentration (mean [Hb]/red cell)a.   

  = (Hg ´ 100)/Hct

a)     MCH– 

mean cell Hb (mean Hg mass/red cell), least impt

a.     MCH = (Hg X 10)/[RBC]

b.     MCHC vs. MCH – 2 RBCs can have same MCHC but diffn’t MCH if one cel is smaller than the other

HDW

a)     Anisochromasia – ↑ variation in Hb concentration

1.     RDW –

 red cell distribution width (variation in RBC volume), reported as % of MCV

Anisocytosis – ↑  RDW, more variation in volume

-       Erythrogram: Quantitative part of CBC

Wintrobe’s Erythrocyte Indices– 

a)     MCV–

 mean RBC volume (avg volume of red cell in patient)

a.     Micro/Normo/Macrocytic  indices 

a)     MCHC – 

mean cell Hb concentration (mean [Hb]/red cell)

a.     MCHC = (Hg ´ 100)/Hct

b.     CHCM – cell Hb concentration mean, only on Advia analyzers, more precise & not affected by artifacts/lipemia

c.     Micro/NormoChromic indicies

d.     Hemoglobin [ ] in blood does not always = the average [ ] in cells.

e.     [Hgb] should be 1/3 of Hct in health THEREFORE MCHC = 33 g/dL

                                                                        i.         If not Artifact, RBC disorder, Instrument erroe

1.     %/volume of blood made up of RBCs (aka hematocrit/PCV/packed cell volume)

a)     Erroneous Values – inadequate mixing or centrifugation, not enough blood in EDTA tube

b)     = (MCV ´ [RBC])/10

1.     [Hgb] –

 blood Hb concentration (g/dL), Hb normally just in RBCs & measured after lysis

a)     Should be 1/3 of Hct in health

b)    Abnormalities – artifact, RBC disorder, lipemia, animal recently ate, instrument error

c)     Decreased = Anemic.  Increased = Erythrocytosis 

[RBC]

1.     RBC concentration in blood (#/μL)

a)     Decreased = Anemic.  Increased = Erythrocytosis

1.     Anemia – 

1.     Erythrocytosis –

a)     Rouleaux 

– rolls of coins

a.     ↑ – from marked hyperglobulinemia (↑ Igs/other globulins)

                                                                        i.         Causes of Hyperglobulinemia – inflmm, dehydration, multiple myeloma

b.     If truly rouleaux, rolls will disperse w/saline

a)     Agglutination 

– grape clusters/not linear

a.     Causes – almost always from Igs bridging RBCs together, IgM is best at it

b.     Grossly – clumps in EDA tube, saline does NOT break up

c.     Causes Errors – RBC parameters other than spun Hct & [Hgb] may be wrong

a)     Rubricytosis –

 ↑ nucleated RBC concentration (normally none-rare)

a.     Appropriate – if BM is responding to anemia

b.     Inappropriate – no anemia/no need for BM response (cannot definitively use as sign BM is responding to anemia)

                                                                        i.         Causes – BM damage, extraBM poiesis, splenic contraction, splenectomy, Pb

c.     Enumeration – count # of nRBCs seen during time to count 100 WBCs, report as     # nRBCs/100 WBCs

d.     Corrected [WBC] – may have to do b/c analyzer will count nRBCs as WBCs

                                                                        i.         Corrected [WBC] = measured [WBC] ´ 100/(100 + #nRBC/100WBC)

a)     – ↑ central pallor/paler, thinner/has less Hb (↓ MCV/MCHC/MCH)

a.     Causes – usually Fe deficiency anemia

a)      – a young anucleate RBC that is more blue/purple b/c it contains RNA that stains blue

a.     Polychromasia – ↑ polychromatophils

b.     Cause – accelerated erythropoiesis, CAN use as indicator that BM is ↑ production

                                                                        i.         Rengenerative anemias often have 

Reticulocytosis 

a.     best evidence of accelerated erythtopoiesis EXCEPT in horses

                                                                        i.         Expected 3-4d after acute blood loss

a)     denatured Hb from oxidative damage, (onions)

a.     appear as small round structures that are same color as RBC, either near membrane or sticking out, can cause severe hemolytic anemia

b.     Cats – few-many are normal in healthy cats

a)     appears just on margin inside cell, intracellular bact, ruminants only

a)     protozoa in all species

a.     Looks like signet rings in RBC 

 – nuclear remnants from mitosis error, darks dots NOT right on margin

a.     Cause – usually from ↑erythropoiesis (more chance for errors), ↓ splenic fxn

a)     variation in RBC volume/fiameter, ↑s RDW

a.     Causes – either macro/microcytosis, from regenerative response to anemia

b.     Macrocytosis w/o polychromasia – NOT regenerative, something wrong w/RBCs

a)     Microcytes – 

RBCs w/↓ vol, can cause ↓ MCV

a.     Cause – usually Fe-deficiency, also hepatic dz, portosystemic shunts

b.     Breed/Age Variation – in Akitas isn’t as abnormal

a)     Macrocytes –

 RBCs w/↑ vol, can cause macrocytosis & ↑ MCV

a.     Cause – accelerated erythropoiesis or abnormal erythropoiesis

a)    
Acanthocyte – 

spur/burr cell, few irregularly shaped/spaced projections

Causes – splenic infiltrates, hemangiosarcoma, lipid abnormalities

a)     Eccentrocyte – 

membranes fused together, Hb squeezed to one side, other side clear

a.     
Causes – oxidative damage/toxicants, defective RBC reducing pathways

b.     Horses with red maple leaf toxicosis 

a)     Echinocyte –

 many regular protections

Causes – usually an artifact, can be from dehydration, drugs, snakebites

a)    
Keratocyte –

 helmet cell, from trauma/blister or Fe-deficiency

Damaged 

fragment, from trauma, DIC< vasculitis, hemangiosarcoma, Fe-deficiency

Spherocyte

a)     Codocyte –

Anemia

-       

-

1.     Blood Loss

2.     RBC Destruction (hemolysis)

3.     ↓ or Ineffective Production

-       Clinical Signs –all relate to ↓ O₂-carrying capacity

-      Classifications of Anemia 

1. Marrow Responsiveness 

2. RBC Indicies 

3. By Pathophysiology 

Either have
1. Blood Loss or 
2. Hemolysis 

Because the bone marrow is healthy 

Mean Cell Volume - MCV

Mean Cell Hemoglobin Concentraion - MCHC 

a)     Normocytic Normochromic – MCV & MCHC normal

a)     Macrocytic Hypochromic – ↑ MCV & ↓ MCHC

a)     Microcytic Hypochromic – ↓ MCV & ↓ MCHC

a)     Microcytic Normochromic – ↓ MCV, MCHC normal

a.     Causes – Fe-deficiency anemia, liver dz, defective erythropoiesis

b.     Breed/Age Variations – Akitas/Shibas & foals/kittens have small RBCs

a.     External – GI/urinary tracts, blood donors

b.     Internal – peritoneal & pleural spaces

a)     Hemolytic Anemias –

a.     extravascular hemolysis  - RBC lysis by macΦ in tissues

b.     intravascaular hemolysis – RBC lysis in Tissues 

a.     extravascular hemolysis  

- RBC lysis by macΦ in tissues

a)     ↓ Erythropoiesis- i

inflmm dz

chronic renal dz

marrow hypoplasia/aplasia

erythroid hypoplasia/aplasia, ineffective erythropoiesis

Leukocytosis with

Neutrophilia (demargination)

Lymphopenia 

Monocytosis 

1.     occurs in a day, lasts weeks

Neutrophilia (Mature)

LYMPHOCYTOSIS (inc recirculation)

Monocytosis (demargination)

occurs in seconds, lasts minutes-hour

1.     hemorrhage → anemia → hypoxia in kidney → stimulates Epo production → erythropoiesis & reticulocyte release

a)     Microcytosis  in iron def 

poor regenerative anemia in iron def

– when not enough Hb, doesn’t allow enough cells to be made

a)     Rubricytosis in iron def  –

Causes decreased erythropoiesis.

 ↓ functional renal mass → ↓ Epo production → less BM stimulation → ↓  red cell precursors

Normochromic, normocytic but anemic

Because decreaded EPO less BM stimulation 

Blood loss or heamolysis b4 it can regenerate (pre-regenerative)

• Reduced erythropoiesis 
• Inflam dz
• Chronic renal dz
• Disease effecting BM
• Defective Erythropoesis 
• Fe deficiency
• Immune-mediated nonregenerative anemia 
• FeLv-induced erythroid neoplasia 

AID - Anemia of Inflammatry Dz

Chronic Inflammation 

Normochromic

Normocytic 

Non-regenerative Anemia 

Decreased erythopoesis 

Immune Hemolytic Anemia 

a)     idiopathic, drugs, neonatal, transfusion, infections

a.     Spherocytes

b.     IMHA 

Hemolytic Anemia from Infections

RBC Metabolic Defects that cause Hemolytic Anemia 

a.     oxidative injury (acquired/congenital),

b.     defective ATP generation (PK/PFK deficiency, low P)

c.     Heinz Bodies, Eccentrocytes

Hemolytic disorder of RBC Fragmentation-

a.     thrombosis, vasculitis, hemangiosarcoma, turbulence

b.     Keratocytes, schizocytes, fragments

Virus + Antibodies --> Form Complexes (IC) in blood --> IC Attach to RBC --> Macrophages will recognize and Phagocytize (Extravascular hemolysis) ALSO --> Compliment can attach to RBC and cause Mphages to phagocytize. 

Another Pathway is that the IC + RBC + Compliment will have a Membrane Attach Complex form and punch holes in RBC (INTRAVASCULAR HEMOLYSIS) 

Anemia ---> Renal Hypoxia --> Inc. EPO --> Inc Erythropoesis --> Wont release early (b/c horse), but will release bigger (Macrocytes) 

Regenerative Anemias 

Cell swelling (prologed storage, hypernatremia)

Agglutination (immune-mediated, Heparin-induced) 

No Anemia or Hypoproteinemia (before Fluid Redistribution) 

Regenerative anemia (5 days later)

Defective Erythropoiesis 

Chronic External blood loss --> Iron Deficiency