CBC’s at UK • Pretty much all CBC’s start with analysis by an automated Pretty much all CBC s start with analysis by an automated CBC analyzer. • ~75% of CBC’s have an automated differential only. 75% of CBC s have an automated differential only. – Clue to automated differential = reported % with tenths values. Neutrophils 54.8% Neutrophils 54 8% Neutrophils 55% Neutrophils 55% Lymphs 23.6% Lymphs 24% Monocytes 15.2% Monocytes 15% Eosinophils 4.3% Eosinophils 4% Basophils 2.1% Basophils 2% Automated Differential Manual Differential • If the automated analysis picks up certain “flags”, then the CBC i CBC is ticked for a manual assessment by the Heme techs. i k d f l b h H h
Nucleated Blast forms High WBC RBC’s (> 50 000) (> 50,000) Platelet count < 30,000 Immature neutrophils Macrocytosis Abnormal Absolute Marked Abnormal lymphocyte Anisocytosis Absolute count monocyte count Variant lymphocytes Low MCV Certain RBC Pl t l t Platelet Morphology clumps Abnormalities
Bottom line Bottom line… • Today’s automated hematology analyzers are very good for most routine applications, but they’re not perfect. • A Automated CBC’s can’t reliably describe the actual morphology of d CBC’ ’ li bl d ib h l h l f WBC’s or RBC’s – Machines can “flag” certain RBC or WBC abnormalities (e.g. 2 + anisocytosis) g ( g y ) • If the diagnosis that you are considering correlates with a specific WBC or RBC morphology, then order a manual slide review. – Leukemia (blasts) – RBC membrane disorder (spherocytes) • Lik Likewise, if the CBC doesn’t “fit” with the clinical picture, then more i if th CBC d ’t “fit” ith th li i l i t th information might be obtained by examining a peripheral blood smear.
CBC’s and Manual Diff’s • Only when a manual differential is performed will anyone from the lab physically look at the peripheral blood smear. p y y p p • Remains the “gold standard” for blood interpretation • Clinical Lab s Heme techs are excellent and reliable • Clinical Lab’s Heme techs are excellent and reliable • Exceptional smears are also reviewed by hematopathologists • The practitioner has the option of requesting a manual differential right from the start (on the general lab order form) the start (on the general lab order form). • For now, but the lab may evaluate this policy (labor ‐ intensive)
Keys to success with blood smears • The smear must be artifact ‐ free and have an adequate region of cell dispersal Too thick! Too thin! Just right • Take your time, and ask the hematology tech his/her impression
Warm-up slides
Describe those RBC’s! Goodness that is a lot of variation in color! This person must have a high retic count!!! 1. Spherocytes 2 2. Polychromasia Polychromasia 3. Sickled forms 4. Nucleated RBC’s
Describe those RBC’s! Describe those RBC s! Just look at all that variation in red cell size! 1. Poikilocytosis 2 2. Polychromasia Polychromasia 3. Anisocytosis 4. Target Forms g
Describe those RBC’s! Describe those RBC s! I say! Have you ever seen such differences in red cell shape?! 1. Polychromasia 2 2. Anisocytosis Anisocytosis 3. Poikilocytosis 4. Target Forms g
Describe those RBC’s! Describe those RBC s! Which term best describes this smear? Which term best describes this smear? 1. Schistocytes 2 2. Sickled Forms Sickled Forms 3. RBC Stippling The spleen: 4. Spherocytes p y final resting place for many g p y a spherocyte…
Describe those RBC’s! Describe those RBC s! S hi Schistocytes are broken red cell fragments b k d ll f that form with microangiopathy and abnormal shearing. 1. Sickled Forms 2 2. Schistocytes Schistocytes 3. Reticulocytosis 4. Spherocytes p y
Describe those RBC’s! Describe those RBC s! Did someone mention targets?! This patient might have hemoglobin C! 1. Polychromasia 2 2. Anisocytosis Anisocytosis 3. Poikilocytosis 4. Target Forms g
Name that Cell! Name that Cell! B C ll B Cells, T Cells T C ll and NK Cells, Oh My! 1. Monocyte 2. Lymphocyte 3. Neutrophil 4. Blast
Name that Cell! Name that Cell! 1. Neutrophil 2. Eosinophil 3. Granular Lymphocyte 4. Basophil Finding basophils in the peripheral g p p p blood is fairly uncommon.
Name that Cell! Name that Cell! Does anyone else think that eosinophils are beautiful? They look like they’re filled look like they’re filled with little rubies! 1. Neutrophil 2. Monocyte 3. Eosinophil 4 4. Basophil hil
Name that Cell! Name that Cell! 1. Band 2. Monocyte 3. Lymphocyte 4. Basophil Now THAT’s a fine band!
Name that Cell! Hey look ‐ it’s a little neutrophil p smiley face! 1. Neutrophil 2. Monocyte 3. Eosinophil 4 4. Basophil hil
Name that Cell! Name that Cell! Prepare to be phagocytized! 1. Neutrophil 2. Monocyte 3. Lymphocyte 4. Basophil
Name that Cell! Name that Cell! 1. Monocyte y 2. Band 3. Reactive lymphocyte 3. Reactive lymphocyte 4. Blast Ka BOOM!!! Ka-BOOM!!!
Cases CBC and peripheral blood smear can clinch the diagnosis. diagnosis.
Hands on clickers… it’s time for d l k ’ f audience participation fun! p p
Toddler with pallor. 6.1 5 8 5.8 478 478 17.9 MCV: 57 fL RDW: 23 % MCHC: 32 fL P 46 L 38 M 12 E 4 P 46 ,L 38 ,M 12 ,E 4 Retic: 1.3% • Pallor seems to have developed gradually. • No jaundice, no dark urine, no fevers • P l Pale but well-appearing, playful b t ll i l f l • No organomegaly
Toddler with pallor. 6.1 25% 25% 25% 25% 5.8 478 17.9 MCV: 57 fL MCV: 57 fL RDW: 23 % MCHC: 32 fL P 46 ,L 38 ,M 12 ,E 4 Retic: 1 3% Retic: 1.3% Which diagnosis is Which diagnosis is most likely? most likely? 1. Iron deficiency anemia 2. Thalassemia a asse a 3. Vitamin B 12 deficiency 4. Autoimmune hemolysis 1 2 3 4
4 y/o immigrant from Nigeria. 7.7 10.8 423 21.8 MCV 83 fL MCV: 83 fL RDW: 19.1 % P 61 ,L 28 ,M 8 ,E 3 Retic: 9.3% • Not previously known to be anemic. • Bloodwork on his initial “well-child” check. • 4-5 cm firm spleen felt on examination. • Muddy sclerae • II/VI systolic murmur left sternal border
4 y/o immigrant from Nigeria. 7.7 10.8 423 21.8 25% 25% 25% 25% 25% 25% 25% 25% MCV: 83 fL MCV: 83 fL RDW: 19.1 % P 61 ,L 28 ,M 8 ,E 3 Retic: 9.3% Which Which diagnosis is diagnosis is most likely? most likely? 1. Trypanosomiasis 2. G6PD deficiency 3. Thalassemia 4. Sickle Cell Disease 1 2 3 4
4 y/o with widespread bruising. 12.1 5 8 5.8 3 3 35.8 MCV: 87 fL RDW: 12.3 % MCHC: 34 fL P 60 ,L 28 ,M 6 ,E 2 ,Atyp 4 60 28 6 2 4 Retic: 1.3% 1. No family history of bruising/bleeding. 2. Normal medical history. 3 3. N No increased bleeding with neonatal circumcision. i d bl di i h l i i i 4. Petechiae and purpura appeared suddenly overnight.
4 y/o with widespread bruising. 12.1 5.8 3 35.8 25% 25% 25% 25% 25% 25% 25% 25% MCV: 87 fL RDW: 12.3 % MCHC: 34 fL P 60 ,L 28 ,M 6 ,E 2 ,Atyp 4 Retic: 1.3% R ti 1 3% Which dia Which diagnosis is g g nosis is most likel most likely? y? y y 1. Acute Leukemia 2. Idiopathic Aplastic Anemia 3. Child abuse 4. ITP (Primary autoimmune thrombocytopenia) y p ) 1 2 3 4
Teenager with Crohn’s disease and pallor. 6.7 2 8 2.8 110 110 19.8 MCV: 107 fL RDW: 19.1 % P 41 ,L 48 ,M 9 ,E 2 Retic: 2 3% Retic: 2.3% • Diagnosed 4 years prior with IBD Diagnosed 4 years prior with IBD • Hospitalized a few times with Crohn’s exacerbations. • Pallor seems to have come on gradually. • Symptoms include some weakness and dizziness.
Teenager with Crohn’s disease and pallor. 8.1 2.8 110 23.8 MCV: 107 fL MCV: 107 fL 25% 25% 25% 25% 25% 25% 25% 25% RDW: 19.1 % Retic: 2.3% Which Which diagnosis is diagnosis is most likely? most likely? 1. Myelogenous leukemia 1 M l l k i 2. Steroid effect 3. Lupus erythematosus 3 L h 4. Vitamin B 12 deficiency 1 2 3 4
Child in ICU with thrombocytopenia 10.1 8.5 8 5 48 48 29.4 Retic: 3.2% MCV: 87 fL MCV: 87 fL P 64 ,L 26 ,M 8 ,E 2 • Admitted yesterday with high fevers and obtundation. • Blood culture positive for gram-negative rods. Bl d lt iti f ti d
Child in ICU with thrombocytopenia 10.1 8.5 48 25% 25% 25% 25% 25% 25% 25% 25% 29.4 29 4 Retic: 3.2% MCV: 87 fL P 64 ,L 26 ,M 8 ,E 2 P ,L ,M ,E Which Which diagnosis is diagnosis is most likely? most likely? 1. Endocarditis 2. Disseminated intravascular coagulation (DIC) coagulation (DIC) 3. Sickle cell anemia 4. Dehydrated red cells 1 2 3 4
5 y/o with fever, pallor and bruising 8.1 133.8 47 23.9 23.9 25% 25% 25% 25% 25% 25% 25% 25% MCV: 79fL RDW: 13 % MCHC: 33.4 fL MCHC 33 4 fL Retic: 0.4% 1. Sepsis 2. Viral infection 3. Acute leukemia 4 4. I fl Inflammatory response t 1 2 3 4
9 y/o with abdominal cramping. 25% 25% 25% 25% 1. Helminth infection 2. Lactose intolerance Clostridium difficile colitis 3. 4. 4. Irritable bowel syndrome Irritable bowel syndrome 1 2 3 4
Another toddler with pallor 7.1 6 8 6.8 348 348 21.9 MCV: 57 fL RDW: 11 % MCHC: 32 fL P 54 L 28 M 17 E 1 P 54 ,L 28 ,M 17 ,E 1 Retic: 10.3% • Family recently immigrated from Turkey • Firm spleen 5 cm below costal margin • Mildly icteric sclerae
Another toddler with pallor 7.1 6.8 348 21.9 MCV: 57 fL MCV: 57 fL 25% 25% 25% 25% 25% 25% 25% 25% RDW: 11 % MCHC: 32 fL P 54 ,L 28 ,M 17 ,E 1 Retic: 10.3% % Which Which diagnosis is diagnosis is most likely? most likely? 1. Iron deficiency anemia 2. Thalassemia 3. Chronic renal failure (EpO deficiency) 4. 4. Anemia of inflammation Anemia of inflammation 1 2 3 4
2 y/o with pneumococcal sepsis 12.1 6 8 6.8 498 498 36.9 MCV: 81 fL RDW: 12 % MCHC: 34 fL P 65 L 23 M 11 E 1 P 65 ,L 23 ,M 11 ,E 1 Retic: 1.8% • Normal full ‐ term baby • Normal growth/development • 1 prior hospitalization for pneumonia
2 y/o with pneumococcal sepsis 12.1 6.8 498 36.9 25% 25% 25% 25% 25% 25% 25% 25% MCV: 81 fL RDW: 12 % MCHC: 34 fL P 65 L 23 M 11 E 1 P ,L ,M ,E Which Which diagnosis is diagnosis is most likely? most likely? 1. Common variable immunodeficiency 2. HIV infection - AIDS 3. Asplenia 4. Congenital neutropenia 1 2 3 4
7 y/o with pallor. 5.1 8 3 8.3 401 401 15.4 MCV: 103 fL RDW: 26 % MCHC: 34.3 fL P 32 L 48 M 14 E 2 nRBC 4 P ,L ,M ,E ,nRBC Retic: 34.5% • Fatigue and pallor seem to have “come out of nowhere” over the past day or two. • Patient is sallow, ill-appearing and has mild scleral icterus. • Hyperdynamic precordium with III-IV/VI systolic murmur • Hyperdynamic precordium with III-IV/VI systolic murmur. • No organomegaly.
7 y/o with pallor. 5.1 8.3 15.4 401 25% 25% 25% 25% 25% 25% 25% 25% MCV: 103 fL RDW: 26 % MCHC: 34.3 fL P 32 ,L 48 ,M 14 ,E 2 ,nRBC 4 , , , , Retic: 34.5% Which diagnosis is Which dia g g nosis is most likel most likely? y? y y 1. Autoimmune hemolytic anemia 2. Disseminated intravascular coagulation (DIC) 3. Acute Leukemia 4. Folic acid deficiency 1 2 3 4
6 y/o with splenomegaly. 8.1 8 1 5.5 288 24.4 Retic: 8.2% Retic: 8.2% MCV: 87 fL MCHC: 36 P 44 L 46 M 8 E 2 P ,L ,M ,E Direct Coomb’s: negative • Child is asymptomatic • Splenomegaly appreciated on routine exam by an apt clinic resident • Ph t th Phototherapy as a newborn b • Mother with history of cholecystectomy as a teenager
6 y/o with splenomegaly. 8.1 5.5 288 25% 25% 25% 25% 25% 25% 25% 25% 24 4 24.4 Retic: 8.2% MCV: 87 fL MCHC: 36 P 44 L 46 M 8 E 2 P 44 ,L 46 ,M 8 ,E 2 Coomb’s neg Wh Wh Whic Wh h ich dia diagnosis is g o sis is most li o st like kely? ke ke y y? 1. B ‐ thal trait 2. 2 G6PD deficiency G6PD deficiency 3. Hereditary spherocytosis 4. Storage disease (Gaucher’s) 1 2 3 4
18 month old well child with anemia 5.3 7.6 7 6 248 248 25% 25% 25% 25% 25% 25% 25% 25% 14.4 Retic: 0.2% MCV: 89 fL MCV: 89 fL RDW: 13.3 P 49 ,L 41 ,M 9 ,E 1 ESR: 6 sec 1 1. Iron deficiency Iron deficiency 2. Folate deficiency 3. Transient erythroblastopenia of childhood (TEC) hildh d (TEC) 4. Aplastic Anemia 1 2 3 4
Teenager with low-grade fevers, malaise and splenomegaly 25% 25% 25% 25% 25% 25% 25% 25% 1. 1 Acute myelogenous leukemia Acute myelogenous leukemia 2. Infectious mononucleosis (EBV) 3. Systemic lupus erythematosus 4 4. Histiocytosis Histiocytosis 1 2 3 4
Teenager with fevers, recently back from a mission trip. 11.2 10.7 448 32.9 25% 25% 25% 25% 25% 25% 25% 25% MCV: 83 fL RDW: 15 % MCHC: 34 fL Retic: 2.3% 1. Sleeping sickness 2. Liver fluke infection 3. 3. Malaria Malaria 4. Chagas disease 1 2 3 4
School-age child with bruising and low- grade fever grade fever 8.3 28.4 28 4 52 52 25% 25% 25% 25% 25% 25% 25% 25% 24.7 MCV: 91 fL RDW: 13 % MCHC: 33.4 fL Retic: 1.7% 1. Acute myelogenous leukemia y g 2. Reactive left shift 3. ITP 4 4. Listeria infection Listeria infection 1 2 3 4
8 y/o girl with swollen cervical lymph node 25% 25% 25% 25% 25% 25% 25% 25% 1. Lymphoma 2. 2. Atypical bacterial infection Atypical bacterial infection 3. Mumps infection 4. An amazing shot of all the major normal WBC’s in the same field 1 2 3 4
Take home • The CBC and peripheral blood smear offer a wealth of information regarding g g pathophysiology. – Heme/onc diagnoses – Atopy, Rheumatology – Infectious disease “John always review the John, always review the – Others primary data yourself…” • There is benefit to reviewing blood Th i b fit t i i bl d smears yourself, especially in difficult or challenging cases or challenging cases. Or ask your friendly hematologist for help! Howard Weinstein, MD Chief, Pediatric Hematology/Oncology, MGH
jdorazio@uky.edu; 323 ‐ 6238
What the !@#$ are they talking about? • Polychromasia, RBC’s of different staining color, Polychromatophilia y p implies reticulocytosis RBC’s of different shape, mixed • Poikilocytosis population (old + transfused cells) RBC s of different size; RBC’s of different size; • Anisocytosis reticulocytosis, high RDW RBC fragments, microangiopathic • Schistocytosis processes (DIC), shearing • Elliptocytes, Ovalocytosis RBC’s look elliptical, membranopathy • Drepanocytosis • Drepanocytosis Sickle cell forms Hgb S disease Sickle cell forms, Hgb S disease • Spherocytosis No central pallor in rbc’s; HS or AIHA • Howell ‐ Jolly bodies Functional asplenia
Spherocytes p y Schistocytes y Acanthocytes y Elliptocytes Drepanocytes Echinocytes Poikilocytes Target cells Stomatocytes
Decreased Increased • Kostmann’s syndrome • Infections Neutrophils • Cyclic neutropenia • Tissue destruction y p • Bone marrow failure • Corticosteroids • Leukemia • Leukemoid reaction • Autoimmune neutropenia • GCSF administration • Benign neutropenia of childhood g p • Infection/sepsis • Drug ‐ induced (Bactrim, chemotherapy) • Myelodysplasia • Congenital immunodeficiency • Viral infection (e.g. EBV) Lymphocytes • Severe infection • Some fungal, parasitic infections • Drugs (Corticosteroids alkylating) • Rare bacterial infection (Pertussis) Drugs (Corticosteroids, alkylating) Rare bacterial infection (Pertussis) • GI disease • Allergic reactions/drug sensitivities • Acquired Immunodeficiency • Immunologic disease • Corticosteroids • Inflammatory responses Monocytes • Recovery phase of neutropenia • Myeloproliferative disorders • Bacterial infection • Parasitic infections Eosinophils • ACTH administration • Allergic conditions ACTH administration Allergic conditions • Drug therapy • Corticosteroids • Bone marrow failure Basophils • Myeloproliferative syndromes
Normal blood cells Normal blood cells band monocyte platelet l t l t lymphocyte neutrophil basophil eosinophil
Anemia ‐ a suggested approach • Does the child look anemic? • Does the child act anemic? • Key physical exam parameters: – Look at the conjunctiva under the lower eye lid – Splenomegaly? Tachycardia? Petechiae/purpurae? • The lab tests to order will be dictated by: – The degree of pallor, presence of symptoms The degree of pallor presence of symptoms – Physical findings (jaundice, HSM, adenopathy, etc) – Apparent pace of the anemia pp p – What diagnoses are being considered • Start out with a CBC, but always order a reticulocyte count too! – The retic count distinguishes between RBC destruction and underproduction
Red blood cells
Mean corpuscular hemoglobin (MCH) Mean corpuscular hemoglobin (MCH) • Average amount of Hgb inside each RBC g g • High MCH’s go along with large (macrocytic) RBC’s • Low MCH’s correlate with microcytosis and anemias caused by impaired Hgb synthesis. th i
Mean corpuscular hemoglobin concentration (MCHC) i (MCHC) • Average concentration of hemoglobin inside a red cell. • Takes red cell volume into account • Decreased MCHC values (hypochromia) seen in conditions where Hgb is abnormally diluted inside RBC’s • Iron deficiency anemia • Thalassemia • Increased MCHC values (hyperchromia) seen when Hgb is abnormally concentrated inside RBC’s concentrated inside RBC s • Spherocytosis • Abnormal Hgb (S, C) g ( , ) If MCHC If MCHC > 35 fL, think spherocytes! 35 fL thi k h t ! • Severe burns
crit (%) hematoc Normal hematologic values depend on age and gender. Therefore, there is no absolute value for “anemia” for all kids. Always check the age-appropriate cut-offs. But in general, suspect anemia for hgb < 10 g/dl or hct < 30%. Source: Vampire Handbook, Boston Children’s Hospital
1 y/o with pallor 1 y/o with pallor 8.2 25% 25% 25% 25% 25% 25% 25% 25% 8.2 8 2 351 351 23.9 MCV: 71 fL RDW: 18.4 % Retic: 2.6% 1. Lead poisoning 2 2. Malaria Malaria 3. Babesiosis 4. Hemoglobin C disease 1 2 3 4
Febrile infant with pancytopenia 25% 25% 25% 25% 25% 25% 25% 25% 1. Lysosomal storage disease 2. Histoplasmosis 3. Chronic Granulomatous Disease (CGD) 4 4. Pneumococcal sepsis P l i 1 2 3 4
12 y/o with fever, bruising and pallor. 12 y/o with fever, bruising and pallor. 25% 25% 25% 25% 25% 25% 25% 25% 1. Candidal infection 2. Histoplasmosis 3 3. Burkitt’s lymphoma/leukemia Burkitt s lymphoma/leukemia 1 2 3 4 4. Gaucher’s disease
Albino child with frequent infections. Albino child with frequent infections. 25% 25% 25% 25% 25% 25% 25% 25% 1. Abetaproteinemia 2. Tyrosinase neutropenia 3 3. Chronic granulomatous disease Chronic granulomatous disease 4. Chediak-Higashi syndrome 1 2 3 4
This is not a staining artifact! This is not a staining artifact! 25% 25% 25% 25% 25% 25% 25% 25% 1. Obstructive liver disease 1 Obstructive liver disease 2. Abetalipoproteinemia 3. Thalassemia 4. Severe burns 1 2 3 4
Common Sources of Error for Automated CBC machine Clotting Carboxyhemoglobin Cryoproteins Giant platelets Smudge cells Heparin Heparin Agglutination Nucleated RBCs Uremia Hyponatremia DIC DIC WBC count >50,000/ μ L Platelet clumping Hemolysis RBC inclusions RBC inclusions Medications Medications Excess EDTA Hyperbilirubinemia Lipemia p Hyperglycemia Infections
Anemia: not enough red cells. I Inadequate oxygen-carrying hemoglobin capacity d t i h l bi it
Howell jolly bodies Howell jolly bodies
Sickle cell disease Sickle cell disease
Elliptocytes Elliptocytes Elliptocytes (ovalocytes) : elongated RBC - Large number (up to 100%) is the hallmark of hereditary elliptocytosis hallmark of hereditary elliptocytosis - Low number (up to 5 - 10%) is observed in various situations, including iron deficiency and megaloblastic anemias - If they are very large = macro ovalocytes (see "macrocytes")
stomatocytes stomatocytes Stomatocytes : folded RBC leading to an aspect mimicking a mouth and its lips (slit- like appearance) like appearance) - Many situations, including hemolytic anemias, either constitutive or acquired
Cold agglutinin disease Cold agglutinin disease Cold agglutinin disease ; aggregates Cold agglutinin disease ; aggregates disappear after the sample is warmed at 37°C
echinocytes echinocytes Echinocytes or crenated or contracted Echinocytes or crenated or contracted cells : up to 50 protrusions (spines or spurs) may be observed - They correspond usually to an artifact (glass slides, old samples, saline solutions) - Excess in lipids (not diet fed samples) - Various congenital haemoglobin and enzymatic disorders enzymatic disorders - Acute renal failure
Rouleaux formation, myeloma Rouleaux formation, myeloma Rouleaux formation : RBC do not stick to Rouleaux formation : RBC do not stick to each other in normal conditions because their external membrane is negatively charged; if neutralization occurs, RBC stick face to face, leading to the so-called "rouleaux formation" - All inflammatory disorders (slide) - monoclonal gammopathies with excess of monoclonal gammopathies with excess of monoclonal immunoglobulin (does not occur in light chain myeloma)
Acanthocytes : crenation is limited (3 to 12 12 spines or spurs) i ) Hereditary acanthocytosis (abetalipoproteinemia) Liver diseases (cirrhosis) with dyslipidemia (c os s) t dys p de a As a part of artefact, mixed to echinocytes
Basophilic stippling Basophilic stippling Basophilic stippling : numerous thin and dark granules scattered throughout the RBC, related to abnormal hemoglobin synthesis th i - Thalassemic syndromes (including thalassemic trait) - Sideroblastic anemias (lead, drugs, ( , g , idiopathic) - Agnogenic myeloid metaplasia - Newborn (stippling is delicate)
babesia babesia
plasmodium plasmodium
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