Diagnosing anemia:

LABORATORY DIAGNOSIS
OF ANEMIA

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How to diagnose anemia?

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Erythropoiesis
(Kinetics)

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Structure dictates function
Bi-concave disk
Flexible
Maximum
surface/volume ratio
Size: uniform

Water: 65%
Hemoglobin: 35%

Absence of:
Nucleus
Cell organelles

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 CLASSIFICATION OF ANEMIA
BASIS CLASSIFICATION OF ANEMIA

SEVERITY Mild; Moderate; Severe;

Very severe

RATE OF Acute vs Chronic

PROGRESSION

BM Hypercellular vs
CELLULARITY Hypocellular 5
 CLASSIFICATION
BASIS OF ANEMIA
CLASSIFICATION OF ANEMIA

ETIOLOGY Impaired production

Increase destruction
Acute Blood loss

MORPHOLOGIC Microcytic Hypochromic

/ CYTOMETRIC Macrocytic
Normocytic Normochromic

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PATHOPHYSIOLOGIC
CLASSIFICATION OF
ANEMIA
1. Impaired production
2. Increased destruction (Hemolytic)
3. Blood loss

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IMPAIRED PRODUCTION
(1) SC PROLIFERATION/
DIFFERENTIATION
APLASTIC ANEMIA
PURE RED CELL APLASIA
ENDOCRINE DEF (PITUITARY, THYROID, ADRENAL,
TESTIS)

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IMPAIRED PRODN PROLIF/ DIFF
(2) RBC-BLAST (2) RBC-BLAST
DELAYED MATURATION MULTIPLE MECHANISMS
MEGALOBLASTIC A ACD (INFLAMMATORY,
(DNA SYNTHESIS) INFECTIOUS,
VIT. B12 DEFICIENCY NEOPLASTIC)
FOLIC ACID DEFICIENCY RDA
HYPOCHROMIC A (Hgb MYELOPHTHISIC A. (BM
SYNTHESIS) INFILTRATION)
HEME: IDA;
SIDEROBLASTIC REFRACTORY ANEMIA
ANEMIA W/ CELLULAR BM
GLOBIN: PROTEIN
THALASSEMIAS MALNUTRITION
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Anemia Pathogenesis

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Erythropoiesis:
Nutrients
Metals:
Iron
Cobalt
Manganese

Other Nutrients:
Vitamins
Vit B 6, 9, 12
Vit C, E
Amino acids
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Erythropoiesis:
Hormones
Hormones:
EPO

Other Hormones: Target Hgb: 11-12 g/dl

EPO dose: 100-150 u /
kg / wk
For EPO to be effective:
Ferritin >100 ng/ml
Fe sat >/=20% 13
Causes of Anemia of Renal Failure
Decrease
EPO *
RBCpoiesis due to toxic effects of uremia to BM
Fe absorption
RBC survival (esp in hemodialysis)
Blood loss due to
Capillary fragility & poor platelet fxn
Stress ulceration leading to chronic blood loss

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Complications of
ERYTHROPOIETIN
Accelerated HTN
Bone aches
Skin rashes
Urticaria
IDA secondary to increased erythropoiesis

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HEMATOPOIESIS
Synchronous vs Asynchronous

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CONCEPT

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Maturation Defects
Cytoplasmic (Hgb): Nuclear (DNA)

Heme iron def: IDA B12 def

PP def: Sideroblastic a Folate def
Globin def: B12 def r/o in folate def
Thalassemias via supplemental folate
improves anemia of
B12 def but NOT
neurologic sequelae

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CBC RBCI PBS IRON HEMOLYSIS
STUDIES
RBC MCV RBC Serum TB B1:B2
Hgb MCH (Morphology) Fe Urobilinogen
Hct MCHC (Urine / Stool)
WBC WBC (#, Serum Haptoglobin
Diff Morphology) Ferritin Hemopexin
Platelet Methemalbumin
MPV Platelets (#, TIBC Hemoglobinuria
RDW Morphology) Hemosidenuria
Special tests
OTHERS Retic ct/ BMA & BMB
RPI
E-
phoresis 21
How to diagnose anemia?

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How to diagnose anemia?

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MORPHOLOGIC
CLASSIFICATION of
ANEMIAS

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MICROCYTIC HYPOCHROMIC
(MCV < 80/ MCH < 27)
IDA
SIDEROBLASTIC A
THALASSEMIA
CHRONIC DSE A (25%)
LEAD POISONING
UNSTABLE HGB

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MACROCYTIC (MCV > 100)
MEGALOBLASTIC BM NORMOBLASTIC BM

VIT B12 DEF RETICULOCYTOSIS

FOLIC ACID DEF LIVER DSE/ OBSTRUCTIVE
JAUNDICE
THIAMINE DEF POSTSPLEENECTOMY
HYPOTHYROIDISM
APLASTIC A
MYELOPROLIFERATIVE DSE
ALCOHOL ABUSE
DRUGS

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NORMOCYTIC NORMOCHROMIC
RPI > 3 RPI < 3
CHRONIC DSE A (75%)
HEMOLYSIS MILD IRON DEF
SIDEROBLASTIC A
ACUTE BLOOD LOSS REFRACTORY A
(NONSIDEROBLASTIC)
RENAL DSE A
MYELOPHTHISIC A
ENDOCRINE DYSFUNCTION
A
HYPOPLASTIC/ APLASTIC A
HEPATIC DISEASE A
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OVERLAPS
MICROCYTIC HYPOCHROMIC NN w/ RPI < 3

IDA MILD IRON DEF

SIDEROBLASTIC A SIDEROBLASTIC A
ACD (25%) ACD (75%)
THALASSEMIA REFRACTORY A
Pb POISONING (NONSIDEROBLASTIC)
UNSTABLE HGB RENAL DSE A
MYELOPHTHISIC A
ENDOCRINE DYSFUNCTION A
HYPOPLASTIC/ APLASTIC A
HEPATIC DISEASE A

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Red Cell Distribution Width
Estimate of RBC
anisocytosis (COV of
individual RBC vol) Reference value: 12-15%
Use: differentiation of
microcytic hypochromic
anemias
Inc in:
IDA of chronic blood
loss
Dialysis patients
Sideroblastic a
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RDW correlated w/ MCV vs PBS
Normal RDW w/ Increase
MCV
Normal RDW w/ Decrease
MCV
Normal RDW w/ Normal
MCV
Increased RDW w/
Increase MCV
Increased RDW w/
Decrease MCV
Increased RDW w/ Normal
MCV
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Classification of Common Causes of
Anemia Based on MCV and RDW

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CBC RBCI PBS IRON HEMOLYSIS
STUDIES
RBC MCV RBC Serum TB B1:B2
Hgb MCH (Morphology) Fe Urobilinogen
Hct MCHC (Urine / Stool)
WBC WBC (#, Serum Haptoglobin
Diff Morphology) Ferritin Hemopexin
Platelet Methemalbumin
MPV Platelets (#, TIBC Hemoglobinuria
RDW Morphology) Hemosidenuria
Special tests
OTHERS Retic ct/ BMA & BMB
RPI
E-
phoresis 36
37
Pre analytics

Causes of spurious
decrease of RBC ct:
Clotting
In-vitro hemolysis

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Case 1: Patient w/ Leucocytosis
&/or Hyperlipidemia
Patie ts o ditio :
WBC > 50,000/uL
Lipemia

Machine result: Spurious Increase:

RBC ct
Hb & Hct
MCH & MCV

How would you resolve the problem?

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Case 2: Patient dx May- Hegglin
Anomaly
Patient condition: May- Hegglin Anomaly

Machine results:
Inc RBC ct
Dec Platelet ct

How would you resolve the problem?

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Case 2: Patient w/ May- Hegglin
Anomaly PBS

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Case 3: Diagnosed patient dx IM
or M. pneumonia
Patient condition: IM or M. pneumonia

Machine results: Spurious:

Decrease RBC
Increase MCV
Increase MCHC
Increase Platelet ct

How would you resolve the problem?

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Case 3: Diagnosed patient w/ IM or
M. pneumonia PBS

Decrease RBC
Macrocytosis
Inc MCV
Inc MCHC
Inc Platelet ct (precipitated cryoglobulin)
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Troubleshoot Cold Agglutinins

Warm sple (37 C heating block & mix) Test sample

while warm
Redrawn in a pre-warmed tube & kept at body T

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 Types of Cryoglobulins
Type RF Monoclonality Associated Dse
(+)
I No Yes (IgG or IgM) MM;
Waldenstroms
Macroglobulinemia
II Yes Yes (Polyclonal IgG Hepatitis C;
Monoclonal IgM Sjogrens Synd; SLE
III Yes No (Polyclonal IgG & Hepatitis C;
IgM) Sjogrens Synd; SLE
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Case 4: Neutropenia in patient w/
malignancy
Patient condition: Cancer w/ neutropenia

Machine results: Spurious:

Decrease PMN / WBC ct

How would you resolve the problem?

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Case 4: Pseudoneutropenia

Seen in malignancies
IgM causes the clumping

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Case 5: Diabetic patient
Patie ts o ditio : Hypergly e ia > 400 g/dl &
hyperosmolality

Machine results: Spurious:

Increase MCV
Decrease MCHC

How would you resolve the problem?

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Case 6: Patient w/ MAHA
Patient condition: MAHA

Machine results: Spurious:

Decrease RBC ct
Inc Platelet ct

How would you resolve the problem?

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Case 6: Patient w/ MAHA PBS

Fragmented RBCs
Severely microcytic
WBC cytoplasmic fragments

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Case 7: Patient w/ Leukemia
Patient condition: Leukemic patient particularly CLL

Machine result: Dec WBC ct

How would you resolve the problem?

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Case 7: Patient w/ Leukemia PBS

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Case 8: Specimen collected in
EDTA
Patient condition: Patient on routine check up

Machine results: Decrease Platelet ct

How would you resolve the problem?

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Case 8: Specimen collected in EDTA
PBS

Platelet clumps/ satellitosis False dec PC

Incipient clotting False dec PC

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Platelet Count / MPV

In EDTA- induced thrombocytopenia - 0.1 to 2% use

citrate

7 - 25 platelets / OIF

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Case 9: Patient w/ Hemolytic
Anemia
Patient condition: Hemolytic anemia patient

Machine results: Inc WBC ct

How would you resolve the problem?

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Case 9: Patient w/ Hemolytic
Anemia PBS

NRBCs = False inc WBC ct

WBC c= WBC ob x 100 / 100 + NRBC (>5)

Per 100 WBC diff count

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Autoa alyzers ca t read
poikilocytes

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Physiologic variations

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Physiologic Variations Hb, Hct& RBC

Male > Female

< in older men, lesser degree in older women or may
even rise slightly
Caucasians / Americans > Asians

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Physiologic Variations Hb, Hct& RBC

Inc upon standing from Highest in AM, lowest at

recumbent position or night
w/ increased muscle
activity

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Physiologic Variations Hb, Hct& RBC

Inc w/ high altitude & smokers

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Physiologic Variations Hb, Hct& RBC
Overhydration- Decrease

Dehydration- Increase

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Physiological Variations
WBC level:

Highest at PM, lowest at

AM at rest
Increased w/ exercise
Black people lower PMN
ct
Increases w/ smoking 67
Aplastic Anemia

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 Causes: Congenital (20%) 1
Fanconi anemia
Dyskeratosis congenital
Cartilage-hair hypoplasia
Pearson syndrome
Amegakaryocytic
thrombocytopenia
Shwachman-Diamond synd
Dubowitz syndrome
Diamond-Blackfan synd
Familial aplastic anemia 69
 Causes: Acquired (80%) 2
Idiopathic factors
Infectious (hepatitis viruses, EBV,
HIV, parvovirus, mycobacteria)
Transfusional GVHD
Orthotopic liver transplantation
for fulminant hepatitis
Pregnancy
Eosinophilic fasciitis
PNH
MDS 70
Causes: Acquired (80%) 3
Toxic exposure to
radiation & chemicals
Insecticides
Benzene
CCl4
Drugs
6 mecaptopurine
Methotrexate
Cyclophosphamide
Chloramphenicol
NSAIDS
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 CAUSES
Aplastic
anemia DAMAGE TO STEM CELL

DEPRESSION OR CESSATION OF ACTIVITY OF

REPEATED
ALL BLOOD PRODUCING ELEMENT
INFECTION;
FREQUENT
SICK DAY

LEUKOPENIA
BLEEDING THROMBOCYTOPENIA
TENDENCY AEB
ECCHYMOSIS,
DECREASE IN FORMATION OF RBC
PURPURA,
PETICHIAE,
BLEEDING FROM NOSE,
MOUTH, PALLOR OF SKIN & MUCOUS
VAGINA, RECTUM MEMBRANE, CYANOSIS
PANCYTOPENIA

APLASTIC ANEMIA
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 Stem cell damage
Leukemia Drugs, Immune
Viral Infections.

Clinical Features:
Anemia, Infections & Bleeding
1. RBC - Anemia,
Normocytic Pancytopenia 2. WBC - Leukopenia
3. PLT - Thrombocytopenia
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Fa co is A e ia:
Acquired Monosomy 7

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Fa co is A:
Clinical Features
(-) HSM & (-) LAD
(presence suggest
underlying leukemia)

Special features:

Small stature
Microcephaly
Micro-ophthalmia
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 Hyperplastic gingivitis
Cleft lip
Leukoplakia

Hyperpigmentation (caf au-lait spots,

erythematous rash) 76
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Treatment

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IMPAIRED PRODUCTION
(2) RBC-BLAST PROLIF / DIFF
MEGALOBLASTIC ANEMIA (DNA SYNTHESIS)
VIT. B12 DEFICIENCY
FOLIC ACID DEFICIENCY
HYPOCHROMIC ANEMIA (HEMOGLOBIN
SYNTHESIS)
HEME: IDA; SIDEROBLASTIC ANEMIA
GLOBIN: THALASSEMIAS
MULTIPLE MECHANISMS
ACD ; RDA ; MYELOPHTHISIC ANEMIA ; RA W/ CELLULAR
BM ; PROTEIN MALNUTRITION

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Megaloblastic Anemia
Impaired DNA synthesis (Asynchronous
Hematopoiesis)
B12 = co-factor
Folate = transfer single carbon groups
Cells affected: Blood cells, GI epithelial cells

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Causes of B12 Def

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Causes of B12 deficiency
Vegetatrian diet Terminal ileal disease:
B12 malabsorption Sprue
Gastric achlorydia Enteritis
Partial / total Resection
gastrectomy Tumors
Drugs that block acid Competition of
secretion cobalamin:
Pernicious anemia Fish tapewor , li d
loop sy dro e

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2. B12
Malabsorption:
Gastric achlorydia
Partial / total
gastrectomy
Drugs that block acid
secretion
Pernicious anemia

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2. B12
Malabsorption:
Terminal ileal disease:
Sprue
Enteritis
Resection
Tumors
Competition of
cobalamin:
Fish tapewor , li d
loop sy dro e

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Causes of B9 Def

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 Causes of Folic acid Def
Inadequate intake: unbalanced diet
Alcoholics Chronic diseases
Teenagers Old agers
Some infants
Increased requirements
Pregnancy Infancy
Malignancy Impaired metabolism
Chronic hemolytic anemias
Chronic exfoliative skin disorders
Hemodialysis
Malabsorption (Tropical sprue, Gluten-sensitive
enteropathy)
Drugs (Methotrexate, oral contraceptives) 92
OTHER Causes of
MEGALOBLASTIC
ANEMIA

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Other causes of Megaloblastic A
Drugs that impair DNA metabolism
Purine antagonists: 6 mercaptopurine, azthioprine
Pyrimidine antagonists: 5FU, cytosine arabinoside,
others
Others: procarbazine, hydroxyurea, zidovudine
Metabolic disorders (rare)
Hereditary orotic aciduria
Lesch Nyhan syndrome
Megaloblastic anemia of unknown etiology
Refractory megaloblastic anemia
DiGugliel os sy dro e
Congenital dyserythropoietic anemia
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B12 versus FA Deficiency
Similarities &
Differences

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 B12 DEF B9 DEF
SOURCE / DAILY Animal Green leafy veggies,
REQUIREMENTS products liver, yeast
Unaffected Destroyed by
by cooking cooking
Need 1-2 ug Need 100-200 ug
CELL TURNOVER DECREASE DECREASE
GASTRIC SX DECREASE DECREASE
PANCREATIC DECREASE NO EFFECT
INSUFFICIENCY
NEUROLOGIC PRESENT ABSENT
SYMPTOMS 96
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Laboratory Dx:
Megaloblastic A

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(1) CBC
NEUTROPENIA

THROMBOCYTOPENIA
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Macrocytic A: Megaloblastic A.
(2) PBS:
MACROOVALOCYTES
B. STIPPLINGS
HJ BODIES
MACROPOLYCYTES
(HYPERSEG. PMN)

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Macrocytic A: Megaloblastic A.
(3)BM
DYSPLASTIC CHANGES
MEGALOBLASTS
GIANT
METAMYELOCYTES

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Macrocytic A: Megaloblastic A.
(4) OTHERS
INC SERUM BILIRUBIN
Inc LDH
INC. SERUM Fe; DEC. TIBC

INEFFECTIVE ERYTHROPOIESIS

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LAB DX B12 DEF B9 DEF
URINARY / INCREASE NORMAL
SERUM MMA
URINARY FIGLU NORMAL INCREASE
SERUM INCREASE INCREASE
HOMOCYSTEIN
GASTRIC HISTAMINE FAST NONE
ANALYSIS ACHLORYDRIA
SEROLOGIC IF Ab test NONE
Parietal cell Ab
test
SCHILLINGS TEST POSITIVE NONE 107
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SCHILLING TEST
Step 1: Non RB12 IM
(binds transcobalamin &
absorbed)
Step 2: RB12 p.o.
Prevents reabsorption of
RB12 due to
unavailability of Step 3: 24h urine to test
transcobalamin for % RB12 reabsorbed
RB12 Forced to be
excreted into the urine
Result: No RB12 in 24h
urine confirms BI2 def

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 Anti-IF antibodies Dietary deficiency

No
Yes
Corrected w/
Schilling test B12 Diet
abnormal

Corrected w/ Corrected w/ Corrected

IF + Oral Post- AB intake w/
RB12 Pancreatic
extract +
Oral RB12
Bacterial overgrowth
P.A.
Chronic pancreatitis
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