KLUBSYBEAR TUTORIALS

Compiled by Irekevin Aytona

RMT2022-KLUBSYBEAR
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 RODAK’S 6TH EDITION
PTT REFERENCE RANGE 26 to 38 seconds
PTT THEURAPEUTIC RANGE 60 to 100 seconds
Experts once recommended a PTT therapeutic range of 1.5 to 2.5
times the mean of the reference interval
PT TYPICAL REFERENCE RANGE 12.6 to 14.6 seconds
TCT THROMBIN CLOTTING TIME 15 to 20 seconds
REFERENCE RANGE
REPTILASE TIME 10-15seconds
DUCKERT’S TEST /5 M UREA Stabilized clot is insoluble to urea for 24 hours
SOLUBILITY TEST
EUGLOBULIN LYSIS TIME Normal fibrinolysis = Lysis time is more than 1 or 2 hours
RELATIVE RETICULOCYTE COUNT 0.5 to 2.5 % Adults (Rodak’s)
0.5 to 1.5% Adults (Other Books)
ABSOLUTE RETICULOCYTE COUNT 20 to 115 x 109/L
CORRECTED RETICULOCYTE COUNT *Patients with a hematocrit of 35% should have an elevated
(CRC) corrected reticulocyte count of 2% to 3% to compensate for the mild
anemia
*Patients with a hematocrit of less than 25%, the count should
increase to 3% to 5% to compensate for the moderate anemia
RETICULOCYTE PRODUCTION INDEX >3 = Adequate bone marrow response
(RPI) <2= Inadequate erythropoietic response
RETICS COUNT USING MILLER DISK The disc is composed of two squares, the area of smaller square(B)
measuring 1/9 of the larger square

A minimum of 112 cells should be counted in the small square

OSMOTIC FRAGILITY TEST Initial hemolysis is in TUBE 21 or 22 (0.42-0.44%)
(Griffin and Sanford) Complete hemolysis is in TUBE 16 or 17 (0.32- 0.34%)
PLATELETS IN CIRCULATION 2/3 or 60-65%
PLATELETS IN SPLEEN 1/3 or 30-35%
NORMAL CENTRAL PALLOR OF RBC RBC with a central pallor that occupies the middle 1/3 of the cell
HYPOCHROMIC RBC with a central pallor of more than 1/3
SPHEROCYTE RBC WITH No or less than 1/3 central pallor
EXTRAVASCULAR HEMOLYSIS Normally constitutes to 90% of total RBC destruction
INTRAVASCULAR HEMOLYSIS Normally constitutes to ≤10% of total RBC destruction
10 to 20% (Rodak’s 5th edition)
MINIMUM BLOOD FOR ESR (Rodak’s) 2ml
LAP SCORE Sample reference interval for the LAP score is 15 to 170
NORMAL M:E RATIO 3:1 (2:1 or 4:1 second option)
ABOUT HEMOGLOBIN Hemoglobin = 4 HEME + TWO DIMERS OF GLOBIN CHAINS (4 globin
chains)

RBC = consists of 4 HEME + 4 globin chains

1 RBC = CAN CARRY 4 MOLECULES OF O2
1 HEME MOLECULE= contains 4 PYRROLE RINGS
1 HEME MOLECULE= contains 1 ferrous iron

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METHEMOGLOBIN LEVEL

Level Signs and Symptoms

Less than 25% Asymptomatic
More than 30% Cyanosis (bluish discoloration of skin and mucous membranes) and symptoms of hypoxia (dyspnea,
headache, vertigo, change in mental status) occur
More than 50% Coma and Death

WBC ESTIMATION FACTOR

TOTAL WHITE CELL COUNT ESTIMATION

Using 40x HPO Average number of WBC per field x 2000 2-5 WBC/ Hpf = 4000-7000 WBC/ul
using 50x OIO) Average number of WBC per field x 3000 4-6 WBC/ Hpf = 7000-10000 WBC/ul
6-10 WBC/ Hpf = 10000-13000 WBC/ul
10-20 WBC/ Hpf = 13000-18000 WBC/ul

Condition Absolute WBC count

Neutropenia <2.0 x 109/ L
Neutrophilia >7.0 x 109/ L (adults), >8.0 x 109/ L(children)
Eosinophilia >0.4 x 109/ L
Eosinopenia <0.09 x 109/ L
Basophilia >0.15 x 109/ L, commonly associated in CML
Monocytosis >1.0 x 109/ L (adults), >3.5 x 109/ L(neonates)
Frequently the first sign of recovery from acute infection or severe neutropenia
Monocytopenia <0.2 x 109/ L
Lymphocytosis >4.5 x 109/ L in adults, >10 x 109/ L in children
Lymphocytopenia <1.0 x 109/ L in adults, <2.0 x 109/ L in children

WBC DIFFERENTIAL COUNT FROM BARBARA BROWN

100 Differential count 50 Differential Count 200 Differential Count
Routinely used If patient WBC count is <1.0 x 109 /L If there are:
✓ Over 10 % eosinophil
-multiply results by 2 ✓ Over 2 % basophils
✓ Over 11% monocytes
✓ More lymphocytes than neutrophils (except
in children)

WBC DIFFERENTIAL COUNT FROM RODAK’S (6th Edition)

100 Differential Count 200 Differential Count 300-400 Differential Count
Routinely used When WBC count is higher than 40x109/L. When WBC count is ≥100x109 /L.

Normocytic RBC Normal RBC size (6-8um)

Normal MCV (80-100fl)
Microcytic RBC Small size (<6 um in size)
MCV= less than 80 femtoliter
Macrocytic RBC Larger size (>9 um in size)
MCV = greater than 100 femtoliter

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NEWBORNS/NEONATES/INFANTS

RBC COUNT Increases during the first 24 hours of life, remains at this plateau for about 2 weeks,
“polycythemia”
of the newborn and then slowly declines

WBC COUNT Leukocytosis is typical at birth for full-term and preterm infants.
Fluctuations in the number of WBCs are common at all ages but are greatest in infants
At birth, preterm infants exhibit a left shift, with promyelocytes and myelocytes commonly
observed

PLATELET Same with adults

COUNT The platelet count ranges from 150 to 400 x 109/L for full-term and preterm infants,
comparable to adult values

HEMOGLOBIN Reference interval for a full-term infant at birth is 16.5 to 21.5 g/dL
Abnormal value= less than 14 g/dl
Minimum /Lower limit= 14g/dl
At birth Hb F constitutes 53% to 95% of the total hemoglobin. Hb F declines from 90% to 95%
at 30 weeks’ gestation to approximately 7% at 12 weeks after birth and stabilizes at 2% to 3%
by6 months of age

HEMATOCRIT At birth of full-term infant = 48 to 68% (average of 61%)

The HCT usually increases approximately 5% during the first 48 postnatal hours; this is followed
by a slow linear decline to 46% to 62% at 2 weeks and 32% to 40% between the second and
fourth months

MCV OF Markedly macrocytic.

NEWBORNS
The average MCV for fullterm infants is 119 6 9.4 fL; however, a sharp decrease occurs
during the first 24 hours of life

MCH MCH is 30 to 42 pg in healthy neonates and 27 to 41 pg in premature infants

MCHC Average approximately 33 g/dL.

It is the same for full-term infants, premature infants, and adults

RDW Elevated in newborns

Reference range = 14.2% to 17.8% the first 30 days of life

LYMPHOCYTES Lymphocytes constitute about 30% of the leukocytes at birth and increase to 60% at 4 to 6
months. They decrease to 50% by 4 years, to 40% by 6 years, and to 30% by 8 years

MONOCYTES The mean monocyte count of neonates is higher than adult values. At birth the average
proportion of monocytes is 6%.

EOSINOPHIL The percentages of eosinophils and basophils remain consistent throughout infancy and
childhood.

NEUTROPHIL Full-term and premature infants have a greater absolute neutrophil count than that found in
older children.

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 Newborn girls have absolute neutrophil counts averaging 2000 cells/ml higher than those of
boys;
Neonates whose mothers have undergone labor have higher counts than neonates delivered by
cesarean section with no preceding maternal labor

BAND CELLS Band forms are also higher for the first 3 to 4 days after birth

Vitamin K At birth, the vitamin K-dependent coagulation factors (factors II, VII, IX, and X) are about
dependent 30% of adult values; they reach adult values after 2 to 6 months.
clotting factors

Contact Levels of factor XI, factor XII, prekallikrein, and highmolecular- weight kininogen are between
Factors 35% and 55% of adult values at birth, reaching adult values after 4 to 6 months

Fibrinogen, the levels of fibrinogen, factor VIII, and von Willebrand factor (VWF) are similar to adult
Factor VIII, values throughout childhood
and vwF

Physiologic The physiologic anticoagulants and inhibitors of coagulation (protein C, protein S, and
anticoagulants antithrombin) and a disintegrin and metalloprotease with a thrombospondin type 1
motif, member 13 (ADAMTS13) are reduced at about 30% to 40% at birth

Plasminogen Lower levels and decreased activity in neonates compared with adults

A2-antiplasmin Lower in neonates compared to adult levels

Plasminogen Tissue plasminogen activator (TPA) and plasminogen activator inhibitor-1 (PAI-1) levels are
activators increased
However, 5 times the amount of TPA is required to activate plasminogen compared with
activation of plasminogen in adults

PT and APTT Prolonged or longer

Bleeding and The risk of bleeding is not increased in a healthy newborn

thrombosis
The risk of thrombosis is considerably less in neonates and children than in adults.

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GERIATRIC/ELDERLY

AGE CATEGORIES Elderly adults can be roughly divided into three age categories:
CATEGORY AGE
Young-old 65 to 74
Old-old 74 to 84
Very old 85 and above
MARROW CELLULARITY Marrow cellularity begins at 80% to
100% in infancy and decreases to about 50% after 30 years, followed
by a decline to 30% after age 65.
RBC PARAMETERS Most RBC parameters (e.g., RBC count, indices, and RDW) for healthy elderly adults
do not show significant deviations from those for younger adults
HEMOGLOBIN There is a gradual decline in hemoglobin starting at middle age, with the mean level
decreasing by about 1 g/dL during the sixth through eighth decades

Men older than 60 years have average hemoglobin levels of 12.4 to 15.3 g/dL.
Elderly women have hemoglobin concentrations ranging from 11.7 to 13.8 g/dL.
Typically, the lowest hemoglobin levels are found in the oldest patients
WBC In the absence of any underlying pathologic condition, there are no statistically
significant differences between the total leukocyte count and WBC
differential for the young-old and old-old and those for middle-aged adults
LYMPHOCYTES Some investigators have reported a lower leukocyte count in elderly adults, owing
primarily to a decrease in the lymphocyte count
The number of naive T cells decreases in elderly adults
WBC FUNCTION Many neutrophil functions are decreased in elderly adults, including chemotaxis,
phagocytosis of microorganisms, and generation of superoxide
MONOCYTE The aging process does not significantly affect the number of monocytes
PLATELET COUNT The platelet count does not significantly change with age.
PLATELET ACTIVITY Platelets increase in activity with age
Decrease in bleeding time in elderly adults
Increase in markers of platelet activation such as b-thromboglobulin and
platelet factor 4 in the a-granules of platelets
Increased platelet phospholipid content

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HEMOSTASIS Age-related changes occur in the vascular and hemostatic systems, including
alterations in platelets, coagulation, and fibrinolytic factors. These changes contribute
to the increased incidence of thrombosis in elderly adults.
Overall, elderly adults demonstrate a shift of the hemostatic balance toward
increased coagulation and decreased fibrinolysis.
Elderly people are at risk to thrombosis
Clotting factors Fibrinogen, factors V, VII, VIII, IX, and XIII, VWF, highmolecular- weight kininogen,
and pre-kallikrein increase in healthy individuals as they age
Fibrinogen Fibrinogen, which has been implicated as a primary risk factor for thrombotic disorders,
including ischemic heart disease, increases approximately 10 mg/dL per decade
in elderly adults (65 to 79 years), from 280 mg/dL to more than 300 mg/dL
Fibrinolytic system Fibrinolysis is impaired in elderly adults, especially as a result of an increase in PAI-1
D-dimer D-dimer levels trend upward in adults 50 years and older.

HEMOGLOBIN LEVEL IN ANEMIA

Mild anemia 9 to 11 g/dl
-Usually more than 10g/dl but less than the normal value of hemoglobin in adults
Moderate anemia 7 to 10g/dl
-May cause pallor of conjunctivae and nail beds but may not produce clinical symptoms if the onset
of anemia is slow. However, depending on the patient’s age and cardiovascular state, symptoms
such as dyspnea, vertigo, headache, muscle weakness, and lethargy can occur
Severe anemia Less than 7 g/dl
-Usually produce tachycardia, hypotension, and other symptoms of volume loss, in addition to the
symptoms of mild and moderate anemia. Thus, severity of the anemia is gauged by the
degree of reduction in hemoglobin, cardiopulmonary adaptation, and the rapidity of
progression of the anemia
Anemia in newborns Less than 14g/dl

Numerical Description
Scale
0 Normal appearance or slight variation in erythrocytes.
1+ Only a small population of erythrocytes displays a particular abnormality; the terms slightly
increased or few would be comparable.
2+ More than occasional numbers of abnormal erythrocytes can be seen in a microscopic field; an
equivalent descriptive term is moderately increased.
3+ Severe increase in abnormal erythrocytes in each microscopic field; an equivalent descriptive term
is many.
4+ The most severe state of erythrocytic abnormality, with the abnormality prevalent throughout each
microscopic field; comparable terms are marked or marked increase

HEMOGLOBINOPATHIES
HB S HB C Hb A1 Hb A2 Hb F
Sickle cell disease 80% - - variable 20%
Sickle cell trait 40% - 60% normal Normal
HB SC disease 50% 50% - - -
HB CC/disease - 90% 2% 7%
Hb C Trait - 40% 60% - -

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THOMAS PLOT INTERPRETATION
True IDA sTfR will rise and the ferritin will drop so that the sTfR/log
ferritin will be high and the hemoglobin content of
reticulocytes will be low
Plot: Lower right quadrant
Functional IDA Ferritin may be falsely elevated by inflammation, the
sTfR/log ferritin will be normal despite reduced availability
of iron for hemoglobin production resulting in a low
hemoglobin content in reticulocytes
Plot: Lower left quadrant
Latent IDA As iron deficiency develops, other cells are starved for iron
before erythrocytes; production of hemoglobin in
reticulocytes remains at a normal level for as long as
possible. However, the body’s other iron-starved cells will
increase sTfR production and systemic iron stores of ferritin
will be depleted, thus elevating the sTfR/log ferritin value.
Plot: Upper right quadrant

CAPILLARY FRAGILITY/TORNIQUE TEST

Grade Description Numbers of petechiae

1+ Few petechiae on the anterior part of the forearm 0-10

2+ Many petechiae on the anterior part of the forearm 10-20

3+ Multiple petechiae over the whole arm and back of the hand 20-50

4+ Confluent petechiae on the arm and back of the hand 50 and above

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PLATELET ESTIMATION FACTOR
1 platelet per 10-40 RBCs
3-10 platelets per 100 RBCs (or in 1 OIF)
5-20 platelets per 200 RBCs
A normal blood smear should demonstrate approximately 8 to 20 platelets per field

SMEAR: Platelet estimate factor (x20,000), counted on 10 OIF

Platelet estimate Report platelet as

0-49 000 / Ul Marked decreased
50,000-99,000 / ul Moderate decreased
100,000-149,000 / ul Slight decreased
150,000-199,000 / ul Low normal
200,000-400,000 /ul Normal
401,000-599,000 /ul Slight increased
600,000-800,00 /ul Moderate increased
Above 800,000 /ul Marked increased

Platelet counted Dilution factor

If fewer than 50 platelets counted on each side of hemacytometer repeat procedure and dilute blood to 1:20
If more than 500platelets counted on each side of hemacytometer repeat procedure and dilute blood to 1:200

STANDARD HEMOGLOBIN CURVE

X-axis / Abscissa Hemoglobin value (g/dl)
Y-axis / Ordinate Optical density (absorbance)

MANUAL CELL COUNTS WITH MOST COMMON DILUTIONS, AND COUNTING AREA
Cells counted Diluting fluid Dilution Objective Area Counted
WBC 1% ammonium oxalate 1:20 (standard) 10x (LPO) 4mm2
3% acetic acid 1:100 10x (LPO) 9mm2
1%HCL
RBC Isotonic saline / NSS 1:100 40x(HPO) 0.2mm2
(5small squares of center square)
Platelets 1% ammonium oxalate 1:100 40x (HPO) 1mm2

OSMOTIC FRAGILITY TEST NORMAL VALUES (GRIFFIN AND SANFORD METHOD)

Initial hemolysis TUBE 21 or 22 (0.42-0.44%)
Complete hemolysis Tube 16 or 17 (0.32- 0.34%)

BLEEDING TIME
Method Reference value

Duke method-skin puncture 2 to 4 minutes

Ivy method – uses BP cuff inflated at 40mmHg 3 to 6 minutes

Modified template method 6 to 9 minutes

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PT INR VALUES
INR CONDITIONS

2.0-2.5 DVT, high risk surgery

2.0-3.0 Hip surgery, femur fracture

2.0-3.0 DVT, Pulmonary embolism, transient ischemic attack

2.5-3.5 Mechanical/ prosthetic heart valves

2.0-4.5 Recurrent DVT and pulmonary embolism, myocardial infarction, arterial disease

Types of Progenitors Mature Cell/s Developed

G-M (Granulocyte-Monocyte) Progenitor Basophil, Neutrophil, Eosinophil, Monocyte, Macrophage
E-B (Eosinophil-Basophil) Progenitor Basophil, and Eosinophil
M-E (Megakaryocyte-Erythrocyte) Progenitor Platelets, and RBCs
CMP (Common Myeloid Progenitor) Granulocytes, Erythrocytes, Monocytes/Macrophages, and Megakaryocytes
CLP (Common Lymphoid Progenitor) Lymphocytes (B cell, T cell, and NK cell), Plasma cells, and Dendritic cells

RMT2022-KLUBSYBEAR
 FORMULA IN HEMATOLOGY
CELL COUNT

CORRECTED WBC
if nRBC is ≥5

ABSOLUTE COUNT

RULE OF THREE

%RETIC COUNT
(RELATIVE Retics (%) = __number of Retics x 100_
COUNT) 1000 RBCs observed

RETIC COUNT
USING MILLER
DISK

ABSOLUTE RETIC
COUNT

CORRECTED RETIC
COUNT

RPI

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 HEMATOCRIT MATURATION OF RETICS IN BLOOD
40-45 % 1.0 Day
35-39 % 1.5 Days
25-34% 2.0 Days
15-24% 2.5 Days
<15% 3 Days
INR

Sodium Citrate
Blood Collection 100-Hct x ml of Whole blood
Anticoagulant 595-Hct
Adjustment for
Hematocrit >55%

RDW Computed by dividing the SD by the mean of the RBC size distribution

Formula = (SD / Mean) x 100

AUTOMATED For Coulter, Abbott, and Siemens= Hematocrit is computed

HEMATOCRIT
RESULT Formula = (RBC X MCV) / 10

For Sysmex = Hematocrit is detected using cumulative RBC pulse height

MEAN CELL MCV =

𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡(%)
𝑥10
VOLUME 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡

MEAN CELL MCH=

𝐻𝐸𝑀𝑂𝐺𝐿𝑂𝐵𝐼𝑁(𝑔/𝑑𝑙)
𝑥10
HEMOGLOBIN 𝑅𝐵𝐶 𝐶𝑂𝑈𝑁𝑇
MEAN CELL MCHC =
𝐻𝐸𝑀𝑂𝐺𝐿𝑂𝐵𝐼𝑁(𝑔/𝑑𝑙)
𝑥100
HEMOGLOBIN 𝐻𝐸𝑀𝐴𝑇𝑂𝐶𝑅𝐼𝑇 (%)
CONCENTRATION
MUST KNOW ABOUT VENIPUNCTURE

Color coded needle ♥ (18gauge: Pink), (21gauge: green), (22gauge: black), (23gauge: blue/torquise)

♥ The larger the gauge the smaller the internal bore diameter (vice versa)

Routinely used gauge of ♥ 19,20, 21 gauge

needles
♥ 20,21,22 gauge (Brown)

♥ IN INSERTING OF NEEDLE, THE BEVEL SHOULD BE UPWARD

♥ THE COMMONLY/STANDARD USED NEEDLE: 21g (Rodak’s and Turgeon)

Length of needle ♥ Standard = 1inch (preferred)

♥ Range = 1 to 1.5inches
The advantage of using a 1-inch needle is that it provides better control during
venipuncture.

Venipuncture angle ♥ Standard=15-degree angle (preferred)

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 ♥ Range =15 to 30-degree angle

Torniquet application ♥ Should not exceed 1 minute/60 seconds,

2mins (If walang 1minute -Turgeon)

♥ 3 to 4 inches (7.5 cm to 10cm) above venipuncture site

Blood pressure cuff as ♥ Standard=60mmHg

tourniquet
♥ Range=40 to 60mmHg

Number of attempts Twice then call another Medtech

Position of the patient ♥ Lying down – hemodilution: ↓ Packed cell Volume by 8 %, ↓ WBC

♥ Standing/Up- hemoconcentration: ↑ Packed Cell Volume 8%, ↑ WBC

Waste disposal (DOH) ♥ Yellow bag = Infectious waste

♥ Orange bag = Radioactive waste

♥ Red bag = Sharp waste

♥ Yellow with black band = Chemical waste

♥ Green bag = Non-infectious Wet waste or Biodegradable waste

♥ Black bag = Non-infectious Dry waste or non-biodegradable waste

INTERPRETATION OF SOME LAB RESULTS

SOLUBILITY
TEST FOR HB S Negative absence of turbidity and lines are visible
Positive presence of turbidity and lines are not visible

AUTOHEMOLYSIS G6PD deficiency = corrected upon addition of glucose

Pyruvate Kinase deficiency = corrected upon addition of ADP
Hereditary spherocytosis= corrected upon addition of glucose and ADP

KLIEHAUER BETKE
TEST (ACID ELUTION Positive Fetal RBC (with Hb F)
Result =RBCs are deeply stained red and resist acid denaturation
TEST)
Negative Maternal RBC (Hb A)
result =RBC appear as pale pink ghost cells and susceptible to acid denaturation

Donath Landsteiner Test

for PCH There is hemolysis to both tubes incubated at cold and 37’C

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KAPLOW COUNT /LAP SCORE (TURGEON)
CELL RATING AMOUNT (%) SIZE OF GRANULES STAIN INTENSITY
0 none - None
1+ 50 small Faint to moderate
2+ 50 t0 80 small Moderate to strong
3+ 80 to 100 Medium to large Strong
4+ 100 Medium to large Brilliant

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RAI AND BINET STAGING FOR CLL

RAI BINET
Low Risk (formerly Stage 0) Stage A
Hemoglobin ≥ 10 g/dL
Lymphocytosis >5 x 109/L platelets ≥ 100 x 109/L
<3 enlarged nodal areas
Intermediate Risk Stage B
(Formerly Stages I and II) Hemoglobin ≥ 10 g/dL
platelets ≥ 100 x 109/L
Lymphocytosis >5 x 109/L ≥3 enlarged nodal areas
(lymphadenopathy + splenomegaly) or hepatomegaly, or both
High Risk (formerly Stages III and IV) Stage C
Hemoglobin <10 g/dL
Lymphocytosis >5 x 109/L Platelets <100 x 109/L
hemoglobin <11 g/dL Any number of enlarged nodal areas

LABORATORY RESULTS OF DIFFERENT POLYCYTHEMIA

TYPE RBC MASS/COUNT HCT EPO LEVEL
Relative polycythemia Normal Increased NORMAL
Primary absolute (PV) Increased Increased DECREASED
Secondary absolute Increased Increased INCREASED
Genetic polycythemia Increased Increased INCREASED

TRADITIONAL FAB COOPERATIVE GROUP CLASSIFICATION OF MDSs

MDS Blasts in Blasts in BM Ringed Peripheral blood
peripheral blood (%) sideroblasts Monocytes
(%) (%)
Refractory anemia (RA) <1% <5% <15 No
Refractory with ringed sideroblasts (RARS) <1% <5% >15 No
Refractory anemia with excess blast (RAEB) >5% 5-20% No No
Refractory anemia with excess blast in <5% 20-30% No No
transformation (RAEB-T)
Chronic myelomonocytic leukemia (CMML) <5% <20% No >1000

Don’t worry about anything; instead, pray about everything. Tell God what you need, and thank him for all
he has done. Then you will experience God’s peace, which exceeds anything we can understand. –
PHILIPPIANS 4:6

R.M.T BATCH 2022

RMT2022-KLUBSYBEAR