This document discusses the process of decalcification for bone specimens. It begins by introducing physiological and pathological calcification. It then describes the stages of decalcification including selection of tissue, fixation, decalcification using various acids, neutralization of acid, and thorough washing. Various decalcifying agents and their properties are compared. Factors that influence the decalcification rate and methods to determine the endpoint of decalcification are also outlined.
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Decalcification: Unveiling Structures Beneath the Mineral Veil
What is it? Decalcification removes calcium salts from tissues like bone and teeth, making them soft and sliceable for microscopic analysis.
Why do it? Hardened tissues can't be sectioned effectively and interfere with staining. Decalcification allows clear visualization of cellular and structural details.
How is it done? Different methods exist, like using weak acids or chelating agents, each with its pros and cons. The choice depends on tissue type, processing time, and desired preservation level.
Knowing when to stop: Monitoring techniques like X-rays or physical assessment help determine the optimal endpoint to avoid over-decalcification and tissue damage.
Beyond bone: Decalcification finds applications in diverse fields like paleontology, pathology, and cancer research.
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New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
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The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
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Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
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Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
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7. SELECTION OFTISSUE
Bone
Calcified tissue
Sawing
Remove soft tissues & dense connective
tissue
3 – 5 mm thick bone slabs
Fixed 24 – 48 hours
Wash bone debris/dust in running tap water/ wet paper
towel
8. FIXATION
Bone pieces cut into 2 – 5 mm
thickness
Complete fixation protects bone from
acid
4 -5 times more damage in unfixed
tissues
Fixative + Decalcification
Bouin’s decalcifying solution
Formic acid - formalin
Fixative
Routine: Neutral buffered formalin
Bone marrow: Zenker formol
For faster fixation
- Reduced size of
bone
- Bone is opened
- Remove soft tissue
9. DECALCIFICATION
Removal of calcium from tissue for
processing
Even after decalcification, the dense
collagen of bone becomes hard after
processing
On H & E staining
Calcified foci appear cracked
Dark purple granular masses
Light purple halos
Choice of decalcifier
Urgency of the case
Degree of mineralisation
Extent of the investigation
Staining techniques required
10. CRITERIA OF GOOD DECALCIFYING AGENT
Completely removes calcium from tissue
Does not damage tissue cells or fibers
Does not impair the subsequent staining techniques
Reasonable fast decalcification
12. STRONG ACID DECALCIFIERS
Solution Aqueous HCl / HNO3 Formal HNO3 Perenyi’s fluid
Prep Conc. HCl / HNO3 – 10 ml
DistilledWater – 90 ml
Formalin – 5 ml
HNO3 – 7.5 to 15 ml
Distilled water – 100 ml
10% HNO3 – 40 ml
Absolute alcohol – 30 ml
0.5% chromic acid – 30 ml
Time
Taken
12 – 24 hrs 1 – 2 days 2 – 4 days (ribs)
10 – 14 days (5 mm femur)
+
Rapid decalcification
Little damage
Allows most staining methods
Rapid decalcification
Inhibits maceration by HNO3
Discoloration prevented by
Urea
Excellent agent for small deposits &
cytological preparations
-
Tissue swelling
Interferes with staining if >48 hrs
IHC antigen damage
Yellow discoloration - HNO3
Prepared freshly everytime
Violet tinge
Slow decalcification
Cannot detect end point chemically
Acid + calcium ->
soluble calcium
salts
13. WEAK/ DILUTE ORGANIC (MINERAL) ACIDS
Solution Aqueous formic acid Formic – acid formalin
Prep Formic acid (90%) – 10 ml
Distilled water – 90 ml
(Gooding Stewart’s fluid)
Formic acid – 10 ml
Formalin – 5 ml
Distilled water – 100 ml
Time
Taken
2 – 4 days
+ Gentler, Used for Small bone/ teeth
IHC staining
Minimal tissue damage
Fixation + Decalcification
Used forVery small tissues
Minimal damage to tissues
- Slower Increase in formic acid – cell damage
14. BOUIN’S DECALCIFICATION SOLUTION
Formula
Saturated aqueous solution of picric acid
(10.5 g/ 500 ml) – 500 ml
Formalin – 167 ml
Formic acid – 33 ml
Duration required for decalcification
depends on the type and size of tissue
Place bone in buffered neutral
formalin – 2 to 3 days
Decalcification agent 100 times the
volume of tissue
Check for end point
15. ION EXCHANGE RESIN
Aids decalcification in simple
decalcifying fluids
Ammonium form of a sulphonated
polystyrene
20 – 30 times decalcifying agent
Layer Ion – exchange resin on the
bottom
Specimen decalcified & placed on resin
End point determined by X ray
Advantages
IER removes Ca from DA
Reduces Decalcification time
Resin can be reused
Disadvantages
Limited to DA that do not contain
mineral acids
16. CHELATING AGENTS
Organic compounds that bind with
certain metals
EDTA-formalin
EDTA: 5.5 g
Formalin: 10 ml
Distilled water: 90 ml
Neutral EDTAEDTA: 250 g
Distilled water: 1750 ml
NaOH: 25 g
Advantage: Minimal artefacts & good
staining
Disadvantage: slow & not used widely
Tissues fixed in 10% formalin
50 times bulk of 5.5% EDTA with phosphate buffer (pH
7.4)
<5mm thickness tissue – change DA every 4 – 5 days
3 changes done
Tissues again placed in formal saline overnight
Dehydrated & embedded
17. ELECTROPHORETICTECHNIQUE
Principle
Attraction of Ca ions in electric current
Composition of electrolyte
8% HCl
10% Formic acid
Time required for decalcification
6 – 8 hours
Advantage: Rapid decalcification
Disadvantage
Cumbersome
Expensive
Heat damage to tissues
Electrolyte placed in glass jar
Negative electrode: Brass
Positive electrode: Platinum
Platinum wire wound around specimen
Move electrodes closer for faster decalcification
Check by X – ray every 2 – 3 hrs
18. FACTORS INFLUENCINGTHE RATE OF
DECALCIFICATION
Concentration & volume of DA
Suspension of calcified tissue
Age of the patient
Type of bone
Size of specimen
Temperature of decalcifying agents
Agitation
19. DETERMINATION OF END-POINT OF
DECALCIFICATION
Radiography
Check evidence of calcium
Efficient & sensitive method
Many tissues can be X rayed
Physical test
Probing/needling/slicing/bending/
squeezing
Inaccurate & damages tissues
Bubble test
Acids + CaCO3 -> CO2
Bubbles form on tissue surface
20. CHEMICALTEST FOR CALCIUM
Calcium oxalate test
Principle
Detects calcium by precipitation of
insoluble Ca(OH)2 and Calcium oxalate
Interpretation
White precipitate immediately – presence
of residual calcium
White precipitate after Step 2 – less calcium
Clear fluid after 30 mins – Decalcification
complete
5 ml DA from the specimen +
litmus paper/ pH meter
Neutralize by adding Conc. NH4OH till pH 7
Add 5 ml of saturatedAmmonium oxalate
Shake well & stand for 30 mins
21.
22. NEUTRALIZATION OF ACID
Treatment with a weak alkali
Saturated lithium carbonate
5 – 10% aqueous NaCO3
Washing in 2 changes of 70% alcohol for 12 – 18 hrs
23. THOROUGH WASHING
Purpose
To remove the acid/ alkali
Better staining
Method
3 – 4 hours wash in alcohol or overnight in water
Tissues are then routinely processed
Longer wax impregnation/ harder paraffin wax
24. SURFACE DECALCIFICATION
Definition
Removal of small focus of calcification on the
surface of the block
Indication
Partially decalcified bone
Unsuspected Calcium/Mineral deposits
Advantage
Prevent knife damage
Prevent torn tissue sections
Trim & expose the tissue
Place the block face down in 1 -
% HCl, 10% formic acid x 15-60
mins
Rinse with water
Resection in the usual manner
